shotgun
During tracheal development, the tip cells, located at the end of
each branch that is going to fuse, extend filopodia to search for targets; later they change their
cell shape into a seamless ring, to allow passage of lumen. The cell adhesion molecule
E-cadherin accumulates at the site of contact to form a ring that marks the site of lumen
entry and is essential for the fusion. DE-cadherin expression in tip cells of a subset of
branches is dependent on escargot, a zinc finger gene expressed in all tip cells. Such escargot
mutant tip cells failed to adhere to one another and continue to search for alternative targets
by extending long filopodia. These cells also develop an abnormal cuticle in their blind-ended structure and their terminus floats freely in the body. escargot positively regulates
transcription of the DE-cadherin gene, shotgun. Overexpression of DE-cadherin rescues the
defect in one of the fusion points in escargot mutants, demonstrating an essential role of
DE-cadherin in target recognition and identifying escargot as a key regulator of cell adhesion
and motility in tracheal morphogenesis (Tanaka-Matakatsu, 1996).
Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity
gene products, Dishevelled (Dsh), Zeste-white 3 (Zw-3), and Armadillo (Arm), work sequentially; Wg
and Dsh negatively regulate Zw-3, which in turn down-regulates Arm. To biochemically analyze
interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which binds to Arm, Dsh, Zw-3, and Arm, were overexpressed in the Drosophila wing disc cell line, clone 8, which responds
to Wg signal. Dsh overexpression leads to accumulation of Arm primarily in the cytosol and elevation of
DE-cadherin at cell junctions. Overexpression of wild-type and dominant-negative forms of Zw-3
decreases and increases Arm levels, respectively, indicating that modulation in Zw-3 activity negatively
regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevates
DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm
accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative Zw-3-, and truncated
Arm-induced accumulation of DE-cadherin protein is accompanied by a marked increase in the
steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by
Wg signaling. In addition, overexpression of DE-cadherin elevates Arm levels by stabilizing Arm at
cell-cell junctions (Yanagawa, 1997).
The JAK/STAT signaling pathway, renowned for its effects on cell proliferation and survival, is constitutively active in various human cancers, including ovarian. JAK (Hopscotch) and STAT are required to convert the border cells in the Drosophila ovary from stationary, epithelial cells to migratory, invasive cells. The ligand for this pathway, Unpaired (Upd: Outstretched), is expressed by two central cells within the migratory cell cluster. Mutations in upd or jak cause defects in migration and a reduction in the number of cells recruited to the cluster. Ectopic expression of either Upd or JAK is sufficient to induce extra epithelial cells to migrate. Thus, a localized signal activates the JAK/STAT pathway in neighboring epithelial cells, causing them to become invasive (Silver, 2001).
In order to gain further insight into the mechanism by which STAT regulates border cell migration, the expression of a number of proteins that are highly expressed in border cells was examined, some of which are also required for migration. The first gene identified as playing a critical role in border cell migration was slow border cells (slbo). slbo encodes Drosophila C/EBP, a basic region/leucine zipper transcription factor. Slbo protein expression is undetectable in stat mutant border cells, which were identified using a positive clone marking system known as MARCM. This result was confirmed by examining several additional proteins, the expression of which is reduced in slbo mutant border cells. In wild-type stage 8 and 9 egg chambers, FAK expression is upregulated in migratory border cells. Border cells that lack stat exhibit reduced levels of FAK. In wild-type stage 9 egg chambers, DE-cadherin (Shotgun) is enriched throughout the border cell cluster and is expressed to the highest level in the polar cells. Stat mutant border cells exhibit reduced DE-cadherin expression compared to wild-type border cells of the same cluster. The polar cells, though mutant, do not show reduced DE-cadherin staining, which is also true of slbo mutants. Additional downstream targets of slbo, including PZ6356 and zinc finger transcription factor jing, are also reduced in stat mosaic clones. Thus, even the few mutant cells that are recruited to the cluster fail to express many border cell proteins required for migration. The effect is specific because expression of Taiman, a protein that is required for border cell migration but is independent of the slbo pathway, was not altered. Mosaic clusters containing a mixture of wild-type and mutant cells show variable migration defects. On average, the extent of migration is proportional to the number of wild-type cells in the cluster (Silver, 2001).
Egg chambers from females heterozygous for any of the stat alleles have a semi-dominant border cell migration phenotype. Advantage was taken of this slight haploinsufficiency to test for dominant genetic interactions with other genes required for border cell migration. Dominant genetic interactions were observed with slbo, hop, and upd alleles. A mutation in the gene coding for DE-cadherin, shotgun, also exhibited a dominant interaction with stat. These interactions appeared to be specific, since stat does not interact with other known border cell migration genes, such as tai, jing, or PZ6356 (Silver, 2001).
At the time of normal border cell migration, expression of
Drosophila E-cadherin (DE-cadherin: Shotgun) increases within the
border cells, and Drosophila C/EBP (Slbo) is required for this
elevation of DE-cadherin expression. Drosophila ß-catenin, known as Armadillo (Arm), colocalizes with DE-cadherin in both wild-type and mutant egg
chambers. To determine whether
jing function is also required for proper accumulation of DE-cadherin
and Arm, egg chambers containing jing mutant
border cells were stained with antibodies against DE-cadherin
or Arm, and the staining was compared to wild-type and slbo
mutant border cells. In wild-type border cell clusters, staining
for DE-cadherin and Arm is strongest in the central cells
known as polar cells, which express FASIII and in the junctions between border
cells. The staining is somewhat less intense and punctate in
appearance at the interfaces between border cells and nurse
cells. In slbo mutant clusters, DE-cadherin and Arm staining
is only detected in the central polar cells. Border cells mutant for jing exhibit normal expression of both DE-cadherin and Arm.
Thus jing function, unlike slbo, is not required for either DE-cadherin or Arm expression. In all cases FASIII staining is
normal, indicating normal polar cell fate (Liu, 2001).
DE-cadherin expression is required for border cell migration
and is reduced in slbo mutant border cells, but not in jing mutant border cells. Yet
expression of Jing is able to rescue the migration defect
associated with the slbo hypomorph. DE-cadherin
expression in the P[hs-jing];slbo/slbo egg chambers
was examined to determine whether the cells were able to migrate despite the
absence of DE-cadherin expression or, alternatively, whether
high levels of Jing were able to restore DE-cadherin
expression. DE-cadherin expression in the
border cells is restored in early stage 9 slbo;hs-jing egg
chambers, following expression of Jing, but not at
later stages (Liu, 2001).
Thus DE-Cadherin expression is not affected in jing mutant
clones, though it is reduced in slbo mutants. DE-cadherin
expression may require the presence of either jing or slbo. In
slbo mutants, expression of a jing-lacZ reporter is also
reduced, and DE-cadherin expression is affected. However in
jing mutants, slbo expression does not appear to be reduced and
DE-cadherin expression is unaffected. However DE-cadherin
expression may require that some Slbo protein is present since
over-expression of Jing does not rescue the strong female sterile
combination of slbo alleles (LY6/e7b) even though it does rescue
the weaker allele (slbo1). This selective rescue has also been
observed with hs-breathless, which rescues the mild but not the
strong female sterile slbo alleles. To date
only hs-slbo has been observed to rescue the border cell
migration defects associated with the strongest female sterile
alleles of the slbo locus. Thus jing
cannot completely substitute for slbo, consistent with the
observation that there are multiple downstream targets of slbo
with essential roles in border cell migration (Liu, 2001).
Among the diverse cellular processes taking place during oogenesis, the delamination and migration of border
cells (BCs), a group of anterior follicle cells, represent a powerful model to study cell invasion in a normal tissue.
During stage 9 of oogenesis, BCs detach from the outer epithelium to invade the germline cyst compartment. The BC cluster contains two
centrally located polar cells surrounded by approximately six outer border cells and undergoes a nearly 6-hour long posteriorward migration to reach
the anterior part of the growing oocyte. Together with centripetal cells, they assemble the micropyle, a specialized structure required for sperm entry. domeless was isolated in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, dome is required in the germarium for the polarization of
follicle cells during encapsulation of germline cells. In this process,
dome controls the expression of the apical determinant Crumbs. In
contrast to the ligand Upd, whose expression is limited to a pair of polar
cells at both ends of the egg chamber, dome is expressed in all
germline and follicle cells. However, Dome protein is specifically
localized at apicolateral membranes and undergoes ligand-dependent
internalization in the follicle cells. dome mutations interact
genetically with JAK/STAT pathway genes in border cell migration and abolish
the nuclear translocation of Stat92E in vivo. dome
functions downstream of upd and both the extracellular and
intracellular domains of Dome are required for JAK/STAT signaling. Altogether,
the data indicate that Dome is an essential receptor molecule for Upd and
JAK/STAT signaling during oogenesis (Ghiglione, 2002).
The dramatic, early follicle cell phenotype contrasts with the essentially
normal phenotype of dome mutant cells observed in later stage egg
chambers. In this case, follicle cells are viable and divide normally. A similar, dual phenotype has been reported in crumbs mutant chambers. After initial polarization of the follicle cells in the germarium, Crumbs is no longer required and its loss has no visible effects. Importantly, dome controls Crumbs expression in follicle cells, thus providing a novel link between the JAK/STAT signaling pathway and epithelial polarity (Ghiglione, 2002).
In addition to its early function in the germarium, dome is
required for the normal expression of several follicle cell markers, including
DE-cadherin and Fas3. It is important to note that despite a clear defect in
the expression of these markers, dome mosaic egg chambers are
morphologically normal. However, because completely mutant egg chambers cannot
be obtained because of the early effect of dome in the germarium, one
cannot rule out the possibility that large mutant clones would lead to
abnormal development of egg chambers (Ghiglione, 2002).
The pattern of epithelial markers in dome mutant cells indicates
that the JAK/STAT pathway is active in all follicle cells, a notion that is
reinforced by the wide expression of nuclear Stat92E. How is Dome activated
during egg chamber development and does this activation follow the same
profile at all stages? Given the restricted pattern of upd expression
in the egg chamber and its dramatic effect upon overexpression, it is unlikely
that Upd is able to signal long distances in the follicular epithelium of late
stage egg chambers. Rather, a model by which the JAK/STAT pathway
plays a pre-patterning function is favored, acting early during egg chamber development to activate DE-cadherin and Crumbs expression. This
view is consistent both with the expression pattern of upd and the
distribution of Dome-containing vesicles described in this study. The formation of endogenous vesicles can be promoted by Upd, and
a gradient of such vesicles is present around polar cells. Strikingly,
these vesicles, which likely indicate active signaling through Dome, are
widespread at early stages and become more restricted later on. It is proposed that during early development, the Upd signal produced by anterior and
posterior polar cells contributes to the differentiation of all follicle
cells. At this stage, Upd would be more diffusible than later, as suggested by
the pattern of Dome intracellular vesicles. The study of the
mechanisms controlling Dome activation and Upd activity will require
additional tools to directly detect Upd, as, for example, Upd-GFP fusion
proteins (Ghiglione, 2002).
This study has revealed several new findings about the function of
dome and the JAK/STAT pathway during oogenesis. Future work will help
to understand how Upd and Dome initially interact at the cell surface and
transduce the signal to downstream JAK/STAT pathway members (Ghiglione, 2002).
In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies.
Genes involved in cell adhesion and/or development of the neural
system (i.e., FasII, mfas, Ama,
Lac, and shg) are two of the main classes
regulated by ash2. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).
Remodelling of tissues depends on the coordinated regulation of multiple cellular processes, such as cell-cell communication, differential cell adhesion and programmed cell death. During pupal development, interommatidial cells (IOCs) of the Drosophila eye initially form two or three cell rows between individual ommatidia, but then rearrange into a single row of cells. The surplus cells are eliminated by programmed cell death, and the definitive hexagonal array of cells is formed, which is the basis for the regular pattern of ommatidia visible in the adult eye. This cell-sorting process depends on the presence of a continuous belt of the homophilic cell adhesion protein DE-cadherin at the apical end of the IOCs. Elimination of this adhesion belt by mutations in shotgun, which encodes DE-cadherin, or its disruption by overexpression of DE-cadherin, the intracellular domain of Crumbs, or by a dominant version of the monomeric GTPase Rho1, prevents localisation of the transmembrane protein IrreC-rst to the border between primary pigment cells and IOCs. As a consequence, the IOCs are not properly sorted and supernumerary cells survive. During the sorting process, Notch-mediated signalling in IOCs acts downstream of DE-cadherin to restrict IrreC-rst to this border. The data are discussed in relation to the roles of selective cell adhesion and cell signalling during tissue reorganisation (Grzeschik, 2005).
To summarise, IrreC-rst is colocalised with DE-cadherin in epithelial cells of pupal eye discs, and misdistribution of adherens junction components induces the mislocalisation of IrreC-rst, which then affects sorting of IOCs. However, although DE-cadherin forms a continuous belt in the apical regions of all cells (including all IOCs) in wild-type discs, IrreC-rst colocalises with DE-cadherin only at the border between 1° pigment cells and IOCs. What factor(s) might be responsible for the spatial restriction of IrreC-rst to this border? It has recently been shown that the removal of Notch or Delta function during cell-sorting results in the ubiquitous distribution of IrreC-rst to all plasma membranes and the prevention of programmed cell death. This study analysed whether this might be the result of defective DE-cadherin localisation. Antibody staining reveals no influence of Notch on the continuous apical localisation of DE-cadherin, but shows that IrreC-rst now colocalises with the latter on all plasma membranes of the IOCs. This suggests that Notch acts downstream of DE-cadherin in the control of IrreC-rst localisation. It is therefore tempting to speculate that it is the Notch pathway, which provides local signalling between the lattice cells to direct cell death, that prevents the accumulation of IrreC-rst at the borders between IOCs and thus restricts its localisation to the 1°/IOC cell boundary (Grzeschik, 2005).
Pattern formation in the Drosophila eye disc depends on a well-balanced system of signals that promote either the survival or the death of cells, mediated by the EGF and Notch receptor pathways, respectively. In addition, the morphogenetic events, which take place in a single-layered epithelium, crucially depend on factors that regulate the maintenance of cell polarity and cell shape, and modulate cell adhesion. Sorting of interommatidial cells (IOCs) during pupal development, which results in the conversion of several parallel rows of cells into a single ring, requires the weakening of pre-existing adhesive cell contacts and the establishment of new ones without interrupting the epithelial integrity of the tissue. During tissue morphogenesis, epithelial cells use different strategies to modify their adhesive contacts. One of these consists of regulating the amount and/or distribution of the homophilic cell-adhesion molecule E-cadherin, one of the central components of the adherens junctions. The first in vivo evidence for this kind of regulation came from the analysis of the Drosophila egg chamber. There, the localisation of the oocyte at the posterior pole depends on a higher level of expression of DE-cadherin in the oocyte and the posterior follicle cells, when compared with the nurse cells and other follicle cells. Differential adhesion can also be regulated by alterations in the composition or activity of intracellular binding partners, or by the integration of various other molecules into the adhesive complexes. No change in the distribution of the adherens junction components DE-cadherin and alpha-catenin could be detected in wild-type discs undergoing rearrangements of the IOCs. This behaviour contrasts with epithelial rearrangements during morphogenesis of the Drosophila tracheal system, which are associated with alterations in the amount of DE-cadherin, controlled by Drosophila Rac, another member of the Rho GTPase family. This in turn suggests that other cytoplasmic or transmembrane proteins are involved in the modulation of adhesion in IOCs. The adhesion protein IrreC-rst is involved in the control of the cell sorting process. Its predominant localisation at the border between primary pigment cells and IOCs (at the 1°/IOC border) has been suggested to provide an attractive interface that controls sorting. According to this proposal, IOCs tend to maximise their contacts with primary pigment cells. Failure to restrict IrreC-rst to this border results in the inability to sort the IOCs properly. Although IrreC-rst behaves as a homophilic adhesion molecule when expressed in cell culture, data from expression analysis argue for the presence of a different, as yet unknown, partner in the primary pigment cell (Grzeschik, 2005).
Of particular interest is the relationship between the localisation of DE-cadherin, a component of the zonula adherens (ZA) and IrreC-rst. In wild-type discs IrreC-rst colocalises with DE-cadherin at the 1°/IOC border in the apical ZA of the cell and removal of DE-cadherin completely abolishes IrreC-rst accumulation. Nothing is yet known about how IrreC-rst may integrate into the ZA at this border. In vertebrates, the Ca++-independent cell adhesion molecule nectin, a transmembrane protein of the immunoglobulin superfamily, has been implicated in the organisation of cadherin-based adherens junctions, tight junctions and synapses. It is recruited into cadherin-based adherens junctions through interactions with the PDZ domain of l-afadin, an F-actin-binding protein. Intriguingly, the C-terminal sequence of IrreC-rst (T-A-V) matches the consensus binding site for class I PDZ domains (S/T-X-V). Interestingly, the protein encoded by the mutant allele irreC-rstCT, which lacks the C-terminal 175 amino acids of the wild-type form, is no longer recruited into the ZA. It is, however, unlikely that IrreC-rst acts as a general adhesion molecule in IOCs of pupal eye discs, because the epithelial tissue structure is stable in the absence of irreC-rst function, as deduced from the formation of the continuous apical belt of DE-cadherin in irreC-rst mutants (Grzeschik, 2005).
The continuous belt of DE-cadherin can be disrupted by a number of different genetic conditions, such as overexpression of the membrane-bound intracellular domain of Crumbs, of DE-cadherin itself, or of a dominant-negative version of the monomeric GTPase Rho1. Overexpression of the membrane-bound intracellular domain of Crumbs in embryonic epithelia has been shown to lead to a redistribution of DE-cadherin throughout the plasma membrane and the formation of multilayered tissues. By contrast, IOCs overexpressing Crbintra exhibit a fragmented DE-cadherin belt, which remains localized in the apical zone of the cells, and apicobasal organisation and tissue integrity are not affected. This suggests that IOCs may contain additional adhesion components which are independent of, or less affected by, Crb. Support for this view comes from the phenotype of discs lacking crb function, in which the apical belt of DE-cadherin expression is fragmented, yet there is no major effect on polarity or adhesion of the epithelium: the cells undergo nearly normal sorting and IrreC-rst is still restricted to the membrane at the 1°/IOC border. Overexpression of CrbintraDeltaERLI (lacking four C-terminal amino acids [ERLI] of its short cytoplasmic domain, which serve to recruit a multiprotein complex that forms apical to the zonula adherens) does not interfere with sorting, suggesting that a protein complex similar to the one that controls apicobasal polarity in embryonic epithelia (which includes Stardust, DPATJ and D-Lin7) contributes to the development of the dominant phenotype (Grzeschik, 2005).
Overexpression of DE-cadherin similarly results in the fragmentation of the adhesion belt and defects in cell sorting. In various tissues, overexpression of full-length DE-cadherin can also reduce Wingless signalling by sequestering Armadillo from the cytoplasmic pool, thus making it unavailable to transduce the Wingless signal. However, the possibility that the defects in sorting are the result of a suppression of Wingless signalling can be excluded. Inactivation of components of the Wingless pathway in eye imaginal discs induces the initiation of ectopic morphogenetic furrows, and this phenotype was not observed upon overexpression of DE-cadherin. Overexpression of DE-cadherin in eye discs therefore seems to interfere with adhesion, rather than Wingless signalling (Grzeschik, 2005).
Rho GTPases play central roles in the organisation of the actin cytoskeleton and in cell adhesion. In mammals, inhibition of Rho activity results in the removal of cadherins from epithelial cell junctions, while increased Rho activity induces an invasive and metastatic phenotype. Members of the Rho GTPase family are recruited into the adherens junctions by direct interactions with junctional components. Thus, in Drosophila, Rho1 localises to the adherens junctions and interacts directly with alpha-catenin and p120ctn, a homologue of ß-catenin. As in pupal epithelia expressing a dominant-negative form of Rho1, Rho1 mutant embryos exhibit a diffuse distribution of components of the ZA, such as DE-cadherin and alpha- and ß-catenin. Rho1 may either act directly on the accumulation of cadherins at the junctions, or indirectly by recruiting accessory proteins, which then modulate the amounts or activity of junctional and/or cytoskeletal proteins. Rho1 plays a different role in tracheal epithelia insofar as its inactivation does not disrupt DE-cadherin localisation, but rather interferes with the formation of the apical surface and the tracheal lumen (Grzeschik, 2005).
Although it is evident that DE-cadherin plays a crucial role in the accumulation of IrreC-rst at the adherens junctions, other mechanisms are required to explain the asymmetric localisation and restriction of the latter to the 1°/IOC boundary. It has been speculated that an as yet unknown ligand expressed in the primary pigment cell may account for this restricted accumulation. As an alternative, but not mutually exclusive model, it is suggested that signalling between the IOCs, mediated by Notch, which is expressed in IOCs during pupal development, prevents the accumulation of IrreC-rst at their borders. Interplay between adhesion and signalling molecules also directs other processes in which cellular polarisation is involved in tissue remodelling. The growth of the wing imaginal disc along the proximodistal axis, for example, is the result of cell shape changes and cell rearrangements during pupal development, which are controlled by the atypical cadherins Fat and Dachsous, as well as Four-Jointed, which is assumed to be a secreted molecule. This process, in turn, is responsible for the asymmetric localisation of components that control planar polarity, such as Frizzled, Dishevelled or Strabismus, that serves to ensure that bristles and hairs adopt a common orientation. During germ band elongation in the Drosophila embryo, adherens junction remodelling in intercalating ectodermal cells is facilitated by the polarised expression of non-muscle myosin II at the anteroposterior and of Bazooka at the dorsoventral cell boundaries. Future experiments will demonstrate whether cell sorting in pupal eye discs makes use of any of the components known to be involved in these processes (Grzeschik, 2005).
Invasive cell migration in both normal development and metastatic cancer is
regulated by various signaling pathways, transcription factors and
cell-adhesion molecules. The coordination between these activities in the
context of cell migration is poorly understood. During Drosophila oogenesis, a
small group of cells called border cells (BCs) exit the follicular epithelium to
perform a stereotypic, invasive migration. The ETS transcription
factor Yan is required for border cell migration and Yan expression is
spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor tyrosine kinase pathways (Egfr and Pvr) in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. These results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells (Schober, 2005).
The dynamic expression of Yan is crucial for BC migration, as indicated by
the migratory defects associated with both gain- and loss-of-function alleles
of yan. Analysis of mutations in the JAK/STAT and Notch
signaling pathways reveals that they are required for the
expression of at least two transcription factors that are crucial for BC
migration and which themselves influence DE-Cad activity. Slbo is specifically
expressed in BCs and enhances shg transcription. Yan, by contrast, is
expressed in anterior terminal cells, but becomes upregulated in BCs at the
time they exit from the epithelium to become migratory. Yan might enhance
DE-Cad turnover to facilitate the transition from an immobile epithelial state
to a migratory one. Enhanced BC migration defects of hypomorphic slbo
mutant egg chambers overexpressing Yan further underscore their interaction to
regulate DE-Cad expression and BC migration (Schober, 2005).
Is the function of Yan to facilitate the transition of BCs from an
epithelial to a migratory state, or to promote their motility? Although
E-Cadherin is often downregulated as cells transit from an epithelial to a
mesenchymal-like migratory state, this may not be the case in BCs, since DE-Cad is strongly
expressed in BCs and shg mutant BCs fail to migrate.
However, BCs mutant for yan or Ecdysone hormone co-receptor taiman
(tai) accumulate ectopic
DE-Cad-containing adhesive complexes. Consistent with these observations, ectopic
stimulation of PVR in BCs, which enhances tai mutant BC migration
defects, also results in elevated, cortical DE-Cad staining. Even
though the observed BC migration defects in these mutants might not be due to
altered surface levels of DE-Cad only, it was found that overexpression of DE-Cad
alone can cause migration impaired BCs. E-cadherin not only mediates
homophilic cell-cell adhesion but also functions together with its binding
partners as a key regulator of the cortical actin cytoskeleton. It is
therefore interesting to note that follicle cells overexpressing DE-Cad show
severely enhanced filamentous actin staining (Schober, 2005).
The experiments revealed that DE-Cad was elevated in yan mutant
BCs and suppressed upon expression of UAS-yanACT,
suggesting that Yan controls, at least in part, DE-Cad expression in BCs.
These observations find further support in the partial rescue of
slbo-Gal4::UAS-yanACT-induced BC migration defects upon
co-expression of UAS-DE-Cad. How does Yan affect DE-Cad expression in
BCs? Although the function of Yan as a transcriptional repressor in various
tissues suggests
that it may act as a transcriptional regulator of shg, no change was detected
in shg transcription in yan mutant follicle
cells. However, increased membrane dye FM1-43 incorporation in Drosophila SL2
cells overexpressing YanACT, and a decrease in incorporation after
yanRNAi, suggests a change in endocytic activity.
E-Cadherin has been found in endocytic compartments and endocytosis
has been speculated to modulate E-Cadherin activity regulation during
morphogenetic movements. Interestingly, blocking endocytosis by the expression of
dominant-negative Rab5 leads to severe BC migration defects and increased
DE-Cad staining. Consistent with these observations, expression of shg
under a heterologous promoter has been shown to rescue shg
mutant BC migration defects, suggesting that the dynamic expression of DE-Cad
in BCs might depend on both transcriptional and post-transcriptional
mechanisms. Based on these results, a model is favored whereby Yan might,
at least in part, function to regulate DE-Cad turnover, possibly through the
transcriptional regulation of as-yet-unidentified components of the endocytic
machinery (Schober, 2005).
Loss of function of the Drosophila exocyst components in epithelial cells results in E-Cadherin (Shotgun) accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits Shotgun delivery to the membrane. Rab11 and Armadillo interact with Sec15 and Sec10, respectively. These results support a model whereby the exocyst regulates E-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located (Langevin, 2005).
In budding yeast, the exocyst has been proposed to tether post-Golgi vesicles to the membrane of the growing bud prior to fusion. This model is supported by several observations. (1) Exocyst components localize both on post-Golgi vesicles and on the bud membrane (Boyd, 2004). Analogously in Drosophila, Sec5 and Sec15 localize along the lateral membrane and on the REs. (2) Mutations in genes encoding components of the exocyst complex lead to the accumulation of post-Golgi vesicles (Novick, 1980). Analogously, Sec5, Sec6, and Sec15 loss of function leads to an enlargement of the recycling endosome (RE) compartment; this enlargement interpreted as an accumulation of RE vesicles. (3) The localization of Sec8p and Exo70p at the growing bud, i.e., the site of polarized exocytosis, depends on the function of the other exocyst components. Analogously, Sec5 is localized along the lateral membrane, where E-Cadherin delivery is affected, and its localization along the cortex depends on Sec6. It is therefore proposed that in Drosophila epithelial cells, Sec5, Sec6, and Sec15 act by tethering vesicles originating from the recycling endosomal compartment to the lateral membrane of epithelial cells, as a prerequisite for their exocytosis (Langevin, 2005).
In epithelial cells, Arm and E-Cadherin colocalize to the AJs of the ZA as well as along the lateral membrane. In the absence of Sec5, Sec6, and Sec15 function, E-Cadherin trafficking is affected and E-Cadherin accumulates in the RE. Similarly, in the absence of arm, E-Cadherin fails to localize at the membrane and localizes in the RE. The identification of an interaction between Arm and Sec10 is therefore consistent with a model whereby this interaction provides a landmark at the site where Arm is enriched in order to deliver E-Cadherin from the recycling endosomes. Nevertheless, Arm may play an additional role in stabilizing E-Cadherin at the AJs. A direct demonstration of the function of Arm in regulating the delivery of E-Cadherin will therefore require the identification of arm mutant alleles that do not perturb its function as a regulator of E-Cadherin stabilization and only affects its interaction with Sec10 (Langevin, 2005).
In the absence of Sec5, Sec6, or Sec15 function, E-Cadherin delivery to the lateral membrane is inhibited and E-Cadherin accumulates in the REs. Furthermore, E-Cadherin was found to transcytose in a Sec5-dependent manner from the lateral membrane of epithelial cells to the apical AJs. Therefore, this study reveals at least a role of the exocyst in the recycling of E-Cadherin from the lateral membrane to the apical AJs. Furthermore, the strong reduction of E-Cadherin present on the lateral membrane is interpreted as a failure to recycle E-Cadherin from the lateral membrane back to the lateral membrane, which cannot be compensated for by the delivery of newly synthesized E-Cadherin to the lateral membrane. The loss of E-Cadherin on the lateral membrane may also lead to a reduction of E-Cadherin delivery at the AJs. This may have also contributed to the loss of epithelial cell polarity observed in some of the sec5 mutant epithelial cells (Langevin, 2005).
In polarized MDCK cells, the apical REs are well known as a site of sorting during endocytic and transcytotic transport. The REs have also been shown to serve as an intermediate during the transport of newly synthesized proteins from the Golgi to the plasma membrane in nonpolarized MDCK cells. Similarly, upon overexpression of GFP-E-Cad in HeLa cells, E-Cad transits from the Golgi to the Rab11 endosomes. Nevertheless, the existence of such a pathway remains to be established in polarized MDCK cells. In fact, the overexpression of a dominant-negative form of Rab11 leads to sequestration of E-Cadherin in the REs, but whether sequestered E-Cadherin represented newly synthesized or recycled E-Cadherin was not determined. The existence of such a Golgi-to-RE pathway also remains to be established in Drosophila epithelial cells. If so, a role of the exocyst in regulating the delivery of newly synthesized E-Cadherin from the Golgi to the lateral membrane via the REs remains plausible (Langevin, 2005).
Whether the exocyst regulates E-Cadherin localization in mammalian cells has not been directly analyzed. However, E-Cadherin is proposed to act as a regulator of the localization of the exocyst complex in polarizing mammalian cells since E-Cad- and Nectin-2α-dependent cell-cell contacts were proposed to recruit the exocyst complex in order to promote the growth of the lateral epithelial cell domain. The current study suggests that upon the recruitment of the exocyst complex by E-Cadherin, the exocyst promotes the delivery of more E-Cadherin to the lateral membrane during the establishment of apico-basal polarity. In fact, several reports can be reconciled with a function of the exocyst in regulating the transport of E-Cadherin in mammalian cells. Thus, polarized exocytosis of E-Cad to the lateral membrane is dependent upon its interaction with Arm. And, as stated above, REs have shown to serve as an intermediate during the transport of E-Cad from the Golgi to the lateral membrane where E-Cadherin, β-Catenin, and α-Catenin form the AJs. Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane. Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11. Finally, E-Cadherin and catenins are associated with exocyst components (Langevin, 2005 and references therein).
In conclusion, this work provides evidence for a conserved role of the exocyst in regulating the delivery of E-Cadherin from REs to sites on the plasma membrane and in thereby contributing to the maintenance of epithelial cell polarity (Langevin, 2005).
Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).
Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).
Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).
The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).
In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).
The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).
The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).
Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation, and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle. However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis, bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).
This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).
This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors, and, in this study, it has been observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).
Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development (Takahashi, 2005). The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).
The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells (reviewed in Gumbiner, 2005). Evidence for such a mechanism has been provided for integrin-mediated focal adhesions (reviewed in Hynes, 2002), and Src activity can alter focal adhesions (Yeatman, 2004). However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).
The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts (Stewart, 2003) in vitro (Vidal, 2006).
However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M (Read, 2004; Stewart, 2003). Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).
This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).
This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis. Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).
While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).
In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).
JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).
JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).
Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).
Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).
It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).
Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).
Although many organ functions rely on epithelial tubes with correct dimensions, mechanisms underlying tube size control are poorly understood. This study analysed the cellular mechanism of tracheal tube elongation in Drosophila, and describes an essential role of the conserved tyrosine kinase Src42A in this process. Src42A was shown to be required for polarized cell shape changes and cell rearrangements that mediate tube elongation. In contrast, diametric expansion is controlled by apical secretion independently of Src42A. Constitutive activation of Src42A induces axial cell stretching and tracheal overelongation, indicating that Src42A acts instructively in this process. It is proposed that Src42A-dependent recycling of E-Cadherin at adherens junctions is limiting for cell shape changes and rearrangements in the axial dimension of the tube. Thus, distinct cellular processes are defined that independently control axial and diametric expansion of a cylindrical epithelium in a developing organ. Whereas exocytosis-dependent membrane growth drives circumferential tube expansion, Src42A is required to orient membrane growth in the axial dimension of the tube (Forster, 2012).
These findings establish a critical role of Src in epithelial tube morphogenesis. Src42A is necessary and sufficient for cell shape changes that specifically mediate tracheal tube elongation, whereas diametric tube expansion is independent of Src42A function. Consistent with previous work, this study found that Src42A regulates E-Cad recycling at adherens junctions. On the basis of these data it is proposed that Src42A-induced adherens-junction remodelling is limiting for polarized cell shape changes that mediate axial, but not diametric, dorsal-trunk expansion. In this model, secretion drives apical membrane growth independently of Src42A function, which is required to direct apical membrane growth in the axial dimension of the tube. However, the signals activating Src42A in tracheal cells and the Src42A substrates mediating polarized cell behaviours remain elusive. No evidence was found for planar-polarized Src activity in tracheal cells or for a role of Src42A in PCP signalling, indicating that potential PCP cues that might direct axial expansion may be downstream or parallel to Src42A activity. In principle, even subtle asymmetries in the distribution of adherens-junction components may result in large-scale tissue transformations. Alternatively, polarized cell behaviour may result from the inherent anisotropy imposed by cylindrical geometry of tubular epithelia. On the basis of Laplace's law, circumferential tension on a pressurized cylinder's surface is larger than longitudinal tension. Anisotropic tissue tension might orient cell behaviour in tubular epithelia. Normal epithelial cells cultured on cylindrical substrates orient circumferentially, whereas Ras-transformed cells orient axially, accompanied by re-orientation of actin filaments, indicating that cells may be able to sense and respond to tissue geometry. It is proposed that Src42A is involved in sensing cylindrical tissue geometry, possibly as a mechanical force sensor, or by translating this information into polarized cell behaviour. The specific effect of Src42A mutations on axial, but not circumferential, cell shape changes may reflect different requirements for Src42A in remodelling axial and circumferential adherens junctions, respectively. A possible explanation for Src42A affecting elongation of the dorsal trunk and transverse connective, but not of dorsal branches, whose elongation relies on adherens-junction remodelling, is that dorsal-branch cell intercalation relies on pulling forces generated by migrating tip cells, which may override the Src42A requirement. Conversely, the absence of tip-cell-mediated pulling forces in the dorsal trunk and transverse connective may imply an increased requirement for Src42A-mediated junction remodelling in these branches. This work provides a genetically tractable paradigm to analyse Src function in regulating polarized cell behaviours that control epithelial tube dimensions during normal organogenesis or under pathological conditions, such as polycystic kidney disease, in which Src has been implicated (Forster, 2012).
Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several 'noncore' AJ components have been identified that modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. dASPP has been identified as a positive regulator of dCsk (Drosophila C-terminal Src kinase) (Langton, 2007). This study shows that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, abnormal E-cadherin localization is observed in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. Because Src is known to regulate AJs in both Drosophila and mammals, it is proposed that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. The dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene (Langton, 2009).
Cell-cell contacts are essential for development and adult life of multicellular organisms. The best-characterized form of cell-cell contact is the adherens junction (AJ), which links neighboring cells via homotypic E-cadherin (E-Cad) interactions. The highly conserved intracellular domain of E-Cad binds to β-catenin, which itself binds to α-catenin. Transient interactions between α-catenin and actin filaments link AJs to the cytoskeleton, though the exact nature of this connection remains controversial. AJs are particularly important for the integrity of epithelial tissues. In addition to establishing and maintaining cell-cell adhesion, AJs regulate several aspects of cellular behavior, including cytoskeletal rearrangement and transcription. Inappropriate disruption of cell-cell contacts can lead to excess proliferation and is a hallmark of the metastatic process (Langton, 2009).
Dynamic remodeling of AJs occurs during all major morphogenetic events involving movement and rearrangement of epithelial cells, including convergent extension and gastrulation. AJ remodeling is necessary for the generation of epithelial structures with extremely precise patterns, such as the hexagonal array of ommatidia in the Drosophila compound eye (Langton, 2009).
SRC signaling is a major cellular pathway known to promote AJ remodeling in development and metastasis. Cellular SRC (c-SRC) is a member of the SRC family kinases (SFKs), which include c-SRC, FYN, and YES. Activated c-SRC is known to regulate AJs by several mechanisms. For example, c-SRC can induce the ubiquitylation of E-Cad by an E3 ubiquitin ligase called Hakai, promoting E-Cad internalization or degradation. In Drosophila, Src42A (one of two c-Src homologs) genetically interacts with E-Cad (encoded by shotgun [shg] in Drosophila), localizes to AJs, and forms a ternary complex with E-Cad and Armadillo (Drosophila β-catenin). Furthermore, Src42A activation leads to decreased E-Cad protein levels and concurrent stimulation of E-Cad transcription by Armadillo and TCF, which is thought to be important for AJ turnover during morphogenesis (Langton, 2009).
The C-terminal region of c-SRC and other SFKs is targeted by C-terminal SRC kinase (CSK), which negatively regulates c-SRC by phosphorylating a conserved tyrosine residue (Tyr527 in avian c-SRC). Drosophila CSK appears to function analogously to mammalian CSK as a negative regulator of SFKs. dCsk is a negative regulator of tissue growth; mutants die as giant pupae and imaginal discs are enlarged as a result of increased proliferation. These observations are seemingly at odds with studies showing that Src activation in Drosophila tissues stimulates proliferation but also leads to considerable apoptosis. A recent report attempted to reconcile this discrepancy, suggesting that lower levels of Src activation induce proliferation and protection from apoptosis, whereas high levels lead to apoptosis and invasive migration (Langton, 2009 and references therein).
It has been shown that dCsk activity is modulated by dASPP, the Drosophila homolog of mammalian ASPP1 and ASPP2, which physically interacts with dCsk and enhances its capacity to phosphorylate Src42A (Langton, 2007). Accordingly, dASPP phenotypes are enhanced by reducing dCsk gene dosage and are rescued by complete removal of Src64B, which functions redundantly with Src42A. This study identifies dRASSF8 as a new dASPP regulator. dRASSF8 is the homolog of mammalian RASSF7/8 (Ras association domain family 7/8). Ras association (RA) domain-containing proteins are putative Ras effectors; they specifically bind the activated (GTP-bound) form of Ras family GTPases, which function in numerous signal transduction pathways regulating proliferation, apoptosis, and differentiation. Mammalian RASSF family members 1–6 are characterized by their domain structure, with a C-terminal RA domain, a C1-like zinc finger, and a SARAH (Salvador-RASSF-Hippo) domain. Mammalian RASSF7–10 are atypical RASSF proteins because they contain an N-terminal RA domain and lack a C1-like or SARAH domain. Recently, Xenopus RASSF7 was shown to be required for completing mitosis. Human RASSF8 is a putative tumor-suppressor gene; when expressed in lung cancer cells, RASSF8 inhibits anchorage-independent growth. Importantly, the molecular function of RASSF8 has not been elucidated (Langton, 2009).
Two RASSF family proteins are encoded by the Drosophila genome. dRASSF is similar to human RASSF1–6 and has been linked to the Hippo pathway. dRASSF8 is similar to human RASSF7 and RASSF8, having an N-terminal RA domain. Published genome-wide yeast two-hybrid data suggested that dRASSF8 interacts with dASPP, prompting an investigation of the relationship between these proteins. Based on genetic and biochemical data, it is suggested that the dASPP-dRASSF8 complex regulates AJs by directing the activity of dCsk and Src (Langton, 2009).
dRASSF8 is the sole Drosophila homolog of mammalian RASSF7 and RASSF8, which are so-called N-terminal RASSF proteins and the least-studied members of the RASSF family. This study demonstrates that dRASSF8 binds to dASPP in Drosophila cells and that RASSF8 binds to ASPP1 and ASPP2 in human cells, indicating that an evolutionarily conserved relationship between these proteins has been uncovered. The function of RASSF8 is currently unknown, and this study thus provides new insights into the function of N-terminal RASSF proteins (Langton, 2009).
Future experiments will determine whether RASSF7 also binds ASPP family proteins or whether this function is specific to RASSF8. RASSF7 has been studied in Xenopus and was found to associate with centrosomes and to be required for completing mitosis. In contrast, the current data suggest that dRASSF8 is not required for cell-cycle progression because null mutants for dRASSF8 are viable. These findings are suggestive of divergent functions for RASSF7 and RASSF8 in vertebrates, with dRASSF8 being functionally analogous to RASSF8 rather than RASSF7. Indeed, GFP-tagged RASSF7 localizes to the nucleus and centrosomes in Xenopus embryos, whereas this study never observed nuclear localization of dRASSF8. Further studies of N-terminal RASSF proteins in vertebrates should clarify whether RASSF7 and RASSF8 have overlapping or independent functions (Langton, 2009).
In vivo data point at a close relationship between dRASSF8 and dASPP, which colocalize and are required for each other's presence at AJs in epithelial cells. dRASSF8 posttranscriptionally regulates the levels of dASPP protein in epithelia. Thus, it seems likely that binding to dRASSF8 stabilizes dASPP and prevents its degradation, which can be observed for many protein complexes. Overall, these data provide compelling evidence for a functional link between dRASSF8 and dASPP, which is likely to be conserved through to their closest mammalian counterparts, RASSF8 and ASPP1/2 (Langton, 2009).
The data suggest that dRASSF8 has some dASPP-independent roles. For example, dRASSF8 mutant wings are large and broadened, whereas dASPP mutant wings are large but of normal shape. In addition, the dRASSF8 adult eye phenotype is more marked than that of dASPP mutants. Accordingly, it was found that dRASSF8, but not dASPP, is required for apoptosis of excess IOCs in the developing pupal retina. It therefore appears that the dRASSF8 eye phenotype results from both reduced apoptosis of IOCs and cell-cell adhesion defects. The subtle differences between the dASPP and dRASSF8 phenotypes indicate unknown functions for dRASSF8, which are not due to its effects on dASPP. Future efforts will be aimed at elucidating these functions (Langton, 2009).
These data are consistent with a model in which dRASSF8 binds to and positively regulates dASPP and, in this way, promotes dCsk activity indirectly. Coimmunoprecipitation experiments support this idea, showing that dRASSF8 and dASPP associate and that dASPP and dCsk associate. However, no detect interaction was detected between dRASSF8 and dCsk, indicating that dRASSF8 does not directly associate with dCsk. The proposed model is also supported by genetic data; the dRASSF8-dCsk genetic interaction is weaker than the dASPP-dCsk interaction, suggesting that dASPP is the primary regulator of dCsk. The weaker genetic relationship between dRASSF8 and dCsk can be explained by the observation that some dASPP protein persists in dRASSF8 mutant tissue. These observations suggest that dRASSF8 regulates dCsk via dASPP (Langton, 2009).
Retinal morphogenesis involves dynamic changes in cell-cell contacts to create the final ordered array of photoreceptors and accessory cells. dASPP and dRASSF8 are required for normal E-Cad localization in 26-27 hr APF retinas, providing an explanation for the patterning defects in mutant eyes. It is proposed that the abnormal E-Cad localization in dASPP mutant eyes results from increased Src activity based on several lines of evidence. dASPP binds to and positively regulates dCsk, leading to Src inhibition; therefore, loss of dASPP increases Src activity, which is known to reduce cell-cell adhesion by promoting the internalization and degradation of E-Cad. In agreement with this, it was shown that loss of dCsk or overexpression of either Drosophila Src leads to loss of AJ material in 26-27 hr APF retinas. This claim is further supported by the fact that the dASPP eye phenotype is suppressed by loss of Src64B. Thus, the presence of the dASPP-dRASSF8 complex at AJs may be required to locally prevent inappropriate Src activation and dissolution of AJs (Langton, 2009).
The fact that dASPP and dRASSF8 mutants are homozygous viable implies that these genes are dispensable for the majority of morphogenetic processes occurring during development. Therefore, the regulation of AJs by dASPP and dRASSF8 may be restricted to the eye. However, as they are expressed in other epithelial tissues, a closer examination of dASPP and dRASSF8 mutants may reveal subtle defects in other morphogenetic processes (Langton, 2009).
It is suggested that dASPP and dRASSF8 are new noncore AJ components and part of the machinery that ensures the fine regulation of AJs by Src during development. This regulation is crucial to provide precisely the right amount of junctional plasticity to allow cell-cell rearrangements and patterning to take place while limiting this plasticity to maintain epithelial coherence and prevent cell delamination. Because the interaction between these proteins is conserved in mammals, this finding is likely to be relevant to mammalian development and to the metastatic process, which is associated with downregulation of E-Cad and loss of cell-cell adhesions. Indeed, ASPP1 knockout mice present defects in the assembly of lymphatic vessels consistent with a potential adhesion defect. This suggests that regulation of cell-cell adhesion may underlie the function of ASPP1/2 and RASSF8 as mammalian tumor suppressors (Langton, 2009).
All stem cells have the ability to balance their production of
self-renewing and differentiating daughter cells. The germline stem cells
(GSCs) of the Drosophila ovary maintain such balance through physical
attachment to anterior niche cap cells and stereotypic cell division, whereby
only one daughter remains attached to the niche. GSCs are attached to cap
cells via adherens junctions, which also appear to orient GSC division through
capture of the fusome, a germline-specific organizer of mitotic spindles. This study shows that the Rab11 GTPase is required in the ovary to maintain GSC-cap
cell junctions and to anchor the fusome to the anterior cortex of the GSC.
Thus, rab11-null GSCs detach from niche cap cells, contain displaced
fusomes and undergo abnormal cell division, leading to an early arrest of GSC
differentiation. Such defects are likely to reflect a role for Rab11 in
E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling
endosomes (REs) and E-cadherin and Armadillo (ß-catenin) are both found
in reduced amounts on the surface of rab11-null GSCs. The
Rab11-positive REs, through which E-cadherin transits, are tightly associated
with the fusome. It is proposed that this association polarizes the trafficking by
Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC,
thus simultaneously fortifying GSC-niche junctions, fusome localization and
asymmetric cell division. These studies bring into focus the important role of
membrane trafficking in stem cell biology (Bogard, 2007).
The first clue that Rab11 plays important roles in early oogenesis in Drosophila came from immunostaining experiments that revealed strong expression of endogenous Rab11 and a fully functional Rab11::GFP in GSCs, cystoblasts and young (2-4- and 8-cell) germline cysts. Strikingly, the proteins were concentrated as discrete dots on the fusome, which electron microscopy and photobleaching studies have shown is highly vesicular and rapidly exchanged with other membrane stores. Triple-stain experiments showed that some of these dots also contained E-cadherin, which has been shown to transit though Rab11-positive recycling endosomes (REs) en route to the plasma membrane in some cells. High-magnification images showed that the Rab11 (and, more rarely, E-cadherin) dots were often nestled into cavities within the fusome. Such Rab11-harboring cavities are visible in the fusomes of all examined GSCs, cystoblasts and young germline cysts, not only in the ovary but also in the testes. In view of the well-described enrichment of Rab11 in REs, it is proposed that these Rab11- and E-cadherin-harboring cavities are REs and are therefore referred to as FREs (fusome-associated REs) (Bogard, 2007).
These studies indicate that Rab11 maintains GSC identity through polarized trafficking of E-cadherin and, possibly, other cargoes that reinforce essential GSC-niche contacts. These studies further indicate that Rab11 is required for fusome localization and asymmetric GSC division and suggest a feedback linkage between these events and E-cadherin trafficking. Although Rab11 has been implicated in the trafficking of E-cadherin in other cells, there are no other cases in which such trafficking has been correlated with a biological response. It will be of interest to determine whether Rab11 is required for the maintenance of stem cells in other systems and whether such maintenance involves E-cadherin trafficking or the trafficking of other adhesion molecules. It will also be of interest to determine the role of Rab11 in other E-cadherin-dependent cell behaviors, particularly as Rab11, at least in Drosophila, is expressed in only a small subset of E-cadherin-expressing cells (Bogard, 2007).
E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. This study shows that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis results in cell intercalation defects. A pathway is delineated that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, evidence is provided that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin (Levayer, 2011).
Epithelial tissues have a robust architecture that is essential for their barrier function. This barrier function depends on their ability to build adhesive contacts at adherens junctions through the recruitment and stabilization of E-cadherin (E-cad), β-catenin (β-cat) and α-catenin (α-cat) by actin filaments (F-actin). During development, epithelia are also extensively reshaped by remodelling of cell contacts. This plasticity is essential for morphogenesis during embryogenesis and organogenesis. Work in the past decade showed that this requires force generation by actomyosin networks and their anchoring at cell junctions by E-cad/β-cat/α-cat complexes. Thus, E-cad plays a pivotal role in junction robustness and plasticity by mediating both adhesion (cohesion) and tension transmission (remodelling). Understanding what controls the distribution and dynamics of E-cad/β-cat/α-cat complexes is therefore key to understanding cell packing and the mechanics of tissue morphogenesis. Disruption of this balance marks key steps in the progression of solid tumours. The loss of epithelial organization during the epithelial to mesenchymal transition is an extreme example in which E-cad endocytosis causes the loss of adhesion and tension transmission at the cell cortex (Levayer, 2011).
The early development of the Drosophila embryo is a powerful system to study epithelial morphogenesis. Spatial regulation of force generation by actomyosin networks and force transmission to adhesion by E-cad both contribute to apical cell constriction in the invaginating mesoderm1, and cell intercalation in the elongating ectoderm called the germ band. Germ-band extension (GBE) is driven by cell intercalation in the ventrolateral region, whereby cells exchange neighbours through planar polarized junction remodelling, namely shrinkage of 'vertical' junctions (that is, junctions oriented along the dorsoventral axis). Intercalation is powered by non-muscle Myosin-II (Myo-II): anisotropic actomyosin contractile flows from the medial apical region to 'vertical' junctions drive junction shrinkage. The shortening of vertical junctions is stabilized by Myo-II at the cortex. Actomyosin contractility is transmitted at the cortex by E-cad complexes through β-cat. Interestingly, E-cad/β-cat/α-cat complexes also exhibit a planar polarized distribution complementary to that of Myo-II: E-cad is less abundant in shrinking 'vertical' junctions. This E-cad polarity is also required to orient actomyosin flows to 'vertical' junctions. It is unknown what controls the planar polarized distribution of E-cad. This may depend on Rho kinase (ROCK), which is required for the polarized distribution of Par3 (Levayer, 2011).
This study shows that the planar polarized distribution of E-cad is also controlled by an upregulation of clathrin- and dynamin-mediated endocytosis at adherens junctions, in particular in 'vertical' junctions of intercalating cells. Blocking endocytosis causes the loss of E-cad planar polarization and a block of intercalation. This led to an investigation of the mechanisms that control planar polarized upregulation of clathrin-mediated endocytosis (CME) of E-cad at adherens junctions. Activation of WASP (Wiscott-Aldrich Syndrome Protein) and the Arp2/3 (Actin-Related Protein 2/3) complex by Cdc42 controls the branched actin polymerization that is required for vesicular scission. This study identified an additional pathway controlling the initiation of E-cad endocytosis through the recruitment of the AP2 (Adaptor Protein 2) complex and clathrin. This recruitment is driven by lateral clustering of E-cad that relies on unbranched actin polymerization induced by Dia, and the presence of Myo-II. Dia and Myo-II are both activated by the guanine exchange factor RhoGEF2 (Levayer, 2011).
This study has delineated two distinct roles for actin in E-cad endocytosis. Dia and Myo-II control the initiation of E-cad endocytosis by enrichment of clathrin and AP2 in an E-cad-dependent manner. This is tightly spatially regulated in the ventrolateral region and in 'vertical' junctions during cell intercalation by cortical RhoGEF2 localization, an activator of Dia and Myo-II in Drosophila embryos. This is distinct from the role of branched actin polymerization by Arp2/3, which promotes vesicular scission similarly to dynamin. At later stages of development, this depends on WASP and is controlled by Cdc42, aPKC (atypical protein kinase C) and Cip4 (Cdc42-interacting protein 4. In early embryos, as WASP is inhibited by the JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription), Scar instead plays a critical role in vesicular scission. Inhibition of Arp2/3 in scar mutants and its constitutive activation (artificially induced by myrWASP) did not affect clathrin and AP2 concentration at adherens junctions, unlike Dia. The different tiers of regulation of E-cad endocytosis by Arp2/3 and Dia may reflect different roles for actin in constitutive versus regulated E-cad endocytosis. Certain situations require a rapid change in the rate of endocytosis, and may do so by tuning the rate of initiation by clathrin and AP2. It will be interesting to see whether rapid collapse of adherens junctions during epithelial to mesenchymal transition relies on a similar process (Levayer, 2011).
Crosslinking E-cad with an IgG is sufficient to promote dorsal endocytosis of E-cad by upregulating the concentration of clathrin, similarly to Dia activation, even following inhibition of Dia, Myo-II or RhoGEF2. Considering the highly correlated localizations of E-cad complexes with AP2 and RhoGEF2, it is proposed that Dia and Myo-II control the initiation of E-cad endocytosis by inducing lateral clustering of E-cad, similar to Fc receptor clustering during phagocytosis or nanoclusters of GPI (glycosylphosphatidylinositol)-anchored proteins. This may have been co-opted by the pathogen Listeria, whose entry into epithelial cells requires E-cad endocytosis. This mechanism may also require specific 'priming' of E-cad, by ubiquitylation as in mammals, although these tyrosines are not conserved in flies. Importantly, the mechanism of AP2 recruitment by E-cad remains unknown in all systems (Levayer, 2011).
Inhibition of E-cad endocytosis increased E-cad levels and disrupted its planar polarized distribution. Myo-II also accumulated in the medial apical region of cells. The GBE defects in shi-ts mutants or following clathrin inhibition are the result of the altered distribution of actomyosin tensile forces. E-cad/β-cat/α-cat complexes affect the lateral flow of medial actomyosin pulses and Myo-II polarized junctional accumulation, presumably through the regulation of tension transmission within the medial network and/or at the junctions. The medial accumulation of Myo-II when E-cad endocytosis is inhibited may thus reflect an inhibition of actomyosin flow towards the cortex. These results emphasize the interplay between actomyosin contractile dynamics and E-cad adhesive complexes during epithelial morphogenesis (Levayer, 2011).
Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. This study shows that activation of the G protein-coupled receptor (GPCR) Trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gβ as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. Uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. These findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion (Kunwar, 2008).
Cell migration plays a important role during a variety of processes such as development, immune defense, and metastasis. The coordinated migration of different kinds of cells in space and time gives rise to the three germ layers and the three-dimensional architecture of different organs and organisms. Cells of the immune system migrate through blood vessels and tissues to reach infected sites; and cancer cells migrate away from their tissues of origin to ectopic places during metastasis. Thus far, the basic mechanisms of cell migration have been elucidated mostly from in vitro studies in solitary cells. Cell migration in living, multicellular organisms, however, is likely much more complex. At the onset of directed migration, cells not only have to acquire motility but also have to be able to perceive specific, directional migration cues. During their journey, migrating cells may be required to detect and interpret multiple, possibly conflicting guidance cues, and must coordinate their adhesion to surrounding cells to reorient, pause, and move in a directed fashion while targets change. Finally, at the end, cells have to know when they have reached their target and cease their motility (Kunwar, 2008).
Significant progress has been made in identifying guidance molecules, receptors, and intracellular mediators that act during directed migration. G protein-coupled receptors (GPCRs) have been widely studied for their role in directional migration. Cells use GPCRs to detect and migrate toward higher concentrations of chemoattractants. Immune cells and germ cells, for example, express the chemokine receptor CXCR4 and follow the distribution of the chemokine SDF1 (stromal cell-derived factor 1) (Kunwar, 2008).
Lymphocytes use sphingosine-1-phosphate receptors to egress from lymphoid tissues, where S1P levels are higher. Despite significant progress in identifying the guidance molecules, receptors, and intracellular mediators that act during directed migration, the cellular and molecular mechanisms that initiate cell migration are only poorly understood. At the start of migration, cells need to acquire motility, may lose cell adhesion with neighboring cells, and are required to gain the ability to respond directionally to external cues. The detailed cellular transformations, the temporal sequence of these events, and the relative influence caused by intrinsic and extrinsic cell information are the focus of this study (Kunwar, 2008).
Drosophila germ cells provide a genetically tractable system to visualize and follow individual germ cells as they start directed migration. The onset of directed germ cell migration coincides with the transepithelial migration of germ cells through the primordium of the future midgut. Evidence for a germ cell autonomous function for transepithelial migration came from the identification of a novel GPCR trapped in endoderm 1. Maternal tre1 RNA is present in germ cells, and tre1 function is required there. General cell motility and the movements of germ cells toward the gonad do not depend on Tre1, which suggests that Tre1 specifically regulates the onset of migration (Kunwar, 2008).
To understand the cellular mechanisms underlying the onset of directed migration, two-photon imaging was used to visualize the cellular transformations that occur in vivo as germ cells migrate through the midgut epithelium. Comparison of wild-type and tre1 mutant germ cells suggests that regulated activation of the Tre1 GPCR controls three phases of early migration: polarization of germ cells, dispersal into individual cells, and transepithelial migration. Germ cell polarization leads to a redistribution of cell-cell adherens proteins, such that Drosophila E-cadherin (DE-cadherin) levels are reduced from the leading edge of the migrating cells and accumulate in the tail region. Tre1 likely signals via the G proteins Gγ1 and Gβ13f as well as Rho-1, since Gβ and Rho-1 protein localization is detected in the tail region, and deletion of their function specifically in germ cells causes the same phenotype as mutation in tre1. These results suggest a novel function for GPCR signaling in initiating cell migration by polarizing the migrating cell. This polarization leads to the redistribution of signaling components and adherens proteins and may trigger cell dispersal and directed migration (Kunwar, 2008).
To visualize germ cell migration in developing embryos, a germ cell-specific expression system, which translates the actin-binding domain of Moesin fused to EGFP under the control of nanos regulatory sequences, was used (Sano, 2005). Germ cells appear motile soon after their formation at the blastoderm stage (stage 5, 2 h and 10 min to 2 h and 50 min after egg laying [AEL]), as they produce small protrusions away from their neighbors. Despite this apparent motility, germ cells only rarely (1-2 germ cells per embryo) separate from their neighbors and migrated directly through the underlying blastoderm cells. Subsequently, during gastrulation (stage 7-8, 3 h to 3 h and 40 min AEL), as germ cells are internalized together with the invaginating posterior midgut primordium, they round up and show less protrusive activity. At stage 9 (3 h and 40 min to 4 h and 20 min AEL), germ cells are found inside the midgut primordium in a tight cluster; they are in close contact with each other and show little contact with the surrounding somatic midgut cells. During this stage, germ cells started to reorganize, changed their shape, and take on a highly polarized morphology. Using electron microscopy, a radial organization of germ cells within the midgut is clearly visible, with the large germ cell nuclei pointed toward the midgut while fine membranous material, apparently corresponding to the tail region, fills the inside of the cluster. This organization orients the leading edge of each germ cell toward the surrounding midgut primordium. Next, the germ cells lose adhesion to each other, and extensions reach from the germ cells toward the midgut epithelium (Kunwar, 2008).
Subsequently, germ cells disperse as they migrated through the midgut primordium to reach the basal side of the midgut cells by stage 10 (4 h and 20 min to 5 h and 20 min AEL). Long cytoplasmic extensions connected germ cells with each other immediately after the onset of transepithelial migration. As germ cells transmigrate through the midgut epithelium, they appear completely individualized, display amoeboid behavior, and are polarized with a broad lagging edge and actin localized at the leading edge. On average, individual germ cells transmigrate the midgut within 40 min from the onset of polarization. Tracking of individual germ cells showed that they disperse radially and transmigrate in all directions through the pocket of the midgut epithelium After transmigration, germ cells reorient on the midgut toward the dorsal side of the embryo, sort into two bilateral groups, and migrate toward the gonadal mesoderm, which forms on either side of the embryo (Kunwar, 2008).
Tre1 encodes an orphan GPCR that is required maternally in germ cells for their migration through the midgut epithelium. In embryos from tre1 mutant females, (hereafter referred to as 'mutant embryos'), germ cells fail to cross the midgut epithelium. This phenotype could result from a defect in the acquisition of motility by germ cells or in their ability to polarize, disperse, or transmigrate. To distinguish between these possibilities, tre1 mutant germ cells were observed live by in vivo imaging. At stage 5, tre1 germ cells show small protrusions and sporadically cross the blastoderm with a similar frequency to the wild type. In striking difference to the wild type, however, the tre1 germ cell cluster does not reorganize at stage 9 and fails to transmigrate to the midgut. Mutant germ cells do not polarize, and remain in a tight, disorganized group in which germ cells fail to interact with the surrounding midgut cells (Kunwar, 2008).
To begin to understand how Tre1, an orphan GPCR, initiates germ cell migration, it was asked whether Tre1 function is mediated by trimeric G protein activation in germ cells. It was found that only a single Gγ (Gγ1) and a single Gβ (Gβ13f) subunit are provided maternally. Loss of maternal Gβ13f or Gγ1 function causes defects in gastrulation, which precluded an immediate analysis of germ cell migration (Kunwar, 2008).
However, it was possible to rescue the gastrulation defect through early zygotic, soma-specific expression of the respective G protein. This genetic manipulation allowed testing for a germ cell-specific role of these G proteins, since early Drosophila germ cells are transcriptionally silent, and germ cells thus depend completely on the maternally provided G proteins. In embryos rescued for the gastrulation defect, Gβ13f mutant germ cells are unable to disperse and migrate through the midgut epithelium, and thus resembled the tre1 phenotype. Gγ1 mutants showed a similar although slightly weaker phenotype likely caused by residual function of the Gγ1N159 allele used, which lacks the C-terminal isoprenylation site required for membrane anchoring. These results suggest that germ cell transepithelial migration requires Tre1-mediated canonical G protein signaling (Kunwar, 2008).
For Gα proteins, focus was placed in particular on the role of the single D. melanogaster Gα12/13A homologue, encoded by concertina (cta), because this subfamily of G proteins has been shown to regulate cell migration and metastatic invasion and to directly interact with E-cadherin and Rho1. Cta protein is present in the germ cells and maternal loss of cta causes a gastrulation defect similar to Gβ13f and Gγ1. Again, it was possible to rescue the gastrulation phenotype by early, somatic Cta expression, as described for Gγ1 and Gβ13f. In contrast to findings with Gβ and Gγ mutants, however, cta mutant germ cells migrated normally to the gonad. To confirm this result, mutant cta germ cells derived from cta mutant mothers were transplanted into wild-type embryos. It was found that cta germ cells migrated to the gonad with similar efficiency as transplanted wild-type control germ cells. Thus, Gα12/13 does not act as the sole mediator of Tre1 GPCR activation. Analysis of the available mutants in other Gα proteins did not reveal a single Gα protein that mediates the Tre1 signal, which perhaps indicates that redundant or overlapping functions of Gα proteins act downstream of Tre1 (Kunwar, 2008).
The observation that both Gβ13f and Gγ1 are required for germ cell dispersal and transepithelial migration suggests that Tre1 function in germ cells is mediated by a G protein-dependent pathway, akin to the requirement for GPCR signaling seen during the directed migration of Dictyostelium discoideum amoeba and in neutrophils toward a chemokine gradient. To determine how Tre1 signaling may affect downstream components, the localization of Gβ13f protein was analyzed, as well as the localization of Rho1, which has been shown to affect germ cell transepithelial migration in wild-type and tre1 mutant germ cells. It was found that Gβ13f and Rho1 proteins were localized uniformly along the cell membrane of wild-type germ cells at the blastoderm stage (Kunwar, 2008).
At stage 9, as wild-type germ cells polarize, Gβ13f and Rho1 proteins are down-regulated along the germ cell membranes facing the midgut, and become highly enriched in the tail region. In early germ cells, Gβ13f and Rho1 proteins are uniformly distributed in tre1 mutants similar to the wild type; in contrast to the wild type, however, this uniform distribution persists during stage 9. These results suggest that Tre1 receptor activation leads to germ cell polarization in part by causing the redistribution of downstream signaling molecules away from the leading edge and accumulation in the tail (Kunwar, 2008).
tre1 mutant germ cells fail to disperse at the onset of the migration, which suggests that tre1 regulates adhesion molecules in germ cells. DE-cadherin is a good candidate, since it is deposited maternally in the early embryo. The role of DE-cadherin was first tested in the adhesion of wild-type germ cells. For this analysis, a newly identified partial loss-of-function allele of Drosophila E-cadherin encoded by the shotgun (shg) gene, which allows normal oogenesis, was used. In embryos derived from shgA9-49 mutant ovaries, germ cells did not organize into a radial cluster. Instead, germ cells separated from one another prematurely, at early stage 8 (3 h and 10 min to 3 h and 40 min AEL) compared with stage 10 in the wild type (4 h and 20 min to 5 h and 20 min AEL). This dispersal phenotype was observed in embryos from homozygous germ line clones, in which embryonic patterning defects were rescued by a wild-type shg+ copy from the father (M-Z+). This suggests that DE-cadherin is required autonomously in germ cells, since they are transcriptionally quiescent and thus likely depend exclusively on maternally contributed DE-cadherin. These results indicate that DE-cadherin is required for germ cell-germ cell adhesion in the wild-type embryo (Kunwar, 2008).
To understand how DE-cadherin is regulated in the dispersal step, the distribution of DE-cadherin was analyzed in wild-type germ cells. It was found that DE-cadherin as well as α and β catenins were initially uniformly present along the germ cell membrane but become enriched in the tail region during germ cell polarization (Kunwar, 2008).
In stark contrast, DE-cadherin remained uniformly distributed along the cell surface in tre1 mutant embryos. To quantitate the levels, the fluorescent intensity of DE-cadherin staining on the cell membrane of wild-type and tre1 mutant germ cells was compared. It was found that DE-cadherin is distributed uniformly and that levels are similar in wild-type and mutant germ cells at stage 5, before migration, whereas the levels are reduced along the leading edge membrane of wild-type germ cells compared with tre1 mutant germ cells at stage 9. These results suggest that tre1 activation leads to a reduction of DE-cadherin along the leading edge and restricts it in the tail region (Kunwar, 2008).
In shg mutants, early dispersal of germ cells does not lead to premature migration through the midgut, as would be expected if release of germ cell-germ cell adhesion via E-cadherin was the only trigger for transepithelial migration. Instead, shg mutant germ cells moved through the midgut slightly later during stage 10 than wild-type germ cells. This delay phenotype is less penetrant compared with the precocious dispersal phenotype and could be caused by an impaired ability of the shgA9-49 mutant germ cell to migrate at this and subsequent stages (Kunwar, 2008).
To test directly if Tre1 acts via DE-cadherin in transepithelial migration, embryos were generated that lacked tre1 and maternal shgA9-49 function. The germ cells in these embryos dispersed early, thus displaying a phenotype similar to shgA9-49 mutants; 80% of tre1, shgA9-49 double mutant embryos showed precocious dispersal as opposed to 0% in the tre1 mutant embryos (Kunwar, 2008).
However, even these dispersed germ cells were not able to transmigrate through the midgut in tre1, shgA9-49 double mutant embryos, thereby resembling tre1 mutant germ cells. This suggests that loss of germ cell-germ cell contact may not be sufficient to trigger transepithelial migration. To test this idea further, germ cell-germ cell contact was disrupted independent of E-cadherin function by reducing the germ cell number. Alleles of the maternal effect gene tudor (tud) were used to reduce the number of germ cells in the embryo to a single germ cell. Such single, tud mutant germ cells migrated through the midgut and invariably reached the gonad. These germ cells had normal morphology and appeared polarized. Next, mutant embryos lacking both tre1 and maternal tud were analyzed. In the absence of tre1, single germ cells were left inside the midgut and did not migrate to the gonad. Thus, whereas germ cell individualization requires Tre1-mediated down-regulation of DE-cadherin, Tre1 activity has additional roles in transepithelial migration (Kunwar, 2008).
This study has used live imaging to explore the mechanisms by which germ cells acquire motility and traverse the midgut epithelium. It was found that before transepithelial migration, germ cells polarize toward the midgut and down-regulate E-cadherin from the leading edge and accumulate E-cadherin in the tail region. This polarization requires Tre1 GPCR activity. It is proposed that GPCR-mediated polarization triggers germ cell dispersal and orients germ cells toward the midgut for directed transepithelial migration (Kunwar, 2008).
A requirement for GPCR signaling during the directed migration toward a chemokine gradient has been described in detail in D. discoideum amoeba and in mammalian neutrophils. The events underlying signal transduction leading to the polarization of migrating cells have been worked out extensively in these cells. The first localized response to receptor activation is the enrichment of the activated G protein βγ subunits, which results in the activation of phosphoinositide 3 (PI3) kinase. As a consequence of chemokine sensing, the PI3 kinase product phosphatidylinositide 3,4,5-tris phosphate (PIP3) becomes localized to the leading edge, and the phosphatase PTEN (phosphatase and tensin homolog) moves to the lagging edge in a Rho dependent manner (for review see Affolter, 2005). These signaling events organize the cytoskeleton leading to cellular polarization and directional movement. The current studies suggest a new mechanism by which GPCR signaling initiates directed cell migration. Activation of Tre1 causes a redistribution of G protein β, the GTPase Rho1, DE-cadherin, and other adherens junction components to a small region in the tail of the germ cells. The decrease in DE-cadherin from the leading edge of germ cells causes a loss of adhesion across the broad leading edge of the germ cells and causes germ cell polarization toward the midgut. This localization event may thereby convert an adherent group of cells into directionally migrating individuals. Tre1 belongs to a family of GPCRs that includes Moody in D. melanogaster and GPR84 in mouse and human (Bainton, 2005; Bouchard, 2007). Based on the results with Tre1, this family may act to regulate cellular polarity and adhesion, a view in line with the proposed function of Moody in epithelial morphology at the blood-brain barrier, and with GPR84, which was recently described to be up-regulated in microglia upon infection (Kunwar, 2008).
How could Tre1 activation cause DE-cadherin redistribution? Regulation of E-cadherin is widely attributed to play an important role in metastasis and in the epithelial-to-mesenchymal transition that occurs during gastrulation and neural crest migration. In these systems, it has been proposed that E-cadherin is regulated by transcriptional repression or by Gα12/13-mediated uptake and turnover. The data suggest the presence of a different mode of regulation, since neither transcriptional regulation nor Gα12/13 activity seem to be required for the regulation of DE-cadherin in germ cells. An attractive mechanism for DE-cadherin down-regulation could be the control of its endocytosis by Tre1. During zebrafish gastrulation, Rab GTPases have been shown to control E-cadherin turnover and the adhesion of mesendodermal cells (Ulrich, 2005). A role for Rab proteins in germ cell migration has yet to be demonstrated. This study found the same localization pattern for Gβ13f, Rho1, and DE-cadherin in the wild type, and this pattern is disrupted in tre1 mutant germ cells. This suggests a role for G protein and Rho1 activation in the polarization of DE-cadherin in germ cells (Kunwar, 2008).
Tre1 also affects transepithelial migration independently of global DE-cadherin regulation. Uniform down-regulation of DE-cadherin or loss of germ cell-germ cell contact in single cells are neither sufficient to trigger precocious transepithelial migration in the wild type nor able to suppress the tre1 transepithelial migration phenotype. One possibility is that the localized activation of Tre1 and polarized down-regulation of DE-cadherin at the leading edge would orient germ cells radially toward the midgut. This radial orientation would allow germ cells to respond to additional guidance cues required for directed transepithelial migration. Although these additional guidance cues may not depend on DE-cadherin, they require G protein signaling and Tre1 (Kunwar, 2008).
A function for E-cadherin in controlling adhesion and migration has been studied extensively in the progression of tumor metastasis and the development of epithelial-mesenchymal transitions (EMTs). Cells undergoing metastasis and EMTs express lower levels of E-cadherin, and the loss of E-cadherin promotes invasion of tumor cells. The loss of E-cadherin in these cases promotes the disruption of E-cadherin-mediated cell adhesion between epithelial cells, allowing these cells to spread and migrate, and is often triggered through induction of the transcriptional repressors Twist and Snail in response to inductive signals. However, in the case of germ cell dispersal, the effect of Tre1 on DE-cadherin is not transcriptional because DE-cadherin is provided maternally in the germ cells. These data suggest that Tre1 GPCR signaling might regulate the turnover or cellular distribution of DE-cadherin-mediated adhesion complexes in a polarized fashion. It is possible that in addition to transcriptional mechanisms, such a polarized regulation also functions during EMT and metastasis (Kunwar, 2008).
The class III phosphatidylinositol-3 kinase [PI3K (III)] regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate [PI(3)P]. While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. This study, in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) were examined in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes (Abe, 2009).
Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive (Abe, 2009).
One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P). PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture. Genetic and pharmacological analysis, using yeast and mammalian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (1) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes; (2) the recycling from early endosomes back to the Golgi complex or other destinations; (3) the entry of proteins into the lysosomal degradation pathway; (4) the formation of internal vesicles of MVBs and (5) autophagy. Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans. In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy. It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes. However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear (Abe, 2009 and references therein).
To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion, although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena (Abe, 2009).
To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 this study analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA), whereas the cell adhesion molecules FasIII and β-integrin are localized in lateral and basal membranes, respectively. This study found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, it was found that dVps15 knockdown results in the accumulation of FasIII at the lateral membrane, whereas it results in intracellular accumulation of DE-cadherin, Fmi and β-integrin. Importantly, inactivation of Rbsn-5 shows accumulation of FasIII and β-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization (see in contrast Mottola, 2010). These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains (Abe, 2009).
This study demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and β-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study (Abe, 2009).
Rbsn-5, a FYVE domain-containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and β-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5C241 null mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface (Abe, 2009).
PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes. However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. The current results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III) (Abe, 2009).
The Drosophila post-embryonic neuroblasts (pNBs) are neural stem cells that persist in the larval nervous system where they proliferate to produce neurons for the adult CNS. These pNBs provide a good model to investigate mechanisms regulating the maintenance and proliferation of stem cells. The transcription factor Grainyhead (Grh), which is required for morphogenesis of epidermal and tracheal cells, is also expressed in all pNBs. This study shows that grh is essential for pNBs to adopt the stem cell program appropriate to their position within the CNS. In grh mutants the abdominal pNBs produced more progeny while the thoracic pNBs, in contrast, divided less and produced fewer progeny than wild type. Three candidates were investigated to determine whether they could mediate these effects; the neuroblast identify gene Castor, the signalling molecule Notch and the adhesion protein E-Cadherin. Neither Castor nor Notch fulfills the criteria for intermediaries, and in particular Notch activity is dispensable for the normal proliferation and survival of the pNBs. In contrast E-Cadherin, which has been shown to regulate pNB proliferation, is present at greatly reduced levels in the grh mutant pNBs. Furthermore, ectopic expression of Grh is sufficient to promote ectopic E-Cadherin and two conserved Grh-binding sites were identified in the E-Cadherin/shotgun flanking sequences, arguing that this gene is a downstream target. Thus one way Grh could regulate pNBs is through expression of E-cadherin, a protein that is thought to mediate interactions with the glial niche (Almeida, 2005).
Previous studies have shown that E-Cadherin is necessary for normal pNB proliferation. These studies show that expression of a dominant negative E-Cadherin in the neural and glial cells reduces the number of progeny produced by pNBs to <25% of wild type. Expression in the ensheathing glia alone led to more minor reduction, arguing that the protein is needed in both glia and pNBs. As reported previously, strong expression of E-Cadherin was detected in the pNBs and their adjacent progeny. In grh370 however, the levels of E-Cadherin in the thoracic region of the CNS were dramatically reduced. Several pNBs lack significant E-Cadherin expression all together, others retained some expression but at much lower levels compared to wild type. Similar effects were seen in clones mutant for another loss of function grh allele, grhB32. In lineages homozygous for grhB32 there was a variable reduction in E-Cadherin compared to neighbouring wild-type lineages (Almeida, 2005).
The effects on Cadherin contrast with those on Notch, where expression in the thoracic pNBs remains robust in grh370, arguing against an indirect effect resulting from changes in size. To further test this, it was asked whether Grh is sufficient to promote E-Cadherin expression when expressed elsewhere in the CNS. A pros::Gal4 driver line was used that directs high levels of expression in neurons and lower expression in the pNB lineages. When this was used to drive expression of the CNS isoform of grh, high levels of ectopic E-Cadherin were detected, particularly in many of the embryo-derived neurons that are normally devoid of E-Cadherin expression at these stages. Neither Castor nor Notch expression was altered under these conditions. Therefore Grh appears to be an activator of E-Cadherin expression. However, ectopic Grh was not sufficient to direct additional proliferation under the conditions tested (Almeida, 2005).
The genomic sequence flanking the E-Cadherin gene (shotgun, shg) was examined for consensus Grh binding sites using two different strategies. Grh binds as a dimer. In recent studies of Grh family proteins a consensus target-site was derived (WCHGGTT). Eight matches to this consensus are present in the genomic region spanning from 1 kb upstream of the shg transcript (another gene, CG10540, starts 944 bp upstream of shg) to 5 kb downstream. A second search using a weighted matrix revealed 13 matches within 5 kb of shg. A comparison of the two sets of putative sites identified four common matches: AAACAGGTTA (−300); AAACAGGTAA (+275); ATACTGGTTT (2650 bp downstream, Shg2); CAACAGGTAG (3131 bp downstream, Shg1). The latter two are 100% conserved between D. melanogaster and the five other Drosophila species for which sequence is available. To confirm that these two sites are recognised by Grh, a stringent assay was used where their ability to compete with a well-characterised, high affinity site, Gbe2 from the Dopa decarboxylase gene, was tested. Both sites were able to compete, reducing the amount of probe bound by 51% (Shg1) and 75% (Shg2) when present at 40× molar excess. The presence of these conserved sites indicates therefore that shg/E-Cadherin is likely to be a direct target of Grh. However, E-Cadherin cannot be the only target, since it was not possible to rescue the grh370 mutant phenotype by supplying E-Cadherin via an exogenous driver (GrhNB::Gal4/UAS::E-Cadherin) (Almeida, 2005).
Although the data show that Cadherin is regulated by Grh, they do not resolve unequivocally whether it is a direct target. Examination of genomic sequence revealed two binding-sites close to the shotgun/E-Cadherin gene that are conserved in other Drosophila species and that are bound by Grh in vitro. Future studies will show whether these sites are essential for shg expression. However, these results are exciting because they provide a link between grh function in the pNBs and in other tissues. Changes were observed in E-Cadherin levels in response to grh in other parts of the animal. There is also an interaction between shg and several genes that act together with grh in epidermal morphogenesis (although no direct genetic interaction was seen between shg and grh itself). Given that the precise levels of E-Cadherin proteins can be critical in shaping the sorting and interactions between cells it will be important to determine whether Grh is required for this regulation. It will also be important to establish whether Cadherins are targets of Grh in other animals, for example in mice where mutations in Grhl3 result in defects in neural tube closure and epidermal integrity (Almeida, 2005).
The defects in the pNB lineages of grh mutants are position dependant. Thus, the thoracic pNBs produce fewer progeny whereas abdominal pNBs proliferate for a more prolonged period. In general, such A/P position dependent patterning is co-ordinated by the homeotic genes and indeed abdA has been shown to regulate the timing of cell death and hence the period of proliferation in the abdominal pNBs, as well as regulating the number of pNBs that persist in abdominal segments. However the phenotype of abdA mutants is significantly different from that of grh; for example the abdominal clusters are much larger and there are no defects in thoracic clusters, so it is unlikely that grh is upstream of abdA. Furthermore, in parallel studies Cenci (2005) has shown that the initiation of AbdA expression still occurs in grh mutants. Therefore it is more likely that grh acts in parallel to the homeotic genes to co-ordinate the pNB program (Almeida, 2005).
Development of organ-specific size and shape demands tight coordination
between tissue growth and cell-cell adhesion. Dynamic regulation of cell
adhesion proteins thus plays an important role during organogenesis. In
Drosophila, the homophilic cell adhesion protein DE-Cadherin
regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study
shows that along the proximodistal (PD) axis of the developing wing epithelium,
apical cell shapes and expression of DE-Cad are graded in response to
Wingless, a morphogen secreted from the dorsoventral (DV) organizer in
distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft)
tumor suppressor, by contrast, represses DE-Cad expression. In
genetic tests, ft behaves as a suppressor of Wg signaling.
Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg
signaling, is negatively correlated with the activity of Ft. Moreover, unlike
that of Wg, signaling by Ft negatively regulates the expression of Distalless
(Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg
signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert
opposing regulation to coordinate cell-cell adhesion and patterning along the
PD axis of Drosophila wing (Jaiswal, 2006).
Cells of the dorsoventral (DV) boundary in the wing imaginal disc
synthesize Wg. The DV boundary marks the distal end of the growing
appendage, while the future hinge region, displaying Wg expression in two
concentric rings, marks the proximal wing. The
lacZ reporter of the quadrant enhancer of vestigial
(vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).
In optical sections of the imaginal disc epithelium, AJs are visualized in
the XY or XZ planes based on
immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with
fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and
DE-Cad display characteristic upregulation across the DV boundary along the PD
axis of the wing imaginal disc. Optical sections along the XY plane reveal
higher levels of DE-Cad localization and narrower apical circumferences in the
AJs of cells flanking the DV boundary when compared with those of the more
proximally located cells. Optical sections along the XZ plane further
confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).
Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, ArmS10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell
autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes
in apical cell shapes. In the presumptive hinge region, Wg
overexpression produces a more striking pattern of non-cell autonomous changes
in cell shapes: cells neighboring the Wg-expressing cells appear to organize
as whorls around the former and display epithelial misfolding (Jaiswal, 2006).
Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which
compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of Nts mutants grown at a restricted
temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).
Somatic clones with altered cell-cell adhesion sort out from their
neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or ArmS10 misexpression sort
out from their neighbors and display smooth clone borders, akin to those
misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels
of catenins appears to be the rate-limiting step of Catenin-Cadherin complex
formation at AJs and cell-cell adhesion. Wild type
ß-catenin/Arm (ArmS2), when overexpressed, does not transduce
Wg signaling. Somatic clones overexpressing ArmS2 display
'wiggly' clone borders, unlike those expressing Dsh or ArmS10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from
Wg signaling (Jaiswal, 2006).
To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined.
Cells receiving high threshold of Wg signaling in the wing imaginal discs, as
in those flanking the DV boundary, displayed higher levels of
DE-Cad-lacZ reporter activity when compared with those further away
from the source of Wg expression. Furthermore, somatic clones expressing
ArmS10 or Dsh display cell-autonomous
activation of the DE-Cad-lacZ. Finally, clones expressing the
secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ:
i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).
Somatic clones lacking Ft (ft-/ft-), marked
by loss of GFP, display overgrowth and altered cell-cell adhesion with
characteristic circular and smooth clone borders, unlike the 'wiggly' borders
of their wild type (ft+/ft+) twins that are
marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of
DE-Cad in their AJs and DE-Cad-lacZ. By
contrast, when Ft was overexpressed, levels of both DE-Cad or
DE-Cad-lacZ were downregulated. Besides, following
overexpression of Ft in the posterior wing compartment, cells flanking the DV
boundary displayed wider apical circumferences when compared
with those of the anterior wing compartment. These results suggest that Ft
regulates DE-Cad expression, cell-cell adhesion and apical cell
shapes in the distal wing (Jaiswal, 2006).
The results suggest that by regulating DE-Cad expression, Wg signaling
integrates cell-cell adhesion with tissue growth and pattern. Regulation of
DE-Cad expression could be a prevalent mechanism for coordination of
the emerging pattern in an organ primordium with the spatial control of its
cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells
flanking the stripe of cells along the AP boundary that express the morphogen
Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad
expression. The Ft tumor suppressor, by contrast,
negatively regulates DE-Cad expression in the distal wing. This may
also explain the inverse correlation between the levels of DE-Cad in AJs and
the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft
controls cell-cell adhesions at AJs by regulating DE-Cad
expression (Jaiswal, 2006).
Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other
cellular processes and developmental mechanisms. E-Cad has
been shown to mark the sites of actin assembly on cell surface.
Cadherin complexes regulate cytoskeletal networks and cell polarity,
while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics.
Interestingly, Ft also regulates orientated cell division (OCD) in imaginal
epithelium, which is mirrored by orientation of the spindles of the dividing
cells; OCD may also regulate organ shape along the PD axis.
Misregulation of DE-Cad may thus affect the cytoskeleton and produce
OCD phenotype in ft mutant discs (Jaiswal, 2006).
In both loss- and gain-of-function assays,
this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the
distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained
to be the direct targets of Wg, all available evidence so far
suggests that these targets positively respond to Wg signaling.
These results also show that Ft and Wg signaling intersect and control distal
wing growth and pattern, presumably through their opposing regulation of a
common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling,
Dpp signaling also regulates Q-vg-lacZ; however,
its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by
its intersection with Wg signaling (Jaiswal, 2006).
The results show that Ft negatively regulates Wg signaling. Loss or gain of
Ft induces a telltale sign of perturbations in Wg signaling, namely, changes
in the cellular pool of ß-catenin/Arm,
consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to
its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also
noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).
One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).
Drosophila wing growth is under dynamic spatial and temporal
regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact
cell proliferation in their characteristic ways and activate distinct sets of
PD markers. Although at a very high threshold, Wg signaling inhibits cell
proliferation, at a modest threshold it has been shown to stimulate growth. It is
noted that loss of Ft fails to activate Wg targets that demand a high threshold
of Wg signaling, e.g., Ac, which is required for wing margin
specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ,
which responds to a high threshold of Wg signaling, is also not upregulated by
loss of Ft. Dll responds to a higher threshold of Wg
signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).
Over-proliferation in ft mutant imaginal discs is induced by
perturbation of as yet unidentified disc-intrinsic mechanisms that determine
the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval
life. By contrast, growth in wild-type imaginal discs is determinate, which ceases
after they attain their predetermined sizes even under conditions of unlimited
developmental time; for example, on transplantation into wild-type adult host
abdomen that can sustain development.
ft mutant imaginal discs thus acquire unlimited proliferative
potential, akin to immortalization, a crucial step during tumorigenesis.
It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling,
a pathway implicated in cancers. Several orthologs of Ft have been identified in
vertebrates with diverse functions. It will thus be interesting to explore if
these orthologs of Ft in higher vertebrates also interact with Wnt signaling
and thereby behave as tumor suppressors (Jaiswal, 2006).
Embryonic gonad formation involves intimate contact between germ cells and
specialized somatic cells along with the complex morphogenetic movements
necessary to create proper gonad architecture. Gonad formation in Drosophila requires the homophilic cell-adhesion
molecule Drosophila E-cadherin (DE-cadherin), and also Fear of
Intimacy (FOI), which is required for stable accumulation of DE-cadherin
protein in the gonad. In vivo structure-function analysis is presented
of FOI that strongly indicates that zinc transport activity of FOI is
essential for gonad development. Mutant forms of FOI that are defective for
zinc transport also fail to rescue morphogenesis and DE-cadherin expression in
the gonad. Expression of DE-cadherin in the gonad is
regulated post-transcriptionally and foi affects this
post-transcriptional control. Expression of DE-cadherin from a ubiquitous
(tubulin) promoter still results in gonad-specific accumulation of
DE-cadherin, which is strongly reduced in foi mutants. This work
indicates that zinc is a crucial regulator of developmental processes and can
affect DE-cadherin expression on multiple levels (Mathews, 2006).
It has been unclear whether ZIP family members regulate
developmental processes by acting as zinc transporters or through some other
unidentified function. These data now indicate that FOI regulates gonad
formation through its zinc transporter activity. The ability of the mutant
forms of FOI to rescue gonad morphogenesis and DE-cadherin expression
corresponds directly with their ability to function as zinc transporters. Mutations that strongly affect the zinc transport activity of FOI (e.g., H554A) also strongly reduce the ability of FOI to rescue gonad morphogenesis and DE-cadherin expression. Mutations that only partially affect the zinc transport activity of FOI (e.g., Y646A) retain some ability to rescue gonad morphogenesis and DE-cadherin expression. If FOI affects gonad formation through a function
separate from zinc transport, identification of conserved
residues that affect these two activities independently would have been expected. This was not the
case. Indeed, even single amino acid changes in very different regions of FOI
(e.g., D308A and H554A) affect both zinc transport and gonad morphogenesis. It is
concluded that the zinc transporter function of FOI is essential for gonad
morphogenesis and regulation of DE-cadherin. This reveals a crucial role for
zinc regulation in development and suggests that other ZIP family members with
developmental roles (e.g., zebrafish LIV1) may also act via zinc transport (Mathews, 2006).
In vivo analysis is also informative for revealing domains that are
essential for FOI function. Even though the N-terminal
extracellular domain of FOI shows little sequence conservation with other
family members, and some ZIP family members lack an extended N-terminal
domain, this domain is nevertheless important for FOI function. In addition to their TM character, the specific sequence of the TM
domains is crucial for FOI function. Mutations that are not predicted to
affect the TM structure of FOI, such as mutating a single acidic residue in
TM2 (D308A) or replacing TM6-8 of FOI with similar TM domains from the related
protein CATSUP (CAT TM6-8), still disrupt the in vivo rescue activity of FOI.
Finally, several characteristics were analyzed of the highly conserved HELP
domain in FOI (which may or may not have TM structure). The
predicted amphipathic alpha-helical nature of this domain appears to be
crucial, since altering the pattern of acidic residues (D551A/D558A and
E584A/E588A/D591A) or inserting a helix contorting proline residue (T557P)
disrupts FOI function. In addition, conserved histidines in this domain are
essential (H554A and H583A/H587A), and mutating even a single histidine has a
dramatic effect in vivo. Since FOI is a zinc transporter, it is likely that the
specific sequences of the TM domains form the proper membrane pore for zinc,
while histidines in the N-terminal and HELP domains act to coordinate zinc
before and during transport (Mathews, 2006).
shg and foi are both required for proper gonad and
tracheal morphogenesis, and foi regulates DE-cadherin expression in the
gonad. DE-cadherin protein levels are not reduced in foi mutants simply
because the gonad has failed to coalesce; other mutations blocking gonad
coalescence do not affect DE-cadherin. Thus, it is likely that foi affects DE-cadherin more directly and this is an important aspect of how foi functions in gonad and tracheal development. In support of this, it was found that expression of DE-cadherin was sufficient to partially rescue foi mutant
gonads (Mathews, 2006).
As both DE-cadherin protein and shg RNA levels are reduced in
foi mutant gonads, whether foi affects
DE-cadherin transcription was investigated. Analysis of a shg enhancer-trap
suggests that some aspects of DE-cadherin regulation by foi may be at
the transcriptional level. Recently, it has been shown that a related ZIP
protein, zebrafish LIV1, can regulate the activity of the Zn-finger
transcription factor SNAIL, which may also influence E-cadherin expression
(Yamashita, 2004; Mathews, 2006).
However, although a majority of studies focus on transcriptional regulation
of E-cadherin, it is likely that this essential cell-adhesion molecule is
often regulated at many levels, including through post-transcriptional and
post-translational mechanisms. This study presents clear evidence that DE-cadherin is regulated at the post-transcriptional level in the embryonic gonad. Expression
of DE-cadherin from a general tubulin promoter
(tub-DE-cad) is sufficient to restore gonad-specific DE-cadherin protein
accumulation in shg mutants. Recent work suggests that DE-cadherin localization within the
ovary is also regulated partly through a post-transcriptional mechanism. Thus,
post-transcriptional regulation may be sufficient to generate tissue-specific
patterns of DE-cadherin expression in many contexts. tub-DE-cad is
much less able to restore DE-cadherin protein to the gonad in foi
mutants. This indicates that FOI is required for positive,
post-transcriptional regulation of DE-cadherin. One component of this
regulation is likely to act on shg RNA stability, since foi
affects the gonad-specific accumulation of shg RNA from
tub-DE-cad,
but does not affect the activity of the tubulin promoter. Thus, the
steady-state pattern of shg RNA accumulation does not merely reflect
shg promoter activity but may have a significant post-transcriptional
component. In principle, zinc could regulate the activity of RNA-binding
proteins that affect RNA stability in the same way it regulates DNA-binding
transcription factors. In addition, DE-cadherin may be further regulated at
the protein level in the gonad, such as through regulation of translation or
protein stability (Mathews, 2006).
Recent in vivo work on several ZIP proteins suggests that these zinc
transporters play essential roles in development and disease that may broadly
involve regulation of cell-cell adhesion. In zebrafish,
regulation of SNAIL by LIV1 is essential for the anterior migration of
zebrafish organizer cells and may regulate E-cadherin expression in this
tissue (Yamashita, 2004). According to this model, LIV1 activates SNAIL activity,
which leads to downregulation of E-cadherin and the decreased cell adhesion
necessary for cell migration (Yamashita, 2004). Interestingly, SNAIL is also thought to be an
important regulator of E-cadherin during the progression and metastasis of
certain cancers, such as breast cancer. As a tumor gains metastatic potential,
SNAIL expression is upregulated and E-cadherin is downregulated. Since human LIV-1
is strongly expressed in breast cancer cell lines, and
has been implicated in breast cancer metastasis, it may
function to activate the activity of SNAIL as a transcriptional repressor of
E-cadherin, again allowing for cell migration and metastasis. A similar, but
opposite, relationship may exist in the Drosophila tracheal system,
where the SNAIL family member Escargot (ESG) is a positive regulator of
E-cadherin during the fusion of neighboring tracheal branches. Since FOI is also required for this process, FOI
may act by promoting ESG activity. In this case, FOI and ESG would activate
DE-cadherin expression, which is necessary for cell-cell attachment during
tracheal branch fusion. In the gonad, FOI is also positively required for
DE-cadherin expression. Although ESG is present in the gonad, no changes have been observed in DE-cadherin expression during gonad coalescence
in esg mutants, indicating that
some other target for regulation by FOI and zinc must exist in this tissue. An
important theme in the action of ZIP proteins may be to influence the activity
of zinc-regulated transcription factors, with cell-cell adhesion molecules
being important targets of such regulation. However, it was
found that additional, post-transcriptional mechanisms are crucial in the
gonad for regulation of DE-cadherin protein expression by FOI. Thus, it will
be very important to analyze the contribution of post-transcriptional
regulation of E-cadherin to other developmental and disease processes. Indeed,
there is even evidence that the same crucial factor, SNAIL, can influence
post-transcriptional regulation (Mathews, 2006).
An important issue relevant to the role of zinc and zinc transporters in
development and disease is whether they play an instructive or permissive
role. Is zinc merely required at a minimum threshold level in various tissues
or does regulation of intracellular zinc concentration play a signaling role
at specific times and places? Existing evidence suggests that zinc may play an
instructive role. Both Drosophila foi and zebrafish LIV1 have highly
tissue-specific patterns of expression and affect the development of selected
tissues, while others remain unaffected.
Mammalian ZIP and Cation Diffusion Facilitator family members also have
tissue-specific expression patterns. Thus,
zinc transporters have the necessary spatial and temporal resolution to play
an instructive role. In addition, zinc transporters have clear roles as
modulators of intracellular signals. They have the capacity to modulate
signaling pathways, for example the ras pathway, and
can influence transcription factor activity and gene expression.
Because the zinc transport activity of FOI is crucial for
its developmental role, it is likely to act by modulating zinc concentration.
Thus, zinc has the potential to be an important and dynamically regulated
signaling molecule during development and adult homeostasis (Mathews, 2006).
The Drosophila dysfusion basic-helix-loop-helix-PAS transcription factor gene is expressed in specialized fusion cells that reside at the tips of migrating tracheal branches. dysfusion mutants were isolated, and genetic analysis of live embryos revealed that mutant tracheal branches migrate to close proximity but fail to recognize and adhere to each other. Misexpression of dysfusion throughout the trachea further indicated that dysfusion has the ability to both inhibit cell migration and promote ectopic tracheal fusion. Nineteen genes whose expression either increases or decreases in fusion cells during development were analyzed in dysfusion mutant embryos. dysfusion upregulates the levels of four genes, including the shotgun cell adhesion protein gene and the zona pellucida family transmembrane protein gene, CG13196. Misexpression experiments with CG13196 result in ectopic tracheal fusion events, suggesting that it also encodes a cell adhesion protein. Another target gene of dysfusion is members only, which inhibits protein nuclear export and influences tracheal fusion. dysfusion also indirectly downregulates protein levels of Trachealess, an important regulator of tracheal development. These results indicate that fusion cells undergo dynamic changes in gene expression as they switch from migratory to fusion modes and that dysfusion regulates a discrete, but important, set of these genes (Jiang, 2006; full text of article).
The isolation of dys mutants allowed detailed phenotypic analysis using time-lapse confocal microscopy of live embryos. Migration and the presence of filopodia during DB branching appeared normal in dys mutant embryos. The two DB fusion cells moved close together, and their filopodia were observed to touch. However, unlike wild-type cells, the dys mutant fusion cells failed to stably adhere. Thus, no fusion occurred, and ultimately the branches retracted from one another. Additional insight into the role of dys emerged from experiments in which dys was misexpressed throughout the trachea. Ectopic fusion events were observed, which is consistent with the dys mutant phenotype and indicates that dys promotes fusion cell recognition/adhesion. In addition, dys misexpression results in a strong reduction in both tracheal branching and formation of MAb 2A12 luminal material at fusion sites. The reduction in branching suggests that another possible function of dys is to inhibit migration in preparation for fusion. The reduction in MAb 2A12 luminal material at sites of normal and ectopic fusion suggests that the fusion process is incomplete. This can be explained for the ectopic fusion results by proposing that Dys activates genes that can mediate fusion but not lumen formation. Since the ectopic cells are not fusion cells, the additional lumen-forming functions would not be present. In contrast, since lumen formation was also defective at normal fusion sites, it is possible that Dys overexpression inhibits lumen formation. In summary, the genetic and misexpression experiments suggest that dys is activated late in the branching process to inhibit migration and promote branch recognition and adhesion. It may also play additional roles after branches join (Jiang, 2006).
Since dys encodes a transcription factor, it is expected that it functions by regulating gene expression. Previous work had identified several genes prominently expressed in fusion cells, as well as additional trachea-expressed genes whose fusion cell expression was low or absent. This paper further shows that a number of prominent trachea-expressed genes are also downregulated in fusion cells, indicating that this is a common occurrence. Expression of 19 genes was assayed in dys mutant embryos to identify Dys target genes. RNA levels of four genes (CG13196, CG15252, mbo, and shg) were reduced. In contrast, Trh protein levels, which normally decline in fusion cells, increased in dys mutants. These results were confirmed by dys misexpression experiments, in which CG13196 and CG15252 were increased and Trh protein levels declined. Despite dys expression in all tracheal fusion cells, there exist branch-specific differences in Dys-regulated gene expression. CG13196 is expressed in all fusion cells, and dys is required for its expression. In contrast, shg is upregulated in DB and DT fusion cells, but only DB upregulation requires Dys, an effect also seen for Esg. CG15252 is expressed only in DT fusion cells, and this restriction may be due to the positive or negative action of branch-specific transcription factors, such as Spalt major (DT specific, positively acting) or Kni and Knrl (non-DT branches, negatively acting) (Jiang, 2006).
All of the dys misexpression defects (and thus probably the mutant defects) require Dys DNA binding, since deletion of the dys basic region, and presumably its ability to bind DNA, abolishes the tracheal defects. Although trh RNA levels decline in fusion cells along with protein levels, the RNA reduction is not dys dependent. Thus, dys likely regulates transcription of a gene that regulates Trh protein levels. Similarly, the requirement of Dys DNA binding to regulate Trh protein levels does not support a model in which Trh levels are reduced as a consequence of Dys competing for their common dimerization partner, Tgo, since this is unlikely to require DNA binding (Jiang, 2006).
The recognition/adhesive properties promoted by dys may be mediated by two Dys target genes, shg and CG13196. Shg is a well-studied adhesion protein, and CG13196 encodes a ZP transmembrane protein, although its function and subcellular localization are unknown. Misexpression of CG13196 results in ectopic fusion events consistent with it playing a role in cell adhesion. Thus, one key role of dys may be to promote tracheal fusion by controlling expression of two or more cell adhesion protein genes. They could work together in the same cellular process or in different aspects of tracheal fusion, lumen formation, or function. The identification of members only (mbo) as a transcriptional target of Dys is intriguing, since mbo mutants have a tracheal fusion defect and it attenuates protein nuclear export. Although the fusion cell protein cargo regulated by mbo is unknown, it presumably includes proteins that are localized to nuclei in fusion cells (Jiang, 2006).
The two major transcription factors studied to date that control fusion cell transcription and development are esg and dys. esg precedes dys during fusion cell development and controls expression of dys in DBs and GBs but not DTs (the case for LTs is unknown, since fusion cells die in esg mutants. dys itself does not affect esg expression. The tracheal fusion phenotypes of both genes are similar. The DT is the one branch that still fuses in both esg and dys mutants, although both show constrictions at the sites of DT fusion. Previous work on esg revealed that, genetically, it is required for both activation of fusion cell gene expression and repression of terminal cell gene expression in fusion cells. In this study, it was found that dys constitutes a transcriptional pathway that carries out a subset of esg functions, focused on upregulating expression of genes involved in cell adhesion and protein localization, although future work may uncover additional target genes. Since a large number of genes are either activated or downregulated in tracheal fusion cells, it will be important to continue genetic and molecular studies to determine which genes are targets of Esg and Dys and whether their control is direct or indirect (Jiang, 2006).
One model of Drosophila dys function is that dys acts as a developmental timer near the end of tracheal branching to inhibit migration and promote cell adhesion and fusion. The adhesion component works, in part, through activation of shg and (possibly) CG13196. The inhibition of migration has only been postulated from misexpression experiments and needs to be confirmed by alternative approaches. It is also important to note that the switch from migration to fusion can also include changes in gene expression that are independent of dys. For example, it is shown here that RNA levels of btl, a gene required for tracheal migration, are downregulated in fusion cells in a dys-independent mode. dys is expressed in a variety of Drosophila embryonic cell types, including leading edge, brain, gut, and anal pad, and the mammalian ortholog is prominently expressed in the brain. However, the function of dys in these cell types is unknown, although a potential connection between tracheal fusion cells and both migrating neuronal axon growth cones and leading-edge cells is worth investigating. The role of dys in controlling fusion cell behavior suggests that it is worthwhile to look in tissues that undergo branching morphogenesis, such as the vertebrate lung and vascular system, for regulatory proteins expressed in tip/fusion cells that control the migration, recognition, and fusion properties of their branches (Jiang, 2006).
In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. This study shows that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, these data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).
The dorsal thorax of Drosophila pupae, the notum, is a single-layered neuroepithelium that produces epidermal and sensory organ (SO) cells. Each adult SO is composed of four cell types and is derived from a single cell, the sensory organ precursor (SOP, also called the pI cell). Notch regulates binary cell fate decisions in the SO lineage. Each SOP undergoes asymmetric cell division to generate two distinct daughter cells; Notch is activated in the SOP daughter cell that adopts the pIIa fate and is inhibited in the other cell, which becomes a pIIb cell. The pIIa cell divides to generate the external cells of the SO, the shaft and socket cells. The pIIb cell undergoes two rounds of asymmetric cell division to generate the internal cells of the SO, the neuron, the sheath cell, and a glial cell. Although Notch-mediated binary cell fate decision in the SO lineage is tightly controlled by intracellular trafficking, the exact subcellular location of where Notch ligand and receptor interact to produce a signal is subject to debate (Benhra, 2011).
To identify new regulators of Notch signaling involved in intracellular trafficking, a double-stranded RNA (dsRNA) screen was carried out for genes affecting SO development and the clathrin adaptor AP-1 complex was identified. AP-1 is an evolutionarily conserved heterotetrameric complex. Drosophila AP-1 complex is composed of AP-1γ (CG9113), β-adaptin (CG12532), AP-1μ1 (encoded by AP-47, CG9388), and AP-1σ (CG5864) subunits. Although mammalian AP-1 is involved in lysosome-related organelle (LRO) biogenesis and in polarized sorting of membrane proteins to the basolateral plasma membrane, the function of Drosophila AP-1 remains largely unknown. Each wild-type SO contains only one socket cell. In contrast, tissue-specific gene silencing of any of the three AP-1 specific subunits, AP-47, AP-1γ, or AP-1σ, gives rise to a Notch gain-of-function phenotype that results in a pIIb-to-pIIa cell fate and/or a shaft-to-socket cell transformation, leading to an excess of socket cells. Following knockdown of AP-1 subunits, 4% to 17% of SO show more than one socket cell. To confirm and extend these dsRNA-induced results, classical mutants were analyzed. Two mutations in AP-47, AP-47SHE11, and AP-47SAE10 were previously recovered as genetic modifiers of presenilin hypomorphic mutations. This stud characterized the AP-47SHE11 allele as a genetic null, whereas the second allele, AP-47SAE10, is hypomorphic. AP-47SHE11/Df(3R)Excel 6264 transheterozygotes die at early first-instar larvae stage, indicating that, as in worms, zebrafish, and mice, AP-47 is essential for viability. To assess the AP-47 loss-of-function phenotype in SO, AP-47 mutant mitotic clones were generated and analyzed in the notum. The same two categories of transformed mutant organs were observed as in the dsRNA experiments. Cell fate transformation was seen in 11% of the mutant organs and in 17% following AP-47dsRNA. The difference could be due to protein perdurance in the mutant clones induced during development. The incomplete penetrance suggests that a compensatory mechanism could bypass the requirement for AP-1. In any case, the results suggest a requirement for the AP-1 complex in Notch-dependent binary cell fate acquisition (Benhra, 2011).
Excess Notch signaling can arise from either disruption of epithelial cell polarity or defects in partitioning of cell fate determinants at mitosis. Because cell polarity relies on the proper apicobasal sorting of membrane proteins, a process requiring both clathrin activity in mammals, this study has analyzed the localization of various polarity markers in AP-47− mutant clones. The Notch gain-of-function phenotype observed in the absence of AP-1 activity cannot be explained by a disruption of epithelial cell polarity, nor by a defect in the partitioning of the cell fate determinants Numb and Neuralized (Neur) at mitosis. Thus, AP-1 activity may be required after unequal segregation of cell fate determinants, possibly at the pIIa/pIIb cell stage to control Notch signaling (Benhra, 2011).
Defects in the endolysosomal degradation, such as in vps25 and erupted mutant cells, result in a Notch gain-of-function phenotype that is caused by ligand-independent mechanisms. Because AP-1 is involved in the biogenesis of LROs in mammals, genetic interaction tests were devised to determine whether excess signaling caused by loss of AP-47 requires the activity of the Notch ligands Delta and Serrate (Ser). Loss of Delta and Ser signaling causes Notch loss-of-function phenotypes, a lateral inhibition defect and a pIIa-to-pIIb cell fate transformation that results in generation of extra neurons, the opposite phenotype to what was observed in AP-47− mutant clones. Loss of external sensory cells accompanied by an excess of neurons is observed in AP-47− Delta− Ser− triple mutant clones, a phenotype indistinguishable from that of Delta− Ser− double mutant clones. The reversal of pIIb-to-pIIa transformation phenotype of AP-47− in AP-47− Delta− Ser− triple mutant clones demonstrates that Delta and Ser are epistatic to AP-47. This finding indicates that the AP-47− mutant phenotype is ligand dependent (Benhra, 2011).
The activity of Delta in the SO lineage is controlled by Neur-dependent endocytosis. Following endocytosis, Delta is recycled, and its trafficking toward apical microvilli requires Arp2/3 and WASp. Mutations in WASp prevent Notch signaling, resulting in a pIIa-to-pIIb cell fate transformation. Excess Notch signaling is observed in AP-47− WASp− clones, as in AP-47− clones. These data demonstrate that AP-47 is required for SO formation even in the absence of WASp. These findings suggest that AP-1 is unlikely to act by preventing Delta recycling and raise the possibility that AP-1 acts on Notch receptor signaling (Benhra, 2011).
Sanpodo (Spdo) is a four-pass transmembrane protein required for Notch signaling in asymmetrically dividing cells. Because mutations in spdo result in reduced Notch signaling, the opposite phenotype to what was observed in AP-47− mutant clones, it could be that AP-1 normally represses Spdo activity. To test this hypothesis, AP-47− spdo− double mutant clones were generated and a phenotype was observed that is indistinguishable from that of spdo− mutant clones. The reversal of the pIIb-to-pIIa transformation phenotype of AP-47− in AP-47− spdo− double mutant clones indicates that AP-1 requires the activity of Spdo to control Notch signaling and suggests that AP-1 might control Spdo trafficking and/or localization (Benhra, 2011).
To test for a role of AP-1 in Spdo localization, the subcellular distribution of Spdo was compared in wild-type and AP-47− SO lineages. In the wild-type SOP, Spdo is found in intracellular compartments. After division, Spdo-positive vesicles remain localized in the pIIb cell as a consequence of the unequal inheritance of Numb during SOP mitosis, whereas Spdo localizes preferentially at the plasma membrane of the posterior pIIa cell. Spdo is also detected at the apical cortex of SOP and pIIa/pIIb cells, albeit at a low level. In contrast, in AP-47− mutant SO cells, Spdo accumulates apically, as well as at the interface between the AP-47− SOP daughter cells, where DE-Cad is present. It is concluded that loss of AP-1 results in the specific accumulation of Spdo at the apical plasma membrane in SO cells, as well as at the level of adherens junction in SOP daughters. It is suggested that this defect in Spdo trafficking could explain the excess Notch signaling (Benhra, 2011).
Because AP-1 is required for proper localization of Spdo, an anti-AP-1γ antibody was generated to investigate the subcellular distribution of AP-1 relative to Spdo. AP-1γ is closely juxtaposed to the trans Golgi network (TGN) marker GalT::RFP and colocalizes partially with Liquid facet related (LqfR; CG42250), the Drosophila ortholog of Epsin related (Epsin-R), recently reported to localize at the TGN. AP-1γ also partially colocalizes with Rab11-positive recycling endosomes (RE). Thus, in epithelial cells of the notum, AP-1 is found on two membrane-bound compartments, the TGN and RE, as previously reported in tissue culture cells. In SOPs, Spdo was previously shown to partially colocalize with Notch, Hrs, and Rab5. This study reports that Spdo also colocalizes with AP-1γ and Rab11-positive endosomes, suggesting that Spdo traffics within the TGN and RE (Benhra, 2011).
Together with the above genetic data, colocalization of AP-1 with Spdo raises the interesting possibility that AP-1 could control the sorting and transport of Spdo. Furthermore, Spdo contains a conserved N-terminal YTNPAF motif that falls into the Y/FxNPxY/F-consensus sorting signal of the LDL receptor whose localization is regulated by clathrin adaptors. If Spdo is an AP-1 cargo, deletion of the sorting motif of Spdo should prevent its interaction with AP-1. To test this prediction, the localization of AP-47-VenusFP (VFP) was analyzed relative to that of Spdo-mChFP versus Spdo-mChFP deleted of its 18 first amino acids containing the YTNPAF motif (SpdoΔ18-mChFP) in the SOP lineage. On average at the two-cell stage, 69% of the AP-47-VFP-positive vesicles are also positive for Spdo-mChFP, whereas only 14% of AP-47-VFP vesicles are positive for SpdoΔ18-mChFP. Thus, the first 18 amino acids of Spdo may be required for its AP-1-mediated sorting. Nonetheless, SpdoΔ18-mChFP does not accumulate at the apical cortex, suggesting that additional sorting motifs or interacting proteins such as Numb, also interacting with Spdo via the YTNPAF motif, contribute to Spdo apical localization. These data reveal that in addition to AP-2, a second clathrin adaptor complex, AP-1, controls the localization of Spdo and regulates Notch signaling. AP-2 and Numb prevent Spdo accumulation at the plasma membrane, whereas AP-1 prevents Spdo accumulation at the apical plasma membrane. Whether AP-1 binds directly to the YTNPAF motif or indirectly via a yet-to-be-discovered clathrin-associated sorting protein (CLASP) like Numb remains unknown. By analogy to Numb and AP-2, the hypothetical CLASP would function together with AP-1 to sort Spdo at the TGN and/or RE (Benhra, 2011).
Based on its localization at the TGN and the RE, AP-1 may ensure sorting of Spdo from the TGN and/or RE. To test whether AP-1 has a role at RE, a functional Spdo construct was generated in which mChFP is inserted in the second extracellular loop of Spdo (SpdoL2::mChFP) and used in a pulse-chase internalization assay with an anti-RFP that recognizes the extracellularly accessible mChFP tag in epithelial cells of the notum. In the control, following a 45 min chase, the anti-RFP has been efficiently internalized and resides primarily in apically localized endosomes. A small pool of anti-RFP is also detected at the level of adherens junctions labeled with DE-cadherin, suggesting that Spdo can be recycled back to adherens junctions, albeit with low efficiency. In cells depleted of AP-1, anti-RFP internalized from the basolateral membrane is efficiently recycled to the adherens junctions, suggesting that AP-1 acts in RE to limit recycling of Spdo toward adherens junctions. In contrast, when AP-2-dependent endocytosis is prevented, anti-RFP remains mostly localized at the basolateral plasma membrane, even after a chase of 45 min, as predicted for a requirement of AP-2 in the internalization of Spdo. Therefore, the data indicate that AP-1 does not regulate endocytosis of Spdo from the basolateral membrane. To test whether AP-1 could regulate apical endocytosis of SpdoL2::mChFP, a pulse-chase internalization assay was conducted in epithelial cells of the wing imaginal discs, a tissue that, in contrast to the pupal notum, allows for access of anti-RFP at the apical plasma membrane. In cells depleted of AP-47, anti-RFP resides predominantly at the apical side at the level of adherens junction at t = 0 and is internalized with similar kinetics as in the control situation. It is concluded that AP-1 does not regulate SpdoL2::mChFP apical internalization. Altogether, these results indicate that AP-1 acts at the RE to prevent or limit apical recycling of Spdo, giving a rationale for why endogenous Spdo accumulates apically in SO mutant for AP-47 (Benhra, 2011).
Does apical accumulation of Spdo cause the Notch gain-of-function phenotype seen in AP-1 mutant SO? Spdo was previously reported to partially colocalize with Notch in large intracellular structures and at the plasma membrane. In wild-type, Notch localizes at the apical membrane of epidermal cells, SOP cells, and SOP daughter cells. Shortly after SOP division, Notch extracellular domain (NECD) is detected apically together with Spdo at the DE-Cad interface between pIIa and pIIb. This specific localization is transient, because NECD and Spdo are detected at the interface of daughter cells in one-third of the cases and are no longer detectable at the pIIa/pIIb interface when the remodeling of the apical cortex of pIIa/pIIb cells takes place. In AP-47− mutant cells, NECD is stabilized with Spdo at the interface of SOP daughter cells, even at a time when control organs have undergone apical cortex remodeling. Similarly, Notch intracellular domain (NICD) is accumulated at the level of adherens junctions in AP-47− mutant cells, whereas it is detected at the interface of wild-type SOP daughters in only half of the cases. To determine whether the stabilization of Notch at the SOP daughter cell interface is caused specifically by AP-47 loss of function, NECD localization was compared in AP-47− versus spdo− or AP-47− spdo− double mutant clones. Although NECD is enriched at the apical surface in these three mutant situations compared to control cells, stabilization of NECD at the interface of SOP daughter cells occurs in AP-47− single and AP-47− spdo− double clones, but not in spdo− single clones. These data indicate that, upon loss of AP-47, Spdo is not required for NECD to accumulate at the junction between SOP daughter cells, which raises the interesting possibility that Notch itself may be an AP-1 cargo. Because Spdo and Notch are transiently detected at the interface of wild-type SOP daughter cells, it is proposed that sustained elevated levels of Spdo and Notch at the interface cause the excess signaling observed in AP-47− mutants. These effects of AP-1 appear to be specific to Spdo and Notch, because Delta is transiently detected in punctuated structures at the level of junctions together with Spdo in a similar manner in both control and AP-47− SOP daughter cells. Furthermore, endocytosis of Delta is unaffected by the loss of AP-1. It is thus concluded that AP-1 regulates the amount of Notch and Spdo at this junctional domain, which could serve as a launching pad from which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).
These data have uncover a novel function for AP-1 complex during development. The observations suggest that AP-1 participates in the polarized sorting of Spdo and Notch from the TGN and/or RE toward the plasma membrane. The correlation between the Notch gain-of-function phenotype and the stabilization of Notch and Spdo at the junctions suggests that adherens junctions may be particularly important for Notch activation. Because the effect of loss of AP-1 on Spdo and Notch localization is completely penetrant, it is proposed that a threshold of Spdo and Notch localized at the junctional domain has to be reached in order to cause the cell fate transformation, explaining why only 10% to 20% exhibit the Notch gain-of-function phenotype (Benhra, 2011).
Previous reports have suggested that trafficking of endocytosed Delta to the apical membrane in the pIIb cell is required for its ability to activate Notch that localizes at the apical side in the pIIa cell. Recently, it was reported that most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Based on phalloidin staining, the ARS appears to be unaffected by the loss of AP-47. Notch and Spdo are stabilized at the junctional domain that is included within the ARS and are therefore poised to receive the Delta signal. This would place this domain of the ARS as an essential site for Delta-Notch interaction, leading to productive ligand-dependent Notch signaling (Benhra, 2011).
Could this novel function for AP-1 be conserved in mammals? Spdo is specifically expressed in Dipterans, and no functional ortholog has been described so far, raising the question of the role of AP-1 in Notch signaling in mammals. Nonetheless, Notch is also mislocalized in AP-1 mutant cells even when Spdo activity is missing. Notch also contains evolutionarily conserved tyrosine-based sorting signals, and it cannot be excluded at present that Notch is itself an AP-1 cargo. Finally, the facts that Notch controls several early steps of T cell development and that mice heterozygous for γ-adaptin exhibit impaired T cell development raise the interesting possibility that Notch-dependent decisions in mammals also required AP-1 function (Benhra, 2011).
Stem cell self-renewal is controlled by concerted actions of extrinsic niche signals and intrinsic factors in a variety of systems. Drosophila ovarian germline stem cells (GSCs) have been one of the most productive systems for identifying the factors controlling self-renewal. The differentiation factor BAM is necessary and sufficient for GSC differentiation, but it still remains expressed in GSCs at low levels. However, it is unclear how its function is repressed in GSCs to maintain self-renewal. This study reports the identification of the translation initiation factor eIF4A for its essential role in self-renewal by directly inactivating BAM function. eIF4A can physically interact with BAM in Drosophila S2 cells and yeast cells. eIF4A exhibits dosage-specific interactions with bam in the regulation of GSC differentiation. It is required intrinsically for controlling GSC self-renewal and proliferation but not survival. In addition, it is required for maintaining E-cadherin expression but not BMP signaling activity. Furthermore, BAM and Bgcn together repress translation of E-cadherin through its 3' UTR in S2 cells. Therefore, it is proposed that BAM functions as a translation repressor by interfering with translation initiation and eIF4A maintains self-renewal by inhibiting BAM function and promoting E-cadherin expression (Shen, 2009).
This study has revealed the biochemical function of the BAM/BGCN complex as a translational repressor. eIF4A in the regulation of GSC self-renewal to be a direct antagonist of BAM function in the Drosophila ovary. A model is proposed explaining how GSC self-renewal is controlled by concerted actions of intrinsic factors and the extrinsic BMP signal. BMP signaling directly represses bam expression, yet leaves low levels of BAM protein expression in the GSC. eIF4A and other unidentified germline factors in the GSC can effectively dismantle BAM/BGCN's repression of GSC maintenance factors, including E-cadherin, through physical interactions, leading to high expression of maintenance factors in the GSC. In the cystoblast (CB), high levels of BAM along with BGCN can keep eIF4A proteins out of the active pool and thus effectively repress GSC maintenance factors, promoting CB differentiation. Therefore, this study has significantly advanced current understanding of how GSC self-renewal and differentiation are regulated by translation factors (Shen, 2009).
bam and bgcn genetically require each other's function to control CB differentiation. Although they are expressed at low levels in GSCs, they have an important role in regulating GSC competition. However, their biochemical functions remained unclear until this study. This study showed that BAM specifically interacts with BGCN, but not other RNA-binding proteins VASA, Rm62, and Me31B, to form a protein complex. In addition, BAM and BGCN are shown to act together; BAM or BGCN alone are not capable of suppressing the expression of the reporter containing the shg 3' UTR. Furthermore, BAM and BGCN do not affect the stability of the reporter mRNA, further supporting that they regulate mRNA translation but not stability. To reveal the role of BGCN in the function of the BAM/BCGN complex, this study showed that direct tethering of BAM to the 3' UTR of the target mRNA can bypass the requirement of BGCN and sufficiently suppress the expression of the reporter. Based on the fact that BGCN contains a putative DEXH RNA binding domain, it is proposed that BGCN helps bring BAM to its target mRNAs to repress their translation. Therefore, this study has revealed the biochemical functions of BAM and BGCN (Shen, 2009).
Previous genetic study showed that BAM and BGCN negatively regulate E-cadherin expression in GSCs to control GSC competition, but the underlying molecular mechanism remains defined. This study showed that in Drosophila S2 cells BAM and BGCN could repress E-cadherin expression through its 3' UTR at the translational level. Along with previous observation that BAM and BGCN negatively regulate E-cadherin expression in GSCs in vivo, it is proposed that BAM and BGCN likely repress E-cadherin expression in GSCs at the translational level. In the future, it will be important to show if BAM and BGCN directly bind to the shg 3' UTR to repress E-cadherin expression in the GSC (Shen, 2009).
eIF4A, an RNA helicase, is one component of the translation initiation complex eIF4F, which is required for loading the small 40S ribosome subunit onto the target mRNA to initiate its translation. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G, another component of eIF4F. Such helicase activity is important to remove the secondary structure of the 5' UTR, facilitating the ribosome scanning along mRNA to find the initiation codon ATG. To reveal how BAM and BGCN confer translation repression, the yeast 2-hybrid screen was used to identify eIF4A as a BAM interacting protein. Then, two pieces of genetic of evidence were provided supporting the idea that eIF4A and bam function together to control the balance between GSC self-renewal and differentiation. First, one copy of the mutations in eIF4A can dramatically promote germ cell differentiation in the hypomorphic bamZ/bamδ86 transheterozygous ovaries. However, a mutation in eIF4A cannot suppress the tumorous phenotype of the bam?86 homozygous ovaries (no bam function at all), suggesting that the reduction of eIF4A dosage helps enhance the remaining BAM function. Second, overexpression of eIF4A can enhance the differentiation defect in the bam?86 heterozygote. These genetic results support the antagonizing relationship between bam and eIF4A (Shen, 2009).
The antagonizing genetic relationship between bam and eIF4A suggests that eIF4A favors GSC maintenance over differentiation. The genetic analysis of the marked eIF4A mutant GSC clones shows that eIF4A is indeed required in GSCs for their self-renewal and division. To uncover the genetic mechanism underlying the function of eIF4A in maintaining GSCs, it was also shown that the marked eIF4A mutant GSC has normal BMP signaling activities in comparison with its neighboring wild-type GSC based on expression results from 2 BMP responses genes, bam and Dad, but has significantly reduced E-cadherin expression in comparison with its neighboring wild-type GSC. These genetic and cell biological results demonstrate that eIF4A controls GSC maintenance at least partly by maintaining E-cadherin expression. In mammalian cells, overexpression of translation initiation factors, such as eIF4A, 4G, and 4E, is implicated in different kinds of cancer due to their ability to increase cell proliferation. In the Drosophila imaginal disc, the block in cell proliferation caused by mutations in eIF4A can be bypassed by E2F overexpression, indicating that eIF4A regulates cell cycle progression and consequently cell proliferation. In this study, it was shown that eIF4A is also required for controlling GSC division. Therefore, it is proposed that eIF4A controls GSC proliferation by regulating cell cycle progression like in Drosophila imaginal tissues (Shen, 2009).
E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. This study shows that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down Capping Protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, these findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours (Jezowska, 2011).
Actin filament (F-actin) turnover and organization is a critical regulator of AJs assembly, maintenance, and remodelling. F-actin growth, stability, disassembly and also their organization into functional higher-order networks are controlled by a plethora of actin-binding proteins (ABPs), strongly conserved between species. Capping protein (CP), composed of an α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer by restricting accessibility of the filament barbed end, inhibiting addition or loss of actin monomers. In Drosophila, removing either cpa or cpb, promotes accumulation of F-actin within the cell and gives rise to identical developmental phenotypes. In the whole larval wing disc epithelium, loss of CP activity reduces Hpo pathway activity and leads to ectopic expression of several Yki target genes that promote cell survival and proliferation. However, inappropriate growth can only be observed in the proximal wing domain. In the distal wing primordium, cpa or cpb mutant cells mislocalize the AJs components DE-Cad and Arm, upregulate puc expression, extrude and die. This indicates that while loss of CP can under certain conditions result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue (Jezowska, 2011).
This study investigated the role of the actin-CP heterodimer in survival of cells in the distal wing disc epithelium. CP has a dual function in regulating DE-Cad: it stabilizes DE-Cad at cell-cell junctions, thereby preventing loss of epithelial integrity and inhibits upregulation of the DE-cad gene. DE-Cad would otherwise provide an active signal, which affects Wg signalling and promotes JNK-mediated apoptosis. However, when cells lacking CP are kept alive, JNK is converted into a potent inducer of proliferation (Jezowska, 2011).
This study demonstrates that in the distal wing disc epithelium, JNK signalling triggers apoptosis of cells with reduced CP expression but induces massive proliferation when apoptosis is blocked with P35. Yki activity is also required to allow overgrowth of 'undead' Cpa-depleted tissues. Induction of apoptosis has been shown to activate Yki through the JNK pathway and triggers compensatory cell proliferation. Thus, in CP-depleted cells kept alive with P35, Yki may act downstream of JNK signalling. Consistent with this, targeting Yki degradation in these tissues fully suppresses ectopic N-Cad expression but not MMP1 upregulation. Because CP also prevents Yki activity in the whole wing disc epithelium, independently of its effect on JNK signalling, (Fernandez, 2011; Sansores-Garcia, 2011), in the distal wing domain, excess Yki activity of 'undead' CP-depleted tissues may result from a dual effect, which involves a JNK-dependent and independent mechanisms (Jezowska, 2011).
JNK signalling has been reported to propagate from cell to cell in the wing disc, where it could trigger apoptosis or Yki-dependent compensatory proliferation. Neither non-autonomous apoptosis nor activation of JNK signalling was observed when patches of CP mutant cells were induced or dsRNA for CP was expressed with or without P35. Therefore, the propagation of JNK activation might be impaired in tissues knocked down for CP. However, increase proliferation was observed of wild-type cells apposed to 'undead' Cpa-depleted tissues. This suggests that JNK propagation is not required to trigger compensatory cell proliferation (Jezowska, 2011).
Several observations argue that in cells lacking CP, a DE-Cad-dependent signal promotes JNK-mediated apoptosis by inhibiting Wg signalling. First, knocking down Cpa affects Wg signalling, which has been shown to prevent JNK-dependent cell death in this region. Second, removing one copy of DE-cad in Cpa-depleted cells partially suppresses apoptosis and ectopic MMP1 expression and restores Wg target genes expression. Third, loss of CP is associated with upregulation of the DE-cad gene and increased levels of the DE-Cad protein. One way by which DE-Cad may block Wg signalling is by tethering Arm. In agreement with this possibility, in the distal wing disc epithelium, overexpression of DE-cad compromises Wg signalling, while co-expression of Arm rescues the DE-cad overexpression phenotype. Moreover, in mouse, overexpression of E-Cad induces apoptosis and sequesters the transcriptionally competent pool of β-cat, effectively shutting off expression of Lef/TCF/β-cat-responsive genes. Interestingly, in Cpa-depleted tissues, the faster mobility form of Arm is enriched. Because this form was proposed to correspond to the cytoplasmic pool of Arm, following CP loss, increase DE-Cad levels might tether and stabilize Arm in the cytoplasm, preventing it to transduce Wg signalling. How a defect in Wg signalling triggers JNK-mediated cell death is not known. In cells lacking CP, JNK activation may occur in response to loss of DIAP1 since overexpressing DIAP1 strongly reduces ectopic MMP1 expression. However, it cannot be excluded that JNK signalling reduces DIAP1 levels since JNK signalling can also function upstream of DIAP1 (Jezowska, 2011).
In the distal wing domain, cells lacking CP mislocalize DE-Cad and Arm at AJs, upregulate expression of DE-cad and extrude from the epithelium (Janody, 2006). DE-cad appears to be a direct transcriptional target of the Hpo signalling pathway. CP inhibits Yki activity (Fernandez, 2011; Sansores-Garcia, 2011) and prevents shg-LacZ upregulation, even in mutant clones that maintain a polarized epithelial architecture in the proximal wing domain. Thus, increased DE-cad expression likely results from inhibition of Hpo pathway activity. However, while mutant clones for Hpo pathway components accumulate DE-Cad, mutant cells do not extrude from the wing disc epithelium. Therefore, the polarity defect of cells lacking CP is unlikely to result from increased DE-Cad levels. Different observations also argue that altered cell-cell adhesion does not result from a defect in Wg signalling or from ectopic activation of JNK signalling, as previously reported. First, reducing DE-cad levels do not restore Arm localization at AJs. Second, in Cpa-depleted tissues in which JNK signalling is blocked, dividing nuclei surrounded by dense F-actin patches are recovered on the basal surface of the distal wing disc epithelium. Third, unlike cells lacking CP, tissues expressing P35 and defective for Wg signalling or overexpressing DE-cad or in which high apoptotic levels were induced maintain a polarized epithelial architecture). Therefore, following loss of CP, the mislocalization of DE-Cad and Arm and the loss of cell-cell contacts are likely upstream or parallel events to DE-cad upregulation and JNK-mediated cell death. Because disruption of apical-basal polarity can trigger JNK activation, a model is favored by which CP prevents JNK-mediated cell death though a dual function on DE-Cad: it promotes DE-Cad-mediated cell adhesion and restricts DE-cad expression (Jezowska, 2011).
While the effect of CP loss on DE-cad transcription is not context dependent, the polarity defect is mainly observed in the distal wing domain. Different regions of the wing disc may have specific requirements in terms of AJs stability and remodelling. Because the distal wing disc is under higher mechanical stress, this epithelium may require higher dynamics of DE-Cad remobilization. CP might be critical to control this kinetic, making distal wing cells lacking CP more prone to lose cell-cell adhesion and extrude from the epithelium (Jezowska, 2011).
Interestingly, the proto-oncogene of the Src family kinases Src42A antagonizes DE-Cad-mediated cell adhesion and stimulates the transcription of DE-cad. Moreover, in the distal wing disc epithelium, the major inhibitor of Src family kinases C-terminal Src kinase (Csk), maintains AJs stability, prevents JNK-mediated apoptosis, whereas halving the genetic dose of DE-cad suppresses the apoptotic phenotype of dCsk-depleted cells. CP and mammalian c-Src both regulate F-actin. Conversely, the control of F-actin impacts on the kinase activity of c-Src. Thus, whether the main role of CP is to regulate Src activity in the distal wing disc is an exciting possibility to be tested in the future (Jezowska, 2011).
This study and others have previously shown that the CP heterodimer acts as tumour suppressor through its control of Hpo pathway activity. This study now shows that in specific epithelia, loss of CP also affects cell-cell adhesion, which is a fundamental step to an epithelial-to-mesenchymal transition (EMT), triggers MMP1 expression, which degrades the basal extracellular matrix, induces cell invasion and promotes massive proliferation of cells that fail to stably retain associations with their neighbours when cell death is blocked with P35. Moreover 'undead' CP-depleted cells show ectopic N-Cad expression, whose de novo expression promotes the transition from a benign to a malignant tumour phenotype. Finally, like other tumour suppressors, loss of CP cooperates with RasV12 in tissue overgrowth. These findings argue that in some epithelia in which CP activity is affected, the appearance of a second mutation that prevents apoptotic cell death may trigger the development of aggressive tumours in humans. However, in contrast to tumour progression, which correlates with loss of overall E-Cad expression and stimulation of canonical Wnt signalling, this study observed increase DE-Cad levels and inhibition of Wg signalling in tissues knocked down for CP. Interestingly, in flies, shg-LacZ expression is also enhanced in response to ectopic expression of the two oncogenes Src42A and Yki. This suggests the interesting hypothesis that transcriptional stimulation of DE-cad is an early mechanism of tumour suppression, which would promote the elimination of deleterious cells, possibly through inhibition of Wg signalling, rather than allowing them to proliferate and form tumours. Malignant cells that become resistant to cell death may compete successfully by losing the overall E-Cad expression and upregulating mesenchymal cadherins such as N-Cad to reinforce their fitness (Jezowska, 2011).
Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. This study shows that mutations in drop out (dop; CG6498) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions
in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. This study shows that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein (Hain, 2014).
This study is the first mutational analysis of a MAST kinase in any organism and demonstrates that the MAST kinase Dop plays an important role in plasma membrane cortex compartmentalisation during the generation of epithelial polarity in the fly. The results reported in this study demonstrate a requirement of Dop in the establishment of the furrow canal and the bAJ
at the cycle 14 transition. The defect in bAJ formation is likely to be a consequence of a failure in the initial specification
of the incipient furrows. It is proposed that Dop acts upstream in furrow canal formation by controlling the formation of F-actin-rich foci, which initiate the assembly of a specific furrow membrane cortex (Hain, 2014).
In mid-cellularisation stages, dop mutant phenotypes are reminiscent of embryos lacking the early zygotic gene bottleneck (bnk). In bnk mutants the initial formation of the cleavage furrows is normal, but then furrows close prematurely. Although it cannot be excluded that bnk might play a role in later defects associated with dop mutations, the primary defect in dop mutants concerned the lack of regular F-actin-rich furrows during the onset of cellularisation. Another early zygotic gene, nullo, is required for the proper recruitment of F-actin during furrow canal formation. Nullo and the actin regulator RhoGEF2 have been proposed to act in parallel pathways controlling processes that are distinct but both essential for F-actin network formation during the establishment of the furrow canal. Since early F-actin rearrangements are largely normal in nullo and RhoGEF2 single mutants, it is proposed that Dop is essential for the initial early focussing of F-actin, whereas Nullo and RhoGEF2 are required to elaborate and maintain F-actin levels to stabilise the furrows. The actin regulator enabled (ena) has been shown to act downstream of Abelson tyrosine kinase (Abl) in the redistribution of F-actin from the plasma membrane cortex into the furrows in both syncytial stages and cellularisation. Although ena would provide a good candidate for acting downstream of dop in the redistribution of F-actin, ena is already required for syncytial cleavages and the F-actin phenotypes in Abl mutants are much more severe than those that were found for dop mutants (Hain, 2014).
The similarity of syncytial cleavage furrows and the cleavage furrows at cellularisation raises the question of how they differ from each other. The molecular basis of the hexagonal pattern of the F-actin-rich cell cortex at the cleavage furrow relies upon the recycling endosome components Rab11 and Nuclear fallout (Nuf) and the actin polymerisation factors Dia and Scar/Arp2/3. In contrast to dop mutants, nuf, dia or Scar mutants indicate that these genes are required also for the dynamic redistribution of F-actin during syncytial development. Since Dop is a maternally supplied protein, its activity might be regulated by events triggered during the cycle 13-14 transition. The major difference between the furrows in syncytial stages and cellularisation is that metaphase furrows are formed during M phase, whereas cellularisation furrows are formed during G2 phase. Since Dop is a maternally supplied gene product, one would have to implicate regulation of Dop by zygotic factors to explain its phenotype at the cycle 13-14 transition. An alternative possibility is that Dop is regulated by phosphorylation or other post-translational modification through the cell cycle machinery and that, in the absence of Cdk1-dependent phosphorylation, its phosphorylation state is changed. This study provided evidence that Dop is indeed differentially post-translationally modified during syncytial versus cellular blastoderm stages. It is proposed that such cell cycle-dependent regulation of Dop may be crucial in transforming syncytial cleavages into persistent cellularisation furrows. Furthermore, the data suggest that this transition could require Dop-dependent regulation of dynein-associated microtubule transport (Hain, 2014).
The mechanisms for the initial localisation of Baz and E-cadherin are still unclear but, interestingly, dop is required for the localisation of both proteins. At the cycle 14 transition, E-cadherin and Arm puncta are associated with apical membrane projections and the homophilic association of these cadherin puncta is strengthened by membrane flow and is dependent on actin. Baz function allows these puncta to become tightened into sAJs. Thus, Dop might affect the stabilisation of the weakly interacting puncta either through cortical actin organisation or membrane flow. In addition to this early requirement for Baz localisation, Dop is also involved in clearing Baz from the basal cytoplasm during late cellularisation. The mechanism that eventually clears Baz from the basal cytoplasm depends on dynein-based transport. Therefore, Dop is required for dynein-based transport of different cargoes during cellularisation: lipid droplets, mRNA particles, Golgi and Baz. It is proposed that the main function of Dop in cellularisation is in regulating dynein-mediated transport of important cargos along microtubules (Hain, 2014).
This study presents the first evidence for regulation of dynein-mediated transport by a MAST family kinase. Dop is shown to controls phospho-Dic levels in a direct or indirect manner. The data are consistent with a model in which the initiation of furrow formation involves dynein-dependent transport that is controlled by Dop. In support of a role in membrane formation, this study found defects in the distribution of the recycling endosome and Golgi compartments in dop mutants. Interference with Rab11 function causes similar defects in Slam distribution as those shown by dop mutants. Therefore, Dop might control the transport of endomembrane compartments, which drive membrane growth. In addition, F-actin redistribution plays a major role in membrane cortical compartmentalisation in the initial stages of cellularisation. The focussing of F-actin to incipient furrows might involve a dynein-dependent shift of actin regulators or existing actin filaments to the furrow. An attractive hypothesis is that the translocation of F-actin and/or its regulators is coupled to an endomembrane compartment that is transported via microtubules towards the incipient furrow canals. Future studies should aim to determine which dynein cargos contribute to furrow formation and how Dop regulates Dic phosphorylation at the molecular level (Hain, 2014).
The Toll/NF-kappaB pathway, first identified in studies of dorsal-ventral polarity in the early Drosophila embryo, is well known for its role in the innate immune response. This study revealed that the Toll/NF-kappaB pathway is essential for wound closure in late Drosophila embryos. Toll mutants and Dif dorsal (NF-kappaB) double mutants are unable to repair epidermal gaps. Dorsal is activated on wounding, and Dif and Dorsal are required for the sustained down-regulation of E-cadherin, an obligatory component of the adherens junctions (AJs), at the wound edge. This remodeling of the AJs promotes the assembly of an actin-myosin cable at the wound margin; contraction of the actin cable, in turn, closes the wound. In the absence of Toll or Dif and dorsal, both E-cadherin down-regulation and actin-cable formation fail, thus resulting in open epidermal gaps. Given the conservation of the Toll/NF-kappaB pathway in mammals and the epithelial expression of many components of the pathway, this function in wound healing is likely to be conserved in vertebrates (Carvalho, 2014).
DE-Cadherin is functionally similar to classic vertebrate cadherins. For example, it is associated with alpha-Catenin and beta-Catenin (Armadillo), and protected from trypsin digestion only in the presence of Ca2+, as is the case for many classic cadherins. Transfection of S2 cells with the DE-Cadherin cDNA enhances their Ca(2+)-dependent cell aggregation. Antibodies to this molecule inhibit aggregation, not only in the transfectants but also of early embryonic cells (Oda, 1994).
A series of Armadillo mutants were generated and examined and expressed in Drosophila embryos. Although DE-cadherin and alpha-catenin bind to Armadillo independent of one another, binding of both is required for the function of
adherens junctions, that is, mutations that block alpha-Catenin and Cadherin-binding block junction formation. E-cadherin appears to bind in the Armadillo repeat region; this region is required for localization to the adherens junction. alpha-Catenin binding is eliminated by deletion of the region between the N-terminus and the repeats. There are two separate regions of Armadillo critical for Wingless signaling. Mutant Arm proteins deleted in the central repeats or lacking the N-terminal alpha-catenin-binding site all localize prominently at higher levels to cell nuclei in cells responding to Wingless signal. Some of the proteins deleted for parts of the central repeat region require Wingless signal to accumulate in the nucleus while others do not. Endogenous Armadillo normally accumulates in the nucleus and it may
act there in transducing Wingless signal. Armadillo's roles in adherens junctions and Wingless signaling are
independent. Mutant proteins lacking the domain between the C-terminal region and the Arm repeats retain function in adherens junctions but lack function in Wingless signal transduction. Phosphorylation changes in the Arm protein are detected only in mutant proteins with deletions in the Arm repeats region. It is concluded that the region essential for alpha-catenin binding is not essential for Wingless signaling. The Arm repeats region are essential both for adherens junction function and for Wingless effector function (Orsulic, 1996).
Cell-cell adherens junctions (AJs), comprised
of the cadherin-catenin adhesion system, contribute to
cell shape changes and cell movements in epithelial morphogenesis.
However, little is known about the dynamic
features of AJs in cells of the developing embryo. In this
study, Dalpha-catenin fused with a green fluorescent
protein (Dalpha-catenin-GFP) was constructed, and found to be targeted
to apically located AJ-based contacts but not other
lateral contacts in epithelial cells of living Drosophila
embryos. Using time-lapse fluorescence microscopy, the dynamic performance of AJs containing
Dalpha-catenin-GFP in epithelial morphogenetic movements was examined.
In the ventral ectoderm of stage 11 embryos, concentration
and deconcentration of Dalpha-catenin-GFP occurs
concomitantly with changes in length of AJ contacts.
In the lateral ectoderm of embryos at the same
stage, dynamic behavior of AJs is concerted with division
and delamination of sensory organ precursor
(SOP) cells. Moreover, changes in patterns of AJ networks
during tracheal extension can be followed. Finally,
Dalpha-catenin-GFP was used to precisely observe
the defects in tracheal fusion in shotgun mutants. Thus,
the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously
observe morphogenetic movements and AJ
dynamics at high spatio-temporal resolution (Oda, 1998b).
The lateral ectoderm of living stage 11 embryos
of arm-GAL4:UAS-DalphaC-GFP was studied. At early stage 11,
many cell divisions occur in the lateral ectoderm. Dividing
epithelial cells show dynamic behavior of Dalpha-catenin-GFP. Before cytokinesis, Dalpha-catenin-GFP is distributed in a line at the equator of
spherical cells. During cytokinesis, Dalpha-catenin
GFP-positive lines of contact between the dividing cell
and surrounding cells can be seen. At the
end of cytokinesis, new AJ contacts where Dalpha-catenin-GFP
has begun to be concentrated appear to be established
between the daughter cells.
Pairs of divided cells that had delaminated
from the surface ectodermal layer were followed. Considering
their positions and behavior,
these cells seemed to be sensory organ precursor (SOP)
cells. After cytokinesis, two daughter cells of a primary
SOP cell recover features characteristic of epithelial
cells. The cells are indistinguishable in the
Dalpha-catenin-GFP distribution pattern from the other ectodermal
cells. About 30 min later, the paired SOP cells
simultaneously begin to reduce their apical surface area. They constrict
their apices smoothly once the
constriction begins. It takes
5-10 min for completion of the apical constriction
phase. Before entering the constriction
phase, Dalpha-catenin-GFP is relatively evenly distributed
at apical cell-cell contacts. During the
constriction phase, however, Dalpha-catenin-GFP is unevenly
distributed at the apical portions of delaminating
SOP cells. After SOP cells disappear from
the embryo surface, high concentrations of Dalpha-catenin-GFP
are left between epithelial cells which have surrounded
SOP cells. Eventually, the remaining
epithelial cells appear to recover characteristic mesh patterns of Dalpha-catenin-GFP on the apical surface (Oda, 1998b).
These observations seem to reveal a typical series of
cellular events in the development of the early embryonic
peripheral nervous system (PNS). Division of an SOP
cell precedes delamination from the surface ectodermal
layer. Although SOP delamination seems to be closely
correlated with mitosis, the two events are temporally
separate. The delamination is coincident with the apical
constriction presumably caused by dynamic functions of
AJs. This kind of cellular behavior is commonly observed
in a variety of morphogenetic events. For example,
the behavior of delaminating SOP cells is reminiscent
of that of invaginating presumptive mesodermal
cells at the beginning of gastrulation. The invagination
process of the mesoderm takes less than 10 min. The
time course of mesoderm invagination is comparable to
that of SOP delamination. During mesoderm invagination,
apically located AJs are broken coinciding with apical
constriction. It is possible that a
similar AJ disruption event occurs in SOP cells during
the apical constriction phase (Oda, 1998b).
Tracheal morphogenesis shows dynamic aspects of DE-cadherin-based cell-cell adhesion. GFP fluorescence
of btl-GAL4;UAS-DaC-GFP#3 embryos were observed to directly
investigate AJ dynamics during tracheal extension.
Concentration of GFP fluorescence can be detected
at portions corresponding to AJs in tracheal primordia
from stage 11, although signals are also seen uniformly
in the cytoplasm but not in nuclei. Tracheal
development is not affected by expression of Dalpha-catenin-GFP.
Tracheal tissues are located on the inside of the embryo
and therefore it has been difficult to examine
changes in cell arrangements in living embryos. However,
targeted expression of Dalpha-catenin-GFP using btl-GAL4
overcomes this difficulty. Time-lapse observation
reveals successive changes in patterns of AJ networks
containing Dalpha-catenin-GFP during tracheal extension, although it was still difficult to precisely follow
changes in the three-dimensional patterns of AJ networks.
Each tracheal primordium
elongates along the dorso-ventral axis and the
distance between the neighboring tracheal primordia
is seen to shorten while the germband is retracting.
Tracheal branches extend, coinciding with elongation
of Dalpha-catenin GFP-labelled lines. Notably, relatively intense
signals for Dalpha-catenin-GFP are often seen
around the tips of extending branches.
Contact of dorsal trunk (DT) cells between
neighboring segments occurs shortly before completion
of germband retraction. Soon after the occurrence
of this contact, lines of weak Dalpha-catenin-GFP signals can be detected
that run through areas of intersegmental DT contact. A previous study has shown that DE-cadherin
and Dalpha-catenin are concentrated in tip cells, contributing to the generation of new AJ-based
contacts between the cells that give rise to a pore
that connects DT lumina. Although it could not be directly determine whether
the lines of Dalpha-catenin-GFP accumulation are in tip
cells, it is possible that Dalpha-catenin-GFP signals are
primary signs of establishing contacts between tip cells
of DT (Oda, 1998b).
Dalpha-catenin-GFP was used to analyse tracheal phenotypes
of zygotic shg mutants. Mutant embryos could be unambiguously
identified by GFP fluorescence in tracheal primordia
without any staining. Moreover, targeted expression of Dalpha-catenin-GFP allows clear visualization of
morphological defects in the trachea of shg mutants.
Epithelial integrity and extension of the tracheal primordia
are relatively normal at earlier stages of tracheal
development (stages 11-13) probably because of the contribution
of maternally supplied functional DE-cadherin molecules. Consistently, considerable amounts of Da-catenin-GFP are detected at apical cell-cell contacts even in
shg zygotic mutants although the levels of its accumulation
at cell contact sites are reduced compared to those
in normal embryos. In normal DT fusion,
DE-cadherin-based cell-cell contacts are newly established
between tip cells, giving rise to ring-like patterns
of cadherin-based junctions, which are visualized by
Dalpha-catenin-GFP. In contrast, the process of DT fusion
is specifically blocked in shg mutants. At fusion
points, tube structures are reduced in diameter or
are not constructed at all. Dalpha-catenin-GFP does not accumulate
between tip cells of DT in shg mutants
probably because a lack of zygotic DE-cadherin
causes failure in establishment of new AJ-based
contacts between the cells. These results indicate that the
accumulation of Dalpha-catenin-GFP at apical contact sites
between tip cells is dependent on zygotic expression of
normal DE-cadherin. Tracheal fusion is a process in which
new apical surface domains facing the lumen are established (Oda, 1998b).
These observations suggested that DE-cadherin is required
not only for establishment of AJ-based contacts but
also for generation and definition of apical cell domains.
In summary, Dalpha-catenin-GFP is a new tool that enables
simultaneous visualization of morphogenetic movements
and behavior of AJs or the cadherin-based adhesion
system in living wild-type and mutant Drosophila embryos.
These observations revealed the dynamic performance
of AJ-based cell contacts. The methods used in this study
will facilitate analysis of the dynamics of cell-cell adhesion
at high spatio-temporal resolution in living animals (Oda, 1998b).
Drosophila Armadillo and its vertebrate homolog beta-catenin
are key effectors of Wingless/Wnt signaling. In the current
model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin,
that then accumulates in nuclei and binds TCF/LEF
family proteins, forming bipartite transcription factors
which activate transcription of Wingless/Wnt responsive
genes. This model was recently challenged. Overexpression
in Xenopus of membrane-tethered beta-catenin or its paralog
plakoglobin activates Wnt signaling, suggesting that
nuclear localization of Armadillo/beta-catenin is not essential
for signaling. Tethered plakoglobin or beta-catenin might
signal on their own or might act indirectly by elevating
levels of endogenous beta-catenin. These hypotheses
were tested in Drosophila by removing endogenous Armadillo. A series of mutant Armadillo proteins with
altered intracellular localizations were generated, and these were expressed in
wild-type and armadillo mutant backgrounds. Membrane-tethered Armadillo cannot signal on its
own; however it can function in adherens junctions. Mutant forms of Armadillo were generated carrying either
heterologous nuclear localization or nuclear export signals.
Although these signals alter the subcellular localization of
Arm when overexpressed in Xenopus, in Drosophila they
have little effect on localization and only subtle effects on
signaling. This supports a model in which Armadillo's
nuclear localization is key for signaling, but in which
Armadillo intracellular localization is controlled by the
availability and affinity of its binding partners (Cox, 1999).
Data in vivo suggest that among
Arm's known partners, cadherins have the highest affinity, with
APC and dTCF (Pangolin) having lower and lowest affinities,
respectively. Thus, in embryos with reduced levels of Arm, the
remaining Arm is exclusively associated with cadherins, as
assayed by immunolocalization and by function. About 70% of cellular Arm is cadherin-associated. When cadherin binding sites are saturated, excess Arm
binds to APC/Axin, leading to its destruction and thus
preventing accumulation of free Arm. While APC levels, at
least in mammalian cells, are low, relative to the total
pool of beta catenin, Arm bound to APC is rapidly targeted for
destruction, thus opening the way for the binding of additional
Arm. Normally the destruction machinery can not only dispose
of all non-junctional Arm, but its resources will not even be fully employed, since
Arm synthesis can be increased several-fold without biological
consequences. However, when the destruction machinery is inactivated
either by Wg signal or mutation, Arm is synthesized
but not destroyed, and thus levels of Arm rise. APC can bind
Arm but in all probability, the APC is rapidly saturated, allowing accumulation of
sufficient Arm to allow dTCF to effectively compete for
binding. DE-cadherin, dAPC, dTCF and any other possible
unknown partners together account for virtually all the Arm in
a normal embryo; little if any free Arm is present.
This model helps explain the differences in localization of
the Armadillo attached to a nuclear localization sequence (Arm-NLS) and Armadillo attached to a nuclear export signal (Arm-NES) in flies and frogs. In Xenopus,
added NLS or NES signals dramatically altered Arm's
intracellular distribution as expected, while in Drosophila the
distribution of wild type Armadillo, Arm-NLS and Arm-NES are
indistinguishable. It is proposed that this reflects differences in
the level of expression. In flies, mutant Arm accumulates at
near wild-type levels, so its binding partners can accommodate
the additional protein. Arm bound to cadherin at the plasma
membrane is unavailable for nuclear import; likewise Arm in
a complex with dTCF is not available for export. Thus Arm-NLS
and Arm-NES localization is primarily determined by
their binding partners, resulting in a near normal localization.
In contrast, Arm-NLS and Arm-NES expression levels in
Xenopus likely exceed those of either endogenous beta-catenin or its
binding partners. Free Arm is thus accessible to the nuclear
import and export machinery, allowing alteration of its localization. Given this, is nuclear localization of Arm a regulated step in
Wg signaling in normal cells? The fact that a subset of cells
accumulate cytoplasmic but not nuclear Arm suggests that
nuclear import may be regulated. In the simplest situation,
addition of an NLS ought to promote Arm nuclear
accumulation and trigger signaling, while addition of an NES
should antagonize signaling. However, heterologous targeting
signals have only subtle effects on signaling. Arm-NES signals in the same fashion as does
Arm-WT, while only a subset of the Arm-NLS lines are
activated for signaling. In the case of Arm-NLS:
in cells in which the destruction machinery is on, no free Arm
is available for nuclear import or export. In cells with
intermediate levels of Wg signaling, the destruction machinery
may be slowed, allowing accumulation of cytoplasmic Arm in
complex with APC, but not to sufficient levels to saturate APC
and allow nuclear import. Only when signaling is fully
activated would sufficient free Arm accumulate for nuclear
import. Addition of an NLS would thus only alter the balance
in cells near the signaling threshold. Further, if nuclear Arm is
bound to dTCF, it may be inaccessible to the nuclear export
machinery. The mechanisms by which Arm/betacat enters nuclei
remain unclear; dTCF-dependent and independent pathways
may exist. The
recent observation that beta Catenin may mediate its own nuclear
transport, independent of importins, further complicates the
issue. Additional levels of regulation may occur,
beyond the simple regulation of Arm/beta Catenin stability (Cox, 1999 and references).
Specialized cell junctions in epithelia serve as cell-cell adhesion sites and thus contribute to the maintenance of tissue integrity. The Drosophila gene crumbs encodes a transmembrane protein that is required for the biogenesis of the zonula adherens, a belt-like structure encircling the apex of epithelial cells. As previously shown, expression of just the short membrane-bound cytoplasmic domain is sufficient to rescue major defects associated with the loss of crumbs function. The cytoplasmic domain of Crumbs is highly conserved in two putative crumbs homologs in C. elegans. To assess the significance of conserved residues, various point mutations and deletions were introduced into this region. Two functional domains were revealed: an amino-terminal region and the carboxy-terminal amino acids EERLI. Both are necessary for rescue of the crumbs phenotype. The EERLI motif interacts with Discs Lost (now redefined as Drosophila Patj), a cytoplasmic protein containing PDZ domains. Overexpression of the Crumbs cytoplasmic domain induces a transition from the single-layered epithelium to a multilayered tissue. This transition is associated with redistribution of the Drosophila homolog of the cell adhesion molecule E-cadherin, and depends on the presence of the EERLI motif (Klebes, 2000).
Data presented here suggest a model in which the Drosophila Crb protein organizes the assembly of an apically localized protein scaffold in epithelial cells that is required for the proper formation and localisation of the ZA. This scaffold includes the protein Dlt (now Patj) and probably other, as yet unidentified, proteins, its assembly depends on the carboxy-terminal segment of Crb. The model further suggests that the Crb-mediated control of DE-cadherin localization depends on interaction between the Crb cytoplasmic domain and the PDZ protein Dlt. Neither DE-cadherin nor Dlt are localized in crb mutant embryos, whereas both proteins are sequestered by mislocalized Crb. However Dlt remains apically localized after overexpression of DE-cadherin. The interaction of Crb with Dlt depends on Crb's carboxy-terminal motif, EERLI. This motif is also necessary for misdistribution of DE-cadherin upon Crb overexpression and for the rescue of crb mutant embryos. The presence of four PDZ domains in Dlt makes it an ideal partner for recruiting other proteins into a hypothetical Crb-dependent, membrane-associated protein network. PDZ domains have been shown to act as versatile organizers of multiprotein complexes. In many cases, the binding site of the interacting protein, often a transmembrane protein, is localized at its carboxyl terminus and ends with a hydrophobic amino-acid residue. Class I PDZ domains bind a conserved S/T-X-V motif (where X is any amino acid), whereas class II domains recognize ligands that carry a hydrophobic amino-acid residue at the -2 position. Since the Dlt-binding site in Crb differs from these motifs, the first PDZ domain of Dlt, which binds to Crb in vitro, may belong to a different class. The presence of the ERLI motif in both C. elegans homologs and the similarities between the phenotypes produced by overexpression of CD2-IntraWT and CD2-IntraCE in the Drosophila embryo suggest that this region might mediate comparable interactions in the nematode. Not surprisingly, a protein similar to Drosophila Dlt has also been detected in the C. elegans database, pointing to the possible conservation of additional components of the postulated protein network (Klebes, 2000).
Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. This study shows that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation (Yamashita, 2003).
Adult stem cells maintain populations of highly differentiated but short-lived cells such as skin, intestinal epithelium, or sperm through a critical balance between alternate fates: Daughter cells either maintain stem cell identity or initiate differentiation. In Drosophila testes, germline stem cells (GSCs) normally divide asymmetrically, giving rise to one stem cell and one gonialblast, which initiates differentiation starting with the spermatogonial transient amplifying divisions. The hub, a cluster of somatic cells at the testis apical tip, functions as a stem cell niche: Apical hub cells express the signaling ligand Unpaired (Upd), which activates the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway within GSCs to maintain stem cell identity (Yamashita, 2003).
Analysis of dividing male GSCs by expression of green fluorescent protein (GFP)-α-tubulin in early germ cells revealed that in 100% of the dividing stem cells observed, the mitotic spindle was oriented perpendicular to the hub-GSC interface throughout mitosis, with one spindle pole positioned within the crescent where the GSC contacted the hub. Stem cell division was rare, averaging one dividing stem cell observed per 5 to 10 testes (~2% of total stem cells) in 0- to 2-day-old adults. Spindles were not oriented toward the hub in gonialblasts (Yamashita, 2003).
Drosophila male GSCs maintained a fixed orientation toward the hub throughout the cell cycle, unlike Drosophila embryonic neuroblasts or the Caenorhabditis elegans P1 cell, in which spindle orientation is established during mitosis by a programmed rotation of the spindle. The single centrosome in early interphase GSCs was consistently located adjacent to the hub. After centrosome duplication, one centrosome remained adjacent to the hub, whereas the other migrated to the opposite side of the nucleus. The mechanisms responsible for Drosophila GSC spindle orientation may differ between sexes. In female GSCs, the spectrosome, a spherical intracellular membranous structure, remains localized next to the apical cap cells, where it may help anchor the spindle pole during mitosis. In interphase male GSCs, in contrast, the spectrosome was often located to the side, whereas at least one centrosome held the stereotyped position adjacent to the hub (Yamashita, 2003).
To investigate centrosome function in orientation of male GSCs, males were analyzed that were null mutant for the integral centrosome component centrosomin (cnn), which is required for normal astral microtubule function. In cnn mutant males, mitotic spindles were not oriented toward the hub in ~30% of the dividing GSCs examined. In an additional 10% to 20%, spindles were properly oriented, but the proximal spindle pole was no longer closely associated with the cell cortex at the hub-GSC interface and the entire spindle was displaced away from the hub. The frequency of spindle orientation defects was highest in metaphase. Loss of function of cnn also partially randomized the interphase centrosome positioning in male GSCs. In more than 35% of the cnn mutant GSCs with duplicated centrosomes that were scored, neither centrosome was positioned next to the hub (Yamashita, 2003).
The number of germ cells associated with the hub was increased 20% to 30% in cnn mutant males, from an average of 8.94 GSCs per hub in the wild type to 11.89 GSCs per hub in cnnHK21/cnnHK21 and 10.69 GSCs per hub in cnnHK21/cnnmfs3. Hub size was not significantly different in cnn compared with wild-type males. In cnn testes with many stem cells, GSCs appeared crowded around the hub and often seemed attached to the hub by only a small region of cell cortex. Finite available physical space around the hub may limit the increase in stem cell number in cnn mutant males (Yamashita, 2003).
As suggested by the increased stem cell number, there were several cases in both live and fixed samples from cnn males in which a stem cell that had recently divided with a mitotic spindle parallel to the hub-GSC interface produced two daughter cells that retained contact with the hub, a finding that was not observed in the wild type. GSCs were also observed dividing with a misoriented/detached spindle that lost attachment to the hub, probably explaining the mild increase in stem cell number relative to the frequency of misoriented spindles (Yamashita, 2003).
The normal close attachment of one spindle pole to a region of the GSC next to the hub and the effects of cnn mutants on centrosome and spindle orientation suggest that a specialized region of the GSC cell cortex touching the hub might provide a polarity cue toward which astral microtubules from the centrosome and spindle pole orient. High levels of DE-cadherin (fly epithelial cadherin) and Armadillo (Arm; fly ß-catenin) colocalized at the hub-GSC interface, as well as at the interface between adjacent hub cells, marked by high levels of Fas III. High levels of DE-cadherin and Arm were not detected around the rest of the GSC surface. Forced expression of DE-cadherin-GFP specifically in early germ cells confirmed that DE-cadherin in GSCs colocalized to the hub-GSC interface (Yamashita, 2003).
DE-cadherin and Armadillo at the hub-GSC interface may provide an anchoring platform for localized concentration of Apc2, one of two Drosophila homologs of the mammalian tumor suppressor gene Adenomatous Polyposis Coli (APC), which in turn may anchor astral microtubules to orient centrosomes and the spindle. Immunofluorescence analysis revealed Apc2 protein localized to the hub-GSC interface. In apc2 mutant males, GSCs were observed with mispositioned centrosomes, misoriented spindles, or detached spindles. Both the average number of stem cells and hub diameter increased in apc2 mutant males compared with that of the wild type. Unlike in cnn mutants, GSCs did not appear crowded around the hub in apc2 males, perhaps as a result of the enlarged hub (Yamashita, 2003).
The second Drosophila APC homolog, apc1, may also contribute to normal orientation of the interphase centrosome and mitotic spindle. Apc1 protein localized to centrosomes in GSCs and spermatogonia during late G2/prophase, after centrosomes were fully separated but before nuclear envelope breakdown. Apc1 was not detected at centrosomes from prometaphase to telophase. Spindle orientation and centrosome position were perturbed in GSCs from apc1 males, and the number of stem cells per testis and the diameter of the hub both slightly increased in apc1 mutant testes compared with those of the wild type (Yamashita, 2003).
It is proposed that the reliably asymmetric outcome of male GSC divisions is controlled by the concerted action of (1) extrinsic factor(s) from the niche that specify stem cell identity, and (2) intrinsic cellular machinery acting at the centrosome and a specialized region of the GSC cortex located at the hub-GSC interface to orient the cell division plane with respect to the signaling microenvironment. Astral microtubules emanating from the centrosome may be captured by a localized protein complex including Apc2 at the GSC cortex where it interfaces with the hub, similar to the way in which cortical Apc2 may orient mitotic spindles in the syncytial embryo or epithelial cells (Yamashita, 2003).
Mechanisms that orient the mitotic spindle by attachment of astral microtubules to specific cortical sites may be evolutionally conserved. In budding yeast, spindle orientation is controlled by capture and tracking of cytoplasmic microtubules to the bud tip, dependent on Kar9, which has weak sequence similarity to APC proteins. Kar9 has been localized to the spindle pole body and the cell cortex of the bud tip, reminiscent of the localization of Drosophila Apc1 at centrosomes and Apc2 at the cell cortex (Yamashita, 2003).
Polarization of Drosophila male GSCs toward the hub could result simply from the geometry of cell-cell adhesion. GSCs appear to be anchored to the hub in part through localized adherens junctions. Homotypic interactions between DE-cadherin on the surface of hub cells and male GSCs could concentrate and stabilize a patch of DE-cadherin. The resulting localized DE-cadherin cytoplasmic domains could then provide localized binding sites for ß-catenin and Apc2 at the GSC cortex. Although binding of E-cadherin and APC to ß-catenin is thought to be mutually exclusive, APC could be anchored at the cortical patch through the actin cytoskeleton, which in turn could interact with ß-catenin/α-catenin (Yamashita, 2003).
Orientation of stem cells toward the niche appears to play a critical role in the mechanism that ensures a reliably asymmetric out-come of Drosophila male GSC divisions, consistently placing one daughter within the reach of short-range signals from the hub and positioning the other away from the niche. Oriented stem cell division may be a general feature of other stem cell systems, helping maintain the correct balance between stem cell self-renewal and initiation of differentiation throughout adult life (Yamashita, 2003).
Src42A is one of the two Src homologs in
Drosophila. Src42A protein accumulates at sites of cell-cell
or cell-matrix adhesion. Anti-Engrailed antibody staining of Src42A
protein-null mutant embryos indicated that Src42A is essential for
proper cell-cell matching during dorsal closure. Src42A, which is
functionally redundant to Src64, was found to interact genetically
with shotgun, a gene encoding E-cadherin, and armadillo, a
Drosophila ß-catenin. Immunoprecipitation and a pull-down assay
indicated that Src42A forms a ternary complex with E-cadherin and Armadillo,
and that Src42A binds to Armadillo repeats via a 14 amino acid region, which
contains the major autophosphorylation site. The leading edge of Src
mutant embryos exhibiting the dorsal open phenotype is frequently kinked and
associated with significant reduction in E-cadherin, Armadillo and F-actin
accumulation. This phenotype suggests that not only Src signaling but also
Src-dependent adherens-junction stabilization are
essential for normal dorsal closure. Src42A and Src64 are required for
Armadillo tyrosine residue phosphorylation but Src activity may not be
directly involved in Armadillo tyrosine residue phosphorylation at the
adherens junction (Takahashi, 2005).
In ectodermal cells, strong Src42A signals in apical or apicolateral
regions were always associated with strong E-cad signals. E-cad is a core
component of the adherens junction that is responsible for cell-cell adhesion
and, hence, most, if not all, E-cad-associated membranous Src42A is probably
related to adherens junction-dependent cell-cell adhesion (Takahashi, 2005).
A considerable fraction of ectodermal cells were also found associated with
the second type of basal Src42A free of E-cad. E-cad-free
Src42A is localized on the ectoderm/mesoderm interface and eliminated from
ectodermal cells that have evaginated or invaginated without mesoderm
association. The
extracellular matrix (ECM) comprises several groups of secreted proteins such
as integrin ligands. During embryogenesis, different cell layers become
properly connected, most probably via cell adhesion to ECM. E-cad-free
Src42A may thus be related to integrin-mediated cell-matrix adhesion. Cell-ECM
adhesion may not be restricted to the interface between ectodermal and
mesodermal cell layers. Strong Src42A signals have actually been found present
on the interface between mesodermal and endodermal cell layers (Takahashi, 2005).
The current study shows that, as with JNK signaling
genes, Src is required not only for thick F-actin
accumulation at the leading edge but proper cell-cell matching along the midline
seam as well. JNK signaling, which includes hemipterous (hep) and
basket (bsk), is essential for dorsal closure of the embryonic epidermis in Drosophila. Based on examination of Tec29 Src42A
mutant phenotypes, it has been suggested that Src42A acts upstream of
bsk (Takahashi, 2005).
The adherens junction is necessary for cell-cell adhesion and thick
F-actin accumulation occurs at the level of the adherens junction at the
leading edge. Since E-cad and Arm signals along with actin signals are
reduced significantly at the leading edge in
Src42A26-1;Src64P1/+ embryos and the leading edge of the mutants is significantly kinked, the absence
of Src protein from the adherens junction may possibly result in destruction
of structural integrity, implying that the adherens junction is also involved in
dorsal closure regulation in a structural way (Takahashi, 2005).
Dorsal closure and CNS defects similar to those in Src mutants
have been observed in abl mutants. In
vertebrates, Abl is tyrosine-phosphorylated with Src and is
capable of interacting with delta-catenin, an E-cad-binding protein. Abl may
thus function as well downstream of Src signaling in Drosophila.
Germ-band retraction and possibly too, head involution, both of which require
Src activity, may be regulated by the two above distinct Src
functions. alpha1,2-laminin and alphaPS3ßPS integrin have clearly been
shown to be essential for spreading a small group of amnioserosa epithelium
cells over the tail end of the germ band during germ-band retraction.
shg activity has also been shown to be essential for
normal germ-band retraction and head involution (Takahashi, 2005).
Src-dependent dynamical regulation of E-cad-dependent cell-cell adhesion
may also be necessary for visual system formation. E-cad overexpression or
elimination of EGFR activity have been shown to render optic placode cells
incapable of invaginating and prevent the separation of Bolwig's organ
precursors from the optic lobe. Virtually identical phenotypes were induced by loss
of Src activity, suggesting involvement of at least the
adherens junction Src in larval visual system formation and that Src should
function either upstream or downstream of EGFR signaling (Takahashi, 2005).
Cadherin-mediated adhesion can be regulated at many levels, as demonstrated by detailed analysis in cell lines. This study examines the requirements for Drosophila epithelial (DE) cadherin regulation in vivo. Investigating Drosophila oogenesis as a model system allowed the dissection of DE-cadherin function in several types of adhesion: cell sorting, cell positioning, epithelial integrity, and the cadherin-dependent process of border cell migration. Multiple fusions were generated between DE-cadherin and alpha-catenin as well as point-mutated ß-catenin and the ability of these fusion proteins to support these types of adhesion was analyzed. It was found that (1) although linking DE-cadherin to alpha-catenin is essential, regulation of the link is not required in any of these types of adhesion; (2) ß-catenin is required only to link DE-cadherin to alpha-catenin, and (3) the cytoplasmic domain of DE-cadherin has an additional specific function for the invasive migration of border cells, which is conserved to other cadherins. The nature of this additional function is discussed (Pacquelet, 2005).
Classic cadherin proteins have multiple essential roles during animal development both in keeping tissues/epithelia intact and in allowing dynamic cell rearrangements. One dramatic example of the latter is the invasive migration of border cells during oogenesis, for which DE-cadherin is essential. This study investigates which features of DE-cadherin are required for migration, and these features are compared with features that are required more generally for other adhesion functions. Cadherin proteins are well conserved from fly to man; the cytoplasmic domain, in particular, is well conserved, and it interacts with the cytoskeleton. Therefore, the in vivo genetic analyses focused on dissecting the functions of DE-cadherin cytoplasmic domain. In the type of in vivo replacement experiments performed, clear conclusions could be made about what is and is not required under physiological conditions. This is the strength of the analysis, and it is thought to be important to further the understanding of the much-studied cadherin molecules. Generally speaking, the idea cannot be excluded that a type of regulation that is not genetically required does, in fact, occur under normal conditions and contribute somewhat to regulation (e.g., to make the system more robust) (Pacquelet, 2005).
Focus was initially placed on a conserved tyrosine of ß-catenin, the phosphorylation of which may induce ß-catenin to dissociate from cadherin, resulting in a decrease of adhesion. This conserved tyrosine (and, hence, its phosphorylation) is not essential even during border cell migration. The idea that phosphorylation of this tyrosine residue happens or that it may induce some dissociation of ß-catenin from DE-cadherin cannot be excluded. What these results show is that such phosphorylation is not an essential mechanism for adhesion regulation in any of the tested types of cadherin-dependent adhesion in vivo. Significant emphasis has been put in the literature on the putative regulatory role of this conserved tyrosine of ß-catenin. However, much of this emphasis is based on correlations between ß-catenin tyrosine phosphorylation and adhesion down-regulation. It is not clear whether ß-catenin phosphorylation is really the cause of adhesion down-regulation. In addition, the tyrosine kinase Src causes a decrease of adhesion in L cells expressing the fusion protein E-cadherin/alpha-catenin. Thus, Src-induced adhesion down-regulation can be independent of ß-catenin phosphorylation. Therefore, the ability to regulate adhesion without phosphorylating ß-catenin tyrosine may be more general (Pacquelet, 2005).
Next, it was found that neither the link between DE-cadherin and ß-catenin nor that between ß- and alpha-catenin need be regulated at all for DE-cadherin function in vivo. A fusion between DE-cadherin-FL and alpha-catenin fully substitutes for endogenous DE-cadherin during oogenesis even in the absence of endogenous ß-catenin. It was surprising to find that there is no need to regulate the link between DE-cadherin and alpha-catenin, since earlier studies using similar fusion proteins had concluded that regulation was required for mouse E-cadherin to support 'intercellular migration' (Nagafuchi, 1994). There are two main explainations for this discrepancy. (1) The previous study did not fuse alpha-catenin to E-cadherin-FL but fused to a truncated E-cadherin (analogous to DE-cadherinDeltaß/alpha-catenin). As was found in the current study, this not only affects the ability to regulate the link to alpha-catenin but also removes additional functionality from cadherin. It was not directly investigated in Nagafuchi (1994) whether the defects were caused by ß-catenin regulation as proposed. (2) Different cell types were analyzed; the previous study overexpressed E-cadherin in mouse fibroblasts that normally have very little of the protein, whereas this study investigated cells that normally depend on DE-cadherin for biological function(Pacquelet, 2005).
It is possible that the link between DE-cadherin and the actin cytoskeleton does need to be regulated but that it occurs downstream of alpha-catenin. More studies of alpha-catenin and of how its interactions are regulated will be of interest, in particular in a physiological context. Alternatively, regulation of adhesion may primarily occur by the turning over of DE-cadherin and/or DE-cadherin complexes via endocytosis. A Cbl-related E3 ligase called Hakai has been identified as a specific regulator of mammalian E-cadherin endocytosis (Fujita, 2002). It is recruited to specific phosphorylated tyrosines on E-cadherin. No evidence was found that the homologous D. melanogaster protein (CG10263) affects DE-cadherin or border cell migration, and the key docking tyrosines are not conserved. However, other regulators may play an analogous role. Finally, adhesive strength could be regulated by lateral clustering of cadherin complexes; for example, by the binding of additional regulatory proteins to the intracellular domain (Pacquelet, 2005).
The full functionality of DE-cadherin-FL/alpha-catenin in the absence of ß-catenin also indicates that ß-catenin has no essential adhesive function other than linking DE-cadherin to alpha-catenin. Based on the abnormal localization of various DE-cadherin mutants, it had been proposed that ß-catenin was required for proper translocation of cadherin to the plasma membrane. However, the relatively normal subcellular localization of DE-cadherin-FL/alpha-catenin that was observed in the absence of ß-catenin suggests that this is not generally the case. It remains possible that ß-catenin also contributes to modifying cadherin localization in D. melanogaster cells, but in a more subtle, nonessential way. This study suggests that parts of the cadherin tail that bind ß-catenin may also have ß-catenin-independent functions. This would complicate the interpretation of how modified cadherin molecules behave unless it is also investigated by ß-catenin loss-of-function experiments (Pacquelet, 2005).
In contrast with DE-cadherin-FL/alpha-catenin, a fusion protein between DE-cadherin and alpha-catenin lacking the DE-cadherin cytoplasmic tail (DE-cadherinDeltaCyt/alpha-catenin) could not substitute for DE-cadherin during border cell migration. It was targeted to the cell surface and was functional in all other contexts. This indicates that the DE-cadherin cytoplasmic tail has a specific function during invasive migration in addition to the basic ß-catenin/alpha-catenin linkage. The function could not be provided by an unrelated cytoplasmic linker (CD2) but could be provided by the corresponding region from mouse E-cadherin or D. melanogaster N-cadherin. Most likely, one or more interactions that are specific to cadherin tails have a critical function in this context. These results raise two questions: (1) why is DE-cadherin tail specifically important for border cell migration and (2) what is the molecular nature of the required function (Pacquelet, 2005)?
With regard to the specific requirement in border cells, the role of DE-cadherin in their migration needs to be considered. Given the absolute requirement for this particular cell-cell interaction to achieve invasive border cell movement, it is likely to be force bearing. DE-cadherin-mediated adhesion between the front of border cells and the attachment point on nurse cells needs to be strong enough to allow border cells to pull themselves into the compact germ line tissue. As the border cell cluster initiates migration using a long, slender cellular extension, the local force application at the tip may be quite high. As an illustration of the forces involved, it was found that mutant border cells with impaired cortical cytoskeleton will break apart when they attempt to invade, whereas other follicle cells (including centripetal cells) with the same defect appear to be relatively normal. It is suggested that the DE-cadherin tail may be required to allow a build-up of sufficiently strong adhesion to withstand forces that are involved in migration (Pacquelet, 2005).
Another important aspect of adhesion during cell movement is that it may need to be effectively down-regulated at the rear of the cells to allow cell translocation along the substrate. Experiments indicated that the primary defect for DE-cadherinDeltaCyt/alpha-catenin in border cells is not a lack of down-regulation; in other words, it is not caused by an excess of adhesion. However, an inability of DE-cadherinDeltaCyt/alpha-catenin to provide sufficient adhesion for migration as discussed above could mask possible additional (migration specific) defects of the fusion protein such as the ability to be down-regulated (Pacquelet, 2005).
The molecular nature of the DE-cadherin tail requirement in migration is in need of further investigation. The function does not simply map to any previously known signal or interaction, suggesting involvement of a novel interaction and/or a redundancy of interactions. The DE-cadherinDeltaß/alpha-catenin fusion results indicate that the most COOH-terminal domain contributes to DE-cadherin function in border cells independently of ß-catenin binding. However, this domain is not essential on its own nor when coexpressed with p120 catenin RNA interference constructs, indicating that additional important signals are located in the more proximal region of the DE-cadherin cytoplasmic domain. A mutant form of Xenopus laevis C-cadherin lacking the 94 proximal amino acids of its cytoplasmic domain can mediate some adhesion but is unable to support strong adhesion. This seems to be caused by its inability to form lateral clusters. Similarly, an absence of the proximal region in DE-cadherinDelta-Cyt/alpha-catenin could prevent its clustering and, thereby, prevent adhesion strengthening (Pacquelet, 2005).
In conclusion, this structure/function analysis of DE-cadherin in different types of cell adhesion has given new information about cadherin regulation in vivo. Several previously defined potential points of regulation that were established through detailed work in tissue culture were found not to be essential for functionality in vivo. The cytoplasmic tail of cadherin was found to have a unique role in the demanding process of invasive cell migration, possibly through a novel interaction (Pacquelet, 2005).
Unconventional myosin proteins of the MyTH-FERM superclass are involved in intrafilopodial trafficking, are thought to be mediators of membrane-cytoskeleton interactions, and are linked to several forms of deafness in mammals. This study shows that the Drosophila myosin XV homolog, Sisyphus, is expressed at high levels in leading edge cells and their cellular protrusions during the morphogenetic process of dorsal closure. Sisyphus is required for the correct alignment of cells on opposing sides of the fusing epithelial sheets, as well as for adhesion of the cells during the final zippering/fusion phase. Several putative Sisyphus cargos have been identifed, including DE-cadherin (also known as Shotgun) and the microtubule-linked proteins Katanin-60, EB1, Milton and aPKC. These cargos bind to the Sisyphus FERM domain, and their binding is in some cases mutually exclusive. These data suggest a mechanism for Sisyphus in which it maintains a balance between actin and microtubule cytoskeleton components, thereby contributing to cytoskeletal cross-talk necessary for regulating filopodial dynamics during dorsal closure (Liu, 2008).
Epithelial movements underlie fundamental physiological processes including embryonic morphogenesis, wound healing and cancer metastasis. During dorsal closure (DC), a morphogenetic event that occurs late in Drosophila embryogenesis, two lateral sheets of epithelial cells move towards one another over a dorsally exposed region of extraembryonic tissue and fuse together at the midline. During this process, the dorsal-most face of leading edge (LE) epithelial cells exhibit dynamic cellular protrusions, lamellipodia and filopodia, required initially for sensing their environment and finding their appropriate counterpart on the opposing epithelial sheet. Subsequently, the opposing protrusions adhere to one another, facilitating the formation of transient cell-cell contacts as the epithelial sheets zipper together, followed by permanent cell-adhesion structures. Filopodia contain a core of organized bundled actin filaments, oriented with their barbed (plus) ends towards the tip. Filopodia continuously assemble and disassemble, with growth occurring via de novo actin nucleation and polymerization locally at their tips. Although the dynamic rearrangements of cells and filopodia during DC are mainly attributed to actin dynamics, a recent report has described a role for microtubules in epithelial zippering, the final step of DC (Liu, 2008).
The coordination of environmental sensing, cell-cell recognition and adhesion mediated by LE cell protrusions must require orchestrated movements of structural, adhesive and regulatory molecules within filopodia. Unconventional myosins have recently been implicated in the movement of such cellular molecules/machineries within filopodia. Unconventional myosins are actin-based motor proteins that can be subdivided into at least 18 distinct classes (I-XVIII) based on their motor and tail domain structural and functional characteristics. One subset, the `MyTH-FERM' unconventional myosins, includes classes VII, X, XII and XV that share structurally conserved features in their tails: MyTH4 (myosin tail homology 4) domains that bind to microtubules and FERM (band 4.1, ezrin, radixin, moesin) domains that are involved in cargo binding. The tail regions are thought to determine where the myosins are located and what cargos they transport (Liu, 2008).
Mutations in MyTH-FERM unconventional myosins result in disorganized stereocilia leading to deafness and vestibular dysfunction in humans and mice: myosin VIIa is responsible for human Usher Syndrome type IB and the mouse shaker 1 mutation, whereas myosin XV is linked to human non-syndromic deafness, DFNB3, and the mouse shaker 2 mutation. In Dictyostelium, Myosin VIIa (MVII) localizes to filopodial tips and is required for the formation of filopodia and cell attachment. In mammalian cells, Myosin X (Myo10) moves bidirectionally within filopodia and accumulates at filopodial tips. Ectopic expression of Myo10 is sufficient to direct assembly of filopodia in cells lacking them. The Myo10 FERM domain has been shown to bind β-integrin and transport it to filopodial tips where it is needed for proper filopodial extension and substratum adherence (Liu, 2008).
Drosophila has three MyTH-FERM myosin homologs: myosin VIIa, VIIb and XV. Functional data has only been reported for crinkled (myosin VIIa), mutants of which are semi-lethal with escaper adults exhibiting defects in actin-rich structures such as bristles and hairs, and deafness due to disruption of scolopidia auditory organ integrity required for transducing auditory signals (Liu, 2008 and references therein).
This study shows that the Drosophila myosin XV homolog, which is here named Sisyphus (Syph), is required for proper DC where it traffics sensory, cytoskeletal, and adhesion cargos within LE cells and their filopodial protrusions (Liu, 2008).
Dorsal closure is a complex morphogenetic process that is dependent on cell protrusions that are highly dynamic, requiring F-actin filaments and microtubules present in the structures to grow and shrink very rapidly. Transport of proteins to and within these protrusions and the leading edge is essential for the environmental sensing, cell-cell recognition and adhesion events that take place during DC. This paper presents the first characterization of the Drosophila myosin XV homolog, Sisyphus, and shows that it is required for DC during at least two key steps: for proper epithelial alignment, and for zippering and fusion of the two epithelial sheets. Live imaging of Syph shows that this motor protein accumulates at the leading edge during DC, whereas mapping and RNAi studies demonstrate interactions with its cargo proteins, consistent with a role as a transport protein. The results also suggest a possible role for Syph in the coordination of the actin and MT networks required for the dynamic protrusions during DC (Liu, 2008).
The MyoX MyTH-FERM myosin has been proposed to play a structural role by facilitating actin polymerization at filopodia tips by pushing the plasma membrane away from the growing actin filament barbed ends to create a space for actin monomer addition. Although Syph is present at filopodium ends and could perform a similar role for actin or MT assembly, unlike MyoX, it is not preferentially found at tips. Instead, Syph moves bi-directionally within LE cells and their protrusions. Reduction of Syph by RNAi disrupts filopodia formation, and this probably contributes to the segment mismatching and zippering/fusion phenotypes observed in syph-deficient embryos. Dictyostelium cells mutant for the myosin-VII MyTH-FERM protein have also been shown to exhibit loss of filopodia and adhesion defects. How filopodial formation is disturbed in syph-deficient embryos remains to be answered, though improper distribution of cargo proteins required for proper filopodial dynamics and integrity of the leading edge is a strong possibility. Another interesting and nonexclusive model is that disruption of the actin-microtubule network caused by Syph knockdown in turn disrupts filopodial formation and/or integrity of the leading edge. Additional studies will be required to differentiate between these two possibilities (Liu, 2008).
Mapping data shows that Syph appears to play a key role in transporting structural, adhesive and regulatory molecules within filopodia via its C-terminal FERM domain. This is consistent with studies in mammals that show that, in addition to the motor domain, the FERM domain of MyoXV is critical for development of stereocilia required for normal hearing and balance). One Syph cargo that binds to the FERM domain of Syph was identified as DE-cadherin. Cadherin is required at filopodium ends where it forms transient cell-cell contacts, followed by more permanent cell adhesion ones. syph-deficient embryos are defective in epithelial fusion during DC, presumably due to the failure of cadherin to correctly accumulate at the dorsal-most edge of LE cells to mediate fusion and adhesion. Interestingly, this 'failure to close' phenotype is reminiscent of that observed in Rac GTPase mutants proposed to interfere with the contact-inhibition machinery. These results may have clinical relevance, since mutations in the X, VIIA and XV classes of MyTH-FERM unconventional myosins have been shown to cause deafness and aberrant morphology of stereocilia in inner-ear hair cells. Interestingly, in addition to MyoVIIa alleles, mapping of recessive mutations for the most common form of hereditary deaf-blindness in humans, Usher syndrome, has identified alleles of cadherin-23 and protocadherin-15. The MyoVIIa MyTH-FERM myosin has also been shown to interact with cadherin complexes through the vezatin adaptor protein. Together, these observations suggest that proper distribution and localization of cadherin is essential for normal stereocilia formation or maintenance and may be a conserved task among MyTH-FERM unconventional myosins (Liu, 2008).
In addition, the binding site for Syph on DE-cadherin was mapped to a C-terminal 40aa fragment of the cadICD. The Drosophila genome contains 17 cadherin proteins in addition to DE-cadherin; three of these show conservation within this C-terminal 40aa motif. Interestingly, this region of DE-cadherin has not been previously assigned a function, but shows conservation with over 15 human cadherins, and thus may define a novel unconventional myosin-binding domain by which cadherins are tethered and transported (Liu, 2008).
A remarkable recent finding is the presence of and requirement for MTs in filopodia, as well as in the final stages of zippering during fly DC. Although unconventional myosins have been generally considered as actin-based motor proteins, members of the MyTH-FERM class were recently shown to bind to and travel along MTs via their MyTH4 domains. Thus, in addition to trafficking on actin, Syph could traffic on MTs through its two MyTH4 domains. Dynamic MTs have been proposed to work by regulating local concentrations of the cadherin needed to establish and maintain cell-cell contacts, or by delivering actin-organizing proteins to filopodia tips. Thus, Syph could use MTs both as a transport substrate, and be involved in their dynamic assembly/disassembly (Liu, 2008).
Another appealing possibility is that Syph may play a role in coordinating the two cytoskeletons. Several of the putative cargos identified for Syph are MT-associated proteins: α-tubulin, Katanin-60, Milt and EB1 are all MT components or binding partners. Transport of the MT subunit, α-tubulin, and the MT-severing protein, Katanin-60, suggest a role for Syph in the assembly and disassembly of MTs, whereas the plus end MT-binding protein EB1 suggests a role in stabilization and regulation. A recent study has shown that ectopic expression of another Drosophila MT-severing protein, spastin, resulted in delayed epithelial hole closure [taking about 9 hours instead of 3 hours to reaching completion]. The similar phenotype that was observe in syph-deficient embryos suggests that Syph modulates MT cytoskeleton regulation by transporting cargo proteins essential for its regulation (Liu, 2008).
Identification of cadherin and several MT-linked proteins as Syph cargos and the mutually exclusive - and perhaps competitive - binding for these cargos on the Syph FERM domain, leads to a proposal that one role of Syph is to coordinate the actin and MT networks during filopodial dynamics through differential cargo transport. Consistent with this possibility, it was found that both α-tubulin and actin overexpression rescues the Syph 'failure to close' phenotype, and in the case of actin, the delayed closure phenotype as well. These results suggest that the actin network can partially compensate for disruption of the MT one, and that Syph, in addition to serving as a delivery system, may play a role in the regulation of actin and MT cytoskeleton cross-talk during processes such as DC. The finding that the binding sites for putative cargo proteins are mutually exclusive implies that Syph itself must be regulated by proteins that help it 'choose' particular proteins to transport. Future studies aimed at uncovering and deciphering the rules governing the choice of cargo and transport substrate for unconventional myosin motors are likely to provide exciting new insight into the coordinate regulation of and cross-talk between the actin and MT cytoskeletons during highly orchestrated morphogenetic events such as DC (Liu, 2008).
Developing tissues require cells to undergo intricate processes to shift into appropriate niches. This requires a functional connection between adhesion-mediating events at the cell surface and a cytoskeletal reorganization to permit directed movement. A small number of proteins are proposed to link these processes. This study identifies one candidate, Cindr, the sole Drosophila melanogaster member of the CD2AP/CIN85 family (this family has been previously implicated in a variety of processes). Using Drosophila retina, it was demonstrated that Cindr links cell surface junctions (E-cadherin) and adhesion (Roughest) with multiple components of the actin cytoskeleton. Reducing cindr activity leads to defects in local cell movement and, consequently, tissue patterning and cell death. Cindr activity is required for normal localization of Drosophila E-cadherin and Roughest, and this study shows additional physical and functional links to multiple components of the actin cytoskeleton, including the actin-capping proteins capping protein alpha and capping protein beta. Together, these data demonstrate that Cindr is involved in dynamic cell rearrangement in an emerging epithelium (Johnson, 2008).
By recruiting proteins into complexes, adaptor proteins create nodes of regulation and activity. The founding member of the CD2AP/CIN85 family of adaptor proteins was initially isolated in a yeast interaction screen as a binding partner of the T cell receptor CD2, independently from the kidney (MET-1) and as a ligand for p130Cas. The homologue CIN85 was identified as a partner of the E3 ubiquitin ligase Cbl and separately as SETA and Ruk. Many roles have been ascribed to the CD2AP/CIN85 family but its function in situ remains poorly understood. This study examine the sole Drosophila melanogaster CD2AP/CIN85 orthologue, cindr (Johnson, 2008 and references therein).
The phenotype of CD2AP knockout mice is chiefly one of tissue degeneration: cardiac hypertrophy, splenic and thymic atrophy, glomerular sclerosis, and a loss of podocyte foot processes. The CD2AP/CIN85 family is primarily proposed to function in endocytosis to down-regulate receptor tyrosine kinase activity. This model arises from coimmunoprecipitation and interaction experiments that have identified a wealth of CD2AP/CIN85 interactors, colocalization studies performed in culture or tissue, and in vitro assays. CIN85 constitutively associates with endophilin and, on growth factor stimulation, complexes with Cbl to mediate receptor down-regulation. Furthermore, interactions between CD2AP/CIN85 and other trafficking proteins have been described including AP-2, Dab2, Rab4, PAK2, Scraps) at the actin-rich cleavage furrow. CD2AP activity is required for migration of rat gastric mucosal cells and polarization of the cytoskeleton during T cell receptor activation. The role and mechanism by which CD2AP/CIN85 regulates cytoskeletal dynamics within an epithelium in situ remains unclear (Johnson, 2008).
Furthermore, the CD2AP/CIN85 family has also been reported to bind the adhesion molecules E-cadherin and nephrin. Nephrin and NEPH-1 form the backbone of the slit diaphragm, a specialized junction that traverses podocyte foot processes in the mammalian kidney. Direct interactions between CD2AP, nephrin, and podocin and between CD2AP and the podocyte-specific actin-bundling protein synaptopodin are essential for slit diaphragm integrity. In addition, a protein complex containing nephrin, cadherin, p120-catenin, ZO-1, and CD2AP has been isolated from Madin-Darby canine kidney cells and mouse glomerular lysates. Collectively, these data suggest that CD2AP may have a role in anchoring junctions to the cytoskeleton or in regulating actin dynamics at this important intersection (Johnson, 2008).
The challenge remains to understand how different roles of CD2AP/CIN85 are integrated in the organism, which interactors are recruited into CD2AP/CIN85 complexes, and how these are regulated. This study shows that targeted reduction of cindr in the pupal fly eye resulted in defects in overall patterning due to aberrant local cell movements. These defects were linked to misregulation of actin dynamics and mislocalization of Drosophila E-Cadherin (DE-Cad) and the fly NEPH-1 orthologue Roughest (Rst) which, with its binding partner Hibris (Hbs; a Drosophila Nephrin orthologue), is a central mediator of cell-cell adhesion in the pupal retina. Cindr was lined functionally and physically to orthologues of capping protein alpha (Cpa) and capping protein beta (Cpb), and this study further explored the role of Cindr in modulating the actin cytoskeleton during eye maturation. The data support a primary role for cindr in linking junction and actin regulation and help account for many of the phenotypes ascribed to mutations in mammalian CD2AP/CIN85 (Johnson, 2008).
The evidence indicates that Cindr provides a functional link between dynamically regulated surface adhesion and the cytoskeletal changes required for normal pupal eye patterning. Loss of cindr activity leads to misplacement of retinal support cells, which adopt shapes uncharacteristic for their niche in the retinal field. The reasons for this became apparent when cell behavior was examined in live tissue. Reducing cindr prevents 1° cells from maintaining enwrapment of the cone cells; instead, cindr 1° cells are unable to firmly establish this niche and frequently retract, allowing neighboring interommatidial precursor cells (IPCs) to have direct contact with the cone cells. Similar instability was observed in the remaining IPCs fated to establish the 2°/3° hexagonal lattice. Histology demonstrated further changes both to AJ components and the actin cytoskeleton. Although additional roles for Cindr such as regulation of endocytosis cannot be ruled out, no evidence was observed for such a role during cell rearrangements (Johnson, 2008).
In wild-type tissue, Hbs and Rst are localized exclusively to 1°-IPC interfaces during IPC patterning; heterophilic interactions between these molecules are thought to direct the rearrangement of IPCs into single rows around each ommatidium. In cindr-IR tissue (an RNAi inverted repeat knockout), a mislocalization was observed of Rst to the entire IPC circumference. Such mislocalization would impede the generation of a preferential adhesive force, disrupting the direction or flow of cell movement and subsequent patterning. Irregularities were detected in the localization of DE-Cad around the circumference of retinal cells when Cindr was reduced. This presumably resulted in uneven or unreliable junctional stability that further destabilized the dynamic switching of cell positions. Genetic interactions with the loci for rst, hbs, and shg confirmed that these loci cooperate with cindr during patterning (Johnson, 2008).
It was also demonstrated that the actin cytoskeleton is dynamically remodeled during pupal eye patterning and that reducing cindr activity leads to a change in the details of cytoskeletal dynamics. In wild-type tissue, polymerized actin was initially detected almost exclusively at AJs of pre-1° cells and IPCs. These actin rings intensify as cells became rearranged and then, remarkably, once patterning is established, membrane-associated F-actin strongly diminishes. The functional significance of these changes is likely linked to concurrent modification of Rst- and AJ-mediated adhesion. For example, the levels of both DE-Cad and Armadillo (β-catenin) decrease between IPCs as they are rearranged, which would serve to facilitate Rst-mediated IPC movements toward 1° cells. Data indicate that the actin cytoskeleton is coordinately reinforced at AJs as adhesion is weakened. This may serve to maintain the surface integrity of the retinal cells while junctional strength decreases and the tissue is remodeled. Once patterning is achieved, the BMP receptor Thickvein then acts to restrengthen AJs; the reduction of F-actin may reflect the reduced need for a dense actin ring. Throughout this process, Cindr acts as a pivotal regulator to coordinate AJ modification and actin polymerization (Johnson, 2008).
Several data presented in this paper suggest that Cindr acts directly to regulate actin. First, the intensity and distribution of cytoplasmic Cindr puncta in retinal cells tracks that of F-actin in IPCs during development. Second, the striking dynamics of F-actin polymerization are lost, presumably helping account for their abnormal cell movements. Third, strong genetic interactions were observed between cindr-IR and multiple components of the actin regulatory machinery. Fourth, the two capping protein subunits Cpa and Cpb coimmunoprecipitated together with Cindr from Drosophila embryos. And, finally, several phenotypes are shared between tissue mutant for cindr-IR and tissue mutant for cpa or cpb: pupal eye mispatterning, gaps in the distribution of DE-Cad around the circumference of retinal cells, bristle malformation, and tissue degeneration (Johnson, 2008).
These results emphasize the important role of the actin cytoskeleton in regulating or maintaining AJ integrity. However, the data also argue that Cindr regulates the localization of transmembrane adhesion proteins at least in part independently of the cytoskeleton: reducing the genetic component of actin regulators enhances the patterning defects of cindr-IR but not the disruption to DE-Cad, and ectopic Arp66B rescues cindr-IR mispatterning but does not rescue aberrant localization of Rst. In the absence of Cindr, miscoordination of the actin machinery together with aberrant localization of junctional complexes is likely the underlying cause of tissue mispatterning during development. Similarly, deregulation of actin and junction instability is apparently cell lethal in more mature tissue, eventually leading to degeneration of mutant tissue. This may be analogous to the degeneration of mammalian podocytes that has been associated with mutations in CD2AP. How Cindr itself is regulated during development remains an open question (Johnson, 2008).
Vertebrates and insects alike use glial cells as intermediate targets to guide growing axons. Similar to vertebrate oligodendrocytes, Drosophila midline glia (MG) ensheath and separate axonal commissures. Neuron-glia interactions are crucial during these events, although the proteins involved remain largely unknown. This study shows that Canoe (Cno), the Drosophila ortholog of AF-6, and the DE-cadherin Shotgun (Shg) are highly restricted to the interface between midline glia and commissural axons. cno mutant analysis, genetic interactions and co-immunoprecipitation assays unveil Cno function as a novel regulator of neuron-glia interactions, forming a complex with Shg, Wrapper and Neurexin IV, the homolog of vertebrate Caspr/paranodin. These results also support additional functions of Cno, independent of adherens junctions, as a regulator of adhesion and signaling events in non-epithelial tissues (Slováková, 2011).
The midline constitutes a key boundary of bilateral organisms. In vertebrates, it is the floorplate and the functionally equivalent structure in Drosophila is the mesectoderm, which gives rise to all midline cells, neurons and glia, in the most ventral part of the embryo. MG are of great relevance at the midline as an intermediate target during axonal pathfinding, providing both attractive and repulsive guidance cues. These signals allow contralateral axons to cross the midline but never to recross, and they also keep ipsilateral axons away from the midline. In addition to this early function in guiding commissural axons towards the midline, MG are also fundamental later on to separate the commissures by enwrapping and subdividing them. This study shows that the PDZ protein Cno and the DE-cadherin Shg participate in, and contribute to, the regulation of these later stage neural differentiation events, in which neuron-glia interactions play a central role (Slováková, 2011).
In Drosophila, Wrapper and Nrx-IV physically interact to promote glia-neuron intercellular adhesion at the MG. This study proposes that Cno and Shg are important components of this adhesion complex and key to its function. Both Cno and Shg are present at the MG, being highly restricted to the interface between MG and commissural axons. Cno and Shg were detected in a complex in vivo with Wrapper at the CNS MG. Nrx-IV, which is located on the surface of commissural axons, was also consistently found in a complex with Cno, although the amount of Cno protein that was co-immunoprecipitated was much lower than that present in Cno-Wrapper complexes. One plausible explanation is that whereas Cno and Wrapper are present in the same cell (MG), Cno and Nrx-IV are in different cell types (MG and neurons, respectively) and, in addition, Cno is a cytoplasmic protein that is indirectly linked to Nrx-IV through other proteins in the same complex (i.e., Shg and Wrapper). Intriguingly, stronger genetic interactions were found between Cno and Nrx-IV than between Cno and Wrapper (double heterozygote analysis). A possible explanation for this is that Nrx-IV is not only acting through Wrapper-Shg-Cno in the MG but also through other partners. In this way, when the dose of Cno and Wrapper was halved, Nrx-IV could still function fully through these other, putative partners. However, halving the dose of Cno and Nrx-IV would impair not only the Nrx-IV-Wrapper-Cno signal but also the other potential pathways. In vertebrates, the ortholog of Nrx-IV, termed contactin-associated protein (Caspr or Cntnap) or paranodin, is located at the septate-like junctions of the axonal paranodes, where it interacts in cis with contactin (at neurons) and in trans with neurofascin (at the glia). The Drosophila homologs of these Ig superfamily proteins, Contactin and Neuroglian, interact in the same way with Nrx-IV at the septate junctions. However, there are no septate junctions at the neuron-MG interface. Hence, other, as yet unknown partners of Nrx-IV might exist at this location (Slováková, 2011).
Cno and its vertebrate orthologs afadin/AF-6/Mllt4 have been shown to localize at epithelial adherens junctions (AJs), where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and Nectin family proteins. However, Cno is not exclusively present at the AJs of epithelial tissues. Indeed, it was found that Cno is also expressed in mesenchymal tissues, where it dynamically regulates three different signaling pathways required for muscle/heart progenitor specification. The asymmetric division of these muscle/heart progenitors and of CNS progenitors also requires an AJ-independent function of Cno to asymmetrically locate cell fate determinants and properly orientate the mitotic spindle. Therefore, Cno seems to act through different mechanisms depending on the cell type. This study describes a novel function of Cno during neural differentiation. In the MG, Cno, through Shg, contributes to the tight adhesion between the MG and the commissural axons and perhaps even to the regulation of some intracellular signaling within the MG. Indeed, Cno has been shown to regulate different signaling cascades during development. No AJs or septate junctions (SJs) have been described at the MG-commissural axon interface. This suggests that the function of Cno in the midline is independent of AJs. In fact, the partner of Cno at this location, the Drosophila Nectin ortholog Echinoid, is not detected at the midline. In this context, it is worth pointing out that Shg is an epithelial cadherin key at AJs. This study has shown that Shg can also be found in non-epithelial tissues with an important function independent of AJs. A similar situation occurs with Nrx-IV. Despite Nrx-IV being a very well established component of SJs, no SJs are formed in the midline and no other known components of SJs are expressed there. Thus, different modes of Cno action, either as an AJ protein or as a signaling pathway regulator, are possible and they are not mutually exclusive: it all depends on the cell type and context (Slováková, 2011).
Frizzled planar cell polarity (PCP) signaling regulates cell motility in several tissues, including ommatidial rotation in Drosophila melanogaster. The Nemo kinase (Nlk in vertebrates) has also been linked to cell-motility regulation and ommatidial rotation but its mechanistic role(s) during rotation remain obscure. This study shows that nemo functions throughout the entire rotation movement, increasing the rotation rate. Genetic and molecular studies indicate that Nemo binds both the core PCP factor complex of Strabismus-Prickle, as well as the E-cadherin-β-catenin (E-cadherin-Armadillo in Drosophila) complex. These two complexes colocalize and, like Nemo, also promote rotation. Strabismus (also called Vang) binds and stabilizes Nemo asymmetrically within the ommatidial precluster; Nemo and β-catenin then act synergistically to promote rotation, which is mediated in vivo by Nemo's phosphorylation of β-catenin. These data suggest that Nemo serves as a conserved molecular link between core PCP factors and E-cadherin-β-catenin complexes, promoting cell motility (Mirkovic, 2011).
The data suggest that Nmo connects the core PCP Stbm-Pk complex to the activity of E-cad-β-cat. Consistent with this, mutations in stbm and pk enhance not only the nmoP rotation defects but also rotation defects of hypomorphic shg (E-cad) backgrounds. As the presence of the Stbm-Pk complex seems to increase the amount of Nmo at R4 membranes and junctional complexes, it is hypothesized that a rise in Stbm levels would increase the ability of sev>Nmo to cause an over-rotation phenotype. This is indeed the case. These data indicate that Nmo serves as a link from PCP factors to the E-cad-catenin complexes. The data are consistent with a model in which the Stbm-Pk complex helps to recruit and/or stabilize Nmo at membrane regions (where the PCP factors partially overlap with E-cad-β-cat complexes (Mirkovic, 2011).
The effect of Nmo on E-cad-β-cat complexes could be mediated either through the dynamics of lateral clustering (for example, formation or disassembly of higher-order E-cad-β-cat complexes) or through changes in the interaction of β-cat with other associated proteins. An E-cad::β-cat fusion protein (which bypasses a β-cat requirement and provides stable adhesion is not influenced by Nmo, suggesting that once β-cat is part of the E-cad-catenin complex Nmo cannot affect their activity. It is thus possible that phosphorylation of β-cat by Nmo affects the E-cad-β-cat complex activity (as an ArmS10AAA isoform with the Nmo target sites mutated no longer cooperates with Nmo) and this phosphorylation may also modulate interactions of the complex with other binding partners, such as β-cat. The interactions of adhesion and planar polarity during the early 'convergence-extension' rearrangements in the fly embryo suggest a mechanism in which a polarized pattern of junction remodeling drives cell intercalation. Polarized activity of RhoA and Myosin II (encoded by zipper) regulates adherens junction disassembly along the anterior-posterior axis, primarily by regulating lateral cadherin clustering without affecting surface levels of cadherins. The specific effect of RhoA on rotation, along with the interaction of nmo with zipper, supports the idea that actin-myosin contractility is downstream of Nmo. Loss of maternal contribution or Nmo overexpression in the embryonic epidermis phenocopies shg alleles or ArmS10 cuticle defects, respectively. Thus, Nmo may be generally required in epithelia undergoing morphogenetic movements, where it modulates polarized remodeling of adherens junctions in response to local asymmetries created by, for example, the activity of PCP signaling complexes (Mirkovic, 2011).
In conclusion, this study defines a framework in which Nmo serves as a link between PCP (Stbm) and the regulation of adhesive cell behavior at the level of adherens junction complexes. Although Nmo is recruited and/or maintained apically by the Stbm-Pk complex, other factors must affect Nmo activity or localization as well, because the set of cells requiring nmo (all outer R-cells) is broader than the set of cells requiring stbm (R4). First, the Nmo could regulate rate of rotation independently of the PCP complexes through Notch (N) signaling and/or Egfr signaling, as suggested by genetic data: N- alleles strongly suppress sev>Nmo, and N functions in all R-cells. Second, an asymmetric input or localization of Nmo by Stbm would provide a direction to rotation. Thus, a Notch-Nmo interaction in all cells and an asymmetric Stbm effect in R4 could combine to regulate both rate and direction of rotation. The observation that zebrafish Nlk enhances the PCP-specific Wnt11 cell migration defects in prechordal plates supports a general Nmo-mediated mechanism in PCP-associated cell movements (Mirkovic, 2011).
In bilateria, positioning and looping of visceral organs requires proper left-right (L/R) asymmetry establishment. Recent work in Drosophila has identified a novel situs inversus gene encoding the unconventional type ID myosin (MyoID). In myoID mutant flies, the L/R axis is inverted, causing reversed looping of organs, such as the gut, spermiduct and genitalia. MyoID has been shown to interact physically with β-Catenin, suggesting a role of the adherens junction in Drosophila L/R asymmetry. This study shows that DE-Cadherin co-immunoprecipitates with MyoID and is required for MyoID L/R activity. It was further demonstrated that MyoIC (Myo61F), a closely related unconventional type I myosin, can antagonize MyoID L/R activity by preventing its binding to adherens junction components, both in vitro and in vivo. Interestingly, DE-Cadherin inhibits MyoIC, providing a protective mechanism to MyoID function. Conditional genetic experiments indicate that DE-Cadherin, MyoIC and MyoID show temporal synchronicity for their function in L/R asymmetry. These data suggest that following MyoID recruitment by β-Catenin at the adherens junction, DE-Cadherin has a twofold effect on Drosophila L/R asymmetry by promoting MyoID activity and repressing that of MyoIC. Interestingly, the product of the vertebrate situs inversus gene inversin also physically interacts with β-Catenin, suggesting that the adherens junction might serve as a conserved platform for determinants to establish L/R asymmetry both in vertebrates and invertebrates (Petzoldt, 2012).
This study showns DE-Cadherin has a dual role in promoting MyoID activity. DE-Cadherin directly stabilizes MyoID at the adherens junctions and inhibits the antagonistic activity of MyoIC, thus eliciting MyoID function. MyoID activity and organization are dependent on the level of MyoIC protein. In MyoIC gain of function, MyoID intracellular pattern is modified with a reduced overall signal and the protein level remains unchanged. In MyoIC loss of function, MyoID is not detected by immunohistochemistry and the protein level measured by western blot is reduced. Nevertheless, in MyoIC loss of function, sufficient MyoID activity remains as confirmed by the wild-type rotation phenotype. Thus, MyoID activity is only impaired when MyoIC is in large excess (Petzoldt, 2012).
It is important to note that these results were obtained through two independent approaches, either by direct modulation of MyoIC expression levels or indirectly by affecting DE-cadherin, silencing of which in the A8 segment leads to an increase of MyoIC (and of MyoID) levels. These results indicate that DE-Cadherin negatively regulates both MyoID and MyoIC levels. Taken together, these data indicate that DE-Cadherin controls both the L/R determinant MyoID and its repressor MyoIC activity and protein levels (Petzoldt, 2012).
Specific depletion of DE-Cadherin in the A8 segment, the L/R organizer, leads to a no-rotation phenotype. Could stronger depletion of the adherens junction components lead to a sinistral phenotype? In other words, does DE-Cadherin depletion also affect sinistral development or pathway, which is taking over in absence of MyoID? To tackle this question, both DE-Cadherin and MyoID were depleted. It was found that, unlike the sole depletion of MyoID that leads to a majority of sinistral flies, the double depletion leads to a majority of non-rotated flies. These data therefore indicate that DE-Cadherin acts both on the dextral (MyoID-dependent) and on the sinistral pathways, making it a general regulator of L/R asymmetry in flies (Petzoldt, 2012).
Adherens junctions have previously been connected to MyoID-mediated L/R asymmetry establishment through biochemical analysis, which identified a physical interaction between the MyoID-tail domain and β-Catenin in vitro. This study demonstrates a role of the adherens junction in the Drosophila L/R pathway and places the adherens junction component DE-Cadherin as a regulator of both unconventional myosins MyoIC and MyoID. DE-Cadherin dually promotes MyoID and β-Catenin association at the adherens junctions and inhibits the antagonistic function of MyoIC. Indeed, a partial colocalization of MyoID and DE-Cadherin was observed and the concomitant exclusion of MyoIC from the adherens junction. Further supporting this model is the additional biochemical evidence that MyoID, but not MyoIC, can directly interact with DE-Cadherin and β-Catenin in vitro. Excess of MyoIC is able to disrupt this interaction both in vivo and in vitro, leading to MyoID loss-of-function phenotypes. This disruption can be rescued by MyoID overexpression. It is proposed that MyoID L/R function depends on its physical interaction with adherens junction through both β-Catenin and DE-Cadherin. Upon adherens junction reduction, MyoID does not associate with β-Catenin, MyoIC is no longer repressed and in turn inhibits MyoID activity, leading to L/R defects. Finally, the temporal synchrony of MyoID, MyoIC and DE-Cadherin requirement in genitalia rotation implies a concomitant activity of these proteins in L/R establishment (Petzoldt, 2012).
Interestingly, MyoIC has been reported to affect L/R looping of the embryonic gut, another marker of L/R asymmetry in Drosophila. It was shown that MyoIC overexpression also causes fully penetrant embryonic gut inversion. Thus, the antagonistic function of MyoIC appears to be conserved in Drosophila L/R tissues. Furthermore, it was recently shown that DE-Cadherin is required for L/R asymmetry establishment of the Drosophila hindgut (Taniguchi, 2011). The dextral curving of the hindgut was lost in a myoID and DE-cadherin mutant background accompanied by a loss of L/R biased asymmetric cell shape and a differential DE-Cadherin localization. It was suggested that both factors are implicated in the creation of asymmetric cortical tension prior to asymmetric curving of the hindgut. Taken together, these data indicate that the Drosophila L/R tissues, hindgut and genitalia, show a similar dependency on adherens junction and type I unconventional myosins for L/R determination (Petzoldt, 2012).
Is the role of adherens junctions in L/R asymmetry establishment conserved among species? Interestingly, despite the apparent differences between vertebrates and invertebrates mechanisms of L/R asymmetry establishment, a common theme can be found in the only two molecularly described situs inversus genes to date. In addition to myoID, the mouse inversin gene also causes constant L/R axis inversion in homozygous mutants. Interestingly, the inversin protein was shown to co-precipitate with β-catenin and N-cadherin and to localize to the adherens junction of polarized epithelial cells (Nurnberger, 2002). Therefore, both situs inversus proteins interact with β-catenin and associate with adherens junction molecules, such as N- or E-cadherin. Additionally, N-cadherin was reported to play a role in L/R asymmetry establishment in chicken, as N-cadherin absence at the Hensen's node leads the randomization of L/R asymmetry, similar to the current results showing a role of DE-Cadherin in L/R determination in Drosophila. The Drosophila inversin homologue diego belongs to the planar cell polarity gene family. Depletion of diego, or of any other of PCP gene, in the L/R organizer does not affect the directionality of genitalia rotation, suggesting that Diego does not play a critical role in Drosophila L/R asymmetry. In conclusion, the situs inversus proteins MyoID and Inversin, which play a central and upstream role in the establishment of L/R asymmetry, both require an interaction with the adherens junctions for their function (Petzoldt, 2012).
It is important to note that the L/R axis is established after and, most importantly, relatively to the other two main axes, the anterior-posterior and dorsal-ventral axes. Indeed, L/R determinants have to be oriented along existing spatial coordinates so that the L/R axis is positioned perpendicular to existing axes. It is proposed that the association of MyoID (and more generally of situs inversus proteins) with the adherens junctions is an essential mechanism to orient MyoID activity along the apical-basal axis, which in epithelia is perpendicular to the anterior-posterior axis and is thus equivalent to a dorsal-ventral axis. Therefore, association of MyoID with the adherens junctions would represent a way to orient and polarize its activity. Following polarization of MyoID in epithelia, the intrinsic chirality of myosins, through their directional activity towards one end of the actin filaments, could create de novo an asymmetric axis similar to the F-molecule model proposed by Brown and Wolpert (Brown, 1990; Petzoldt, 2012 and references therein).
In conclusion, the current findings reveal an important link between the adherens junction and L/R asymmetry determinant in Drosophila and suggest an evolutionary conservation in vertebrates and invertebrates of the interactions between the adherens junction and the situs inversus proteins linking cell architecture and polarity to the patterning of the L/R axis. Future work on the molecular targets of MyoID and Inversin will help understand how the L/R axis becomes oriented to lead to stereotyped asymmetric organogenesis (Petzoldt, 2012).
α-catenin associates the cadherin-catenin complex with the actin cytoskeleton. α-catenin binds to β-catenin, which links it to the cadherin cytoplasmic tail, and F-actin, but also to a multitude of actin-associated proteins. These interactions suggest a highly complex cadherin-actin interface. Moreover, mammalian αE-catenin has been implicated in a cadherin-independent cytoplasmic function in Arp2/3-dependent actin regulation, and in cell signaling. The function and regulation of individual molecular interactions of α-catenin, in particular during development, are not well understood. This study has generated mutations in Drosophila α-Catenin (α-Cat) to investigate α-Catenin function in this model, and to establish a setup for testing α-Catenin-related constructs in α-Cat-null mutant cells in vivo. This analysis of α-Cat mutants in embryogenesis, imaginal discs and oogenesis reveals defects consistent with a loss of cadherin function. Compromising components of the Arp2/3 complex or its regulator SCAR ameliorate the α-Cat loss-of-function phenotype in embryos but not in ovaries, suggesting negative regulatory interactions between α-Catenin and the Arp2/3 complex in some tissues. It was also shown that the α-Cat mutant phenotype can be rescued by the expression of a DE-cadherin::α-Catenin fusion protein, which argues against an essential cytosolic, cadherin-independent role of Drosophila α-Catenin (Sarpal, 2012).
Drosophila is used intensively to investigate the function of adherins junctions (AJs) in development. This analysis is facilitated by the availability of mutations in three of the four core components of the cadherin-catenin complex: the classic cadherin (DEcad or DN-cadherin) and the catenins Arm/β-catenin and p120 catenin. This study reports the isolation of null mutations in Drosophila α-Cat, expanding the fly tool kit to address AJ function. α-Cat mutants display defects that are indicative of loss of cadherin-mediated adhesion in late embryos, imaginal discs and ovaries. Zygotic α-Cat mutant embryos display defects in head morphogenesis, which resemble those caused by weak mutations affecting shg/DEcad. This mild phenotype is probably due to the presence of maternal α-Catenin, which is still detectable in late embryos and supports most DEcad-dependent processes during embryogenesis (Sarpal, 2012).
Analysis of head morphogenesis revealed two defects in α-Cat mutants. First, tissue breaks in the head epithelium are apparent in close proximity to clusters of cells that undergo apical constriction. It is likely that the reduced levels of the cadherin-catenin complex in α-Cat mutants do not provide sufficient adhesive strength to withstand the mechanical force exerted by those apically constricting cells. A correlation between the degree of morphogenetic stress that the epithelium is exposed to and the level of expression of the cadherin-catenin complex needed to maintain tissue integrity has been well established in embryos with reduced DEcad and in other systems. The second defect seen in α-Cat mutants is a failure in head involution. Despite any obvious defects in epithelial integrity, the dorsal fold fails to move forward and to envelope the anterior-dorsal head. As the mechanisms of head involution are unclear, there is currently no basis for speculation on how α-Catenin might contribute to this process. Breaks in the head epithelium are variable in frequency and strength, whereas the failure in head involution is a robust defect, suggesting that the breaks in the epithelium might not be the immediate cause for the problems in head involution (Sarpal, 2012).
Quantification of fluorescent intensities of α-Catenin at AJs of α-Cat zygotic mutant embryos suggested that the levels of α-Catenin had dropped to a small percentage of levels in wild-type embryos by late embryogenesis (early stage 17). DEcad and Arm were also strongly reduced but not to the same degree as α-Catenin. That the loss of α-Catenin does not lead to an immediate corresponding loss of other components of the cadherin-catenin complex was also seen during mesoderm formation in early Drosophila embryos (Oda, 1998). This suggests that the cadherin-β-catenin complex has significant α-catenin-independent membrane stability, perhaps mediated by the association with p120catenin, which regulates cadherin endocytosis. By contrast, normal protein levels of Ed and Baz were retained at AJs in α-Cat mutants, suggesting that the concentrations of these proteins at AJs do not depend on the levels of the cadherin-catenin complex. This is consistent with work in early Drosophila embryos that suggested that Baz acts upstream of the cadherin-catenin complex in AJ assembly. Ultrastructurally, AJs can retain normal appearance and size even though the level of the cadherin-catenin complex is strongly depleted. Collectively, these findings suggest that although the adhesive strength of AJs correlates with the cadherin-catenin complex content, AJ maintenance is largely unaffected by variations in cadherin-catenin complex concentration (Sarpal, 2012).
α-Cat mutant cell clones in the adult ovary display several defects previously seen in shg/DEcad or arm mutant cell clones. These include a mis-localization of the oocyte, a block of border cell migration, and lack of follicle formation and separation. α-Cat mutant cells in the follicular epithelium (FE) flatten and detach from each other and their wild-type neighbors, indicating a reduction or loss in lateral cell adhesion. Even in very large cell clones, α-Cat mutant cells retain a monolayered arrangement, being sandwiched between the germline cells and the basement membrane. Interestingly, α-Cat mutant cells display apical-basal polarity, as indicated by a tuft of microvilli positive for the microvillus cadherin Cad99C. However, apical-basal axis orientation between cells appears uncoordinated, suggesting that the cadherin-catenin complex is not required for all aspects of apical-basal polarity in follicle cells but is required for intercellular adhesion and axis alignment of neighboring cells to form a proper epithelium (Sarpal, 2012).
One interesting feature of α-Cat mutant follicle cells is the formation of prominent cytoplasmic clusters of α-Spectrin, probably the result of a collapse of the lateral cytocortex. Previous work with S2 cells suggested that the cadherin-catenin complex might not be engaged in recruiting and stabilizing cytocortical spectrin in Drosophila. However, the current data and observations made in Drosophila embryos depleted of α-Catenin with RNAi (Magie, 2002) suggest that the cadherin-catenin complex plays a crucial role in the formation of the spectrin-based lateral cytocortex, similar to its role in mammalian cells. Cytoplasmic spectrin clusters also become enriched in other basolateral and apical markers including DEcad and Crb. Notably, these clusters are also enriched in the recycling endosome GTPase Rab11. Rab11 and its effector, the exocyst, are required for surface delivery of DEcad and Crb. AJs can act as docking stations for exocyst-mediated membrane delivery. These observations raise the possibility that DEcad and Crb are retained in Rab11-positive compartments that fail to fuse with the plasma membrane due to the lack of a cadherin- or catenin-dependent docking mechanism (Sarpal, 2012).
Mammalian αE-catenin and the Arp2/3 complex can act as competitive regulators of actin polymerization. Three modes of interaction between the cadherin-catenin complex and Arp2/3 have been proposed. Model 1 proposes that the observed enrichment of αE-catenin at AJs, resulting from the interaction of αE-catenin with β-catenin, fosters local αE-catenin dimerization. Dimer formation and binding to β-catenin are mutually exclusive due to overlapping binding sites, so that dimerization of αE-catenin is thought to occur after dissociation from β-catenin. αE-catenin dimers then bind to F-actin and prevent Arp2/3 interaction with F-actin (Drees, 2005; Benjamin, 2010). αE-catenin therefore promotes the formation of F-actin bundles that are normally associated with AJs, rather than Arp2/3-dependent actin networks, because αE-catenin itself has actin bundling activity and can interact with other actin bundling proteins such as formin (Kobielak, 2004). Model 2 proposes the existence of independent junctional and cytosolic pools of αE-catenin, and that the cytosolic pool of αE-catenin shows negative regulatory interactions with Arp2/3 and, consequently, counteracts lamellipodia formation and cell motility (Benjamin, 2010). A third model for functional interactions between Arp2/3 and the cadherin-catenin complex is based on evidence suggesting that Arp2/3 is required for cadherin endocytosi. Compromised Arp2/3 activity could enhance surface abundance of the cadherin-catenin complex and therefore counteract a genetic reduction of cadherin or catenin levels (Sarpal, 2012).
Each of these models predicts that defects arising from a reduction in α-Catenin function could be suppressed by a concurrent reduction in Arp2/3 activity. This genetic interaction was observed in embryos mutant for α-Cat in which Arp2/3 or SCAR function was reduced. This study also observed that reduced Arp2/3 activity ameliorates defects seen in embryos mutant for intermediate arm alleles. This finding is consistent with models 1 and 3 but difficult to reconcile with model 2 because the loss of Arm should enhance the cytosolic pool of α-Catenin as less α-Catenin is recruited to the junction. However, the observed genetic interactions could be reconciled with model 2 by assuming that the Arm-α-Catenin interaction is required to make α-Catenin competent to interact with F-actin, either by promoting dimerization as suggested (Drees, 2005), or through promoting a post-translational modification of α-Catenin such as phosphorylation (Sarpal, 2012 and references therein).
To explore potential interactions between α-Catenin and Arp2/3 in follicle cells, advantage was taken of RNAi, and α-Cat-RNAi was coexpressed with either Sop2-RNAi or SCAR-RNAi in follicle cell clones. Sop2 or SCAR mutant or knockdown cells showed defects similar to those reported for cells in the pupal wing disc epithelium that lacked Arp2/3 function, and which apparently disrupts cadherin endocytosis. Defects in follicle cells with compromised Arp2/3 are seen only at late stages of follicle development, suggesting that the early FE does not require Arp2/3 function. α-Cat-RNAi expression caused defects similar to those observed in mutant cells, characterized by a loss of cell contact and the formation of spectrin aggregates. Expression of either Sop2-RNAi or SCAR-RNAi in α-Cat-RNAi cells did not noticeably modify the α-Cat phenotype. One potential explanation for the lack of interactions in this context might be that the disruption of adhesion by α-Cat-RNAi is too strong to be overcome significantly by lowering Arp2/3 activity, assuming that the interaction between α-Catenin and Arp2/3 is one among several α-Catenin activities. Alternatively, α-Catenin might not functionally interact with the Arp2/3 complex in follicle cells (Sarpal, 2012).
To reveal a potential function for a cytosolic pool of α-Catenin, a DEcad::αCat fusion protein was expressed in α-Cat mutants. Previously, it was shown that a mammalian E-cadherin:: αE-catenin fusion protein could function in cell adhesion, and that in Drosophila the DEcad::αCat protein can fully substitute for the loss of DEcad or Arm in all cadherin-dependent processes during oogenesis, and for the loss of DEcad (but not Arm) during dorsal closure of the embryo. However, the Drosophila rescue experiments were carried out in the presence of endogenous α-Catenin, leaving open the possibility that α-Catenin has a cytosolic function or interacts with the DEcad::αCat protein through the α-Catenin dimerization domain, which could create a dimer that interacts with actin. It was found that expression of DEcad::αCat rescued head morphogenesis and embryonic lethality of α-Cat mutants, α-Cat mutant cell clones in imaginal discs, the FE, and border cell migration. These effects were similar to those for expression of exogenous α-Catenin. It was suggested that the expression of DEcad::αCat increases the surface abundance of cadherin, which could restore cell adhesion. However, overexpression of DEcad in α-Cat mutants did not show any rescue activity, arguing against this notion. The possibility was also considered that DEcad::αCat undergoes proteolytic cleavage to release α-Catenin. However, no cleavage product was detected on immunoblots. Moreover, whereas expression of a truncated form of α-Catenin that lacks the N-terminal β-catenin-binding and dimerization domain (deletion of amino acids 1-233) does not result in any rescue of α-Cat mutant defects, expression of the same truncated form of α-Catenin fused to DEcad rescues α-Cat mutants similarly to DEcad::αCat (Sarpal, 2012).
Collectively, these data indicate that a cytosolic form of α-Catenin is not required for α-Catenin function in several Drosophila tissues that were have investigated, and that all essential aspects of α-Catenin function during morphogenesis are executed in the immediate vicinity of the plasma membrane. These data do not rule out the possibility that α-Catenin directly interferes with the Arp2/3-actin interaction, but confines the potential for this interaction to the immediate submembranous space (Sarpal, 2012).
The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin-catenin complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the function of α-catenin, which binds to cadherin through β-catenin and can bind F-actin directly or indirectly. This study delineates regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic morphogenetic processes. Moreover, whether persistent α-catenin-mediated physical linkage between cadherin and F-actin is crucial for cell adhesion is addressed, and the functions of α-catenin monomers and dimers at adherens junctions is characterized. The data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin-β-catenin complex and the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin (Desai, 2013).
α-catenin is conserved across the eukaryotic kingdom, where it functions broadly in intercellular adhesion during development and differentiation. In Drosophila melanogaster, cell adhesion is disrupted when α-catenin contains a mutation in the binding site for Armadillo, which is the D. melanogastor homologue of β-catenin. Adherens junctions are also present in the nematode Caenorhabditis elegans, which expresses the homologues HMR-1 (cadherin), HMP-1 (α-catenin) and HMP-2 (β-catenin). In mice, there are three α-catenins and one close relative, which all share substantial amino-acid sequence identity: αE-catenin is most prevalent in epithelial tissues; αN-catenin is restricted to neural tissues; αT-catenin is expressed primarily in heart tissue; and α-catulin, which is an α-catenin-like protein, is ubiquitously expressed. A more distant relative is vinculin, which is ubiquitously expressed and localizes to both focal adhesions and adherens junctions (Desai, 2013 and references therein).
Adherens junctions and their core constituents, the classic cadherin adhesion molecules, contribute significantly to animal development and tissue homeostasis. Adherens junction defects can lead to various human pathologies, including cancer. Adherens junction function relies on the association of cadherins with the microtubule and actin cytoskeleton through their cytoplasmic binding partners, the catenins. Elucidating the function of α-catenin, which operates at the interface of the cadherin- β-catenin complex and F-actin, is a major goal in the field (Desai, 2013).
Studies on mammalian αE-catenin have given rise to two models for α-catenin function: the physical linkage and the allosteric regulation model. αE-catenin can bind both β-catenin and F-actin suggesting that it can physically link the cadherin- β-catenin complex directly to F-actin. This simple model lacks direct experimental support because a quaternary complex between cadherin, β-catenin, αE-catenin and F-actin could not be documented. Complex formation with F-actin could be demonstrated in vitro only in the presence of EPLIN, one of several F-actin-associated proteins that bind to αE-catenin, such as vinculin, α-actinin, afadin, ZO-1 and formin. Thus, a more complex physical linkage model poses that αE-catenin links the cadherin/β-catenin complex to F-actin indirectly by interacting with actin-binding proteins. A role for αE-catenin as a physical linker between cadherin and actin is consistent with the discovery that αE-catenin acts as a tension sensor that is responsive to actomyosin contraction at adherens junctions (Desai, 2013 and references therein).
Alternatively, α-catenin was proposed to regulate actin organization to support adherens junction formation, rather than act as a physical linker. αE-catenin binds β-catenin as a monomer but shows high affinity for F-actin only as a homodimer. The β-catenin binding site and homodimerization domain of αE-catenin overlap, suggesting that it cannot interact with β-catenin and F-actin simultaneously. These findings precipitated the view that αE-catenin may act allosterically by binding β-catenin to increase its own local concentration at adherens junctions, which is required to promote αE-catenin dimerization after dissociation from β-catenin. This model does not adequately address how adherens junctions are physically linked to actin and resist tensile forces. One question that results from these contradictory models is whether α-catenin dimerization is critical for adherens junction function (Desai, 2013).
This study reports an in vivo structure-function analysis of Drosophila α-Catenin (α-Cat) to assess the roles of its domains in several developmental processes and to distinguish between the physical linkage and allosteric regulation models for α-catenin function (Desai, 2013).
The model that α-catenin acts as a physical linker between cadherin and the actin cytoskeleton seems strongly supported by the ability of cadherin- α-catenin chimaeras to substitute for the cadherin-catenin complex, both in tissue culture assays and during morphogenesis (see Sarpal, 2012). The analysis of these fusion proteins emphasizes that: α-catenin acts in the immediate sub-membranous space, excluding an essential cytosolic function suggested by other studies in the tissues this laboratory has examined (Sarpal, 2012). Further, a dynamic interaction between α-catenin and the cadherin-β-catenin complex is not required to support normal adherens junctions. Although recent examples suggest that β-catenin modulation can contribute to the dynamic regulation of the cadherin-catenin complex in certain situations, this is not a basic requirement of adherens junction assembly and function. That cadherin- α-catenin chimaeras can replace the endogenous complex suggests instead that much of the regulation of cadherin-catenin complex function takes place at the interface between α-catenin and the actin cytoskeleton. Physical linkage of α-catenin to cadherin does not interfere with its function. This does not imply, however, that α-catenin normally needs to be physically linked to the cadherin-β-catenin complex to function. Indeed, the estimated dissociation constant (Kd) of the α-catenin β-catenin interaction (~1 µM) is much weaker than the cadherin-β-catenin interaction. The allosteric regulation model for α-catenin suggests that an α-catenin homodimer modulates actin organization through interference with the Arp2/3 complex at adherens junctions. Homodimerization and β-catenin binding require the same binding interface and are mutually exclusive. Cadherins can dimerize or cluster and could therefore promote α-catenin dimerization even in chimaeric proteins that lack the α-catenin dimerization domain (for example, DEcad::αCatΔVH1). Several membrane-bound regulators of actin organization exist, including WASp, indicating that α-catenin could retain its Arp2/3 regulating activity despite its covalent linkage to cadherin. To gain further evidence into the molecular mechanism of α-catenin function this study investigated whether α-catenin function at adherens junctions can be decoupled from β-catenin binding, whether monomeric α-catenin can support adherens junctions and whether α-catenin dimerization promotes or inhibits adherens junction function (Desai, 2013).
To address the first point αCatΔVH1, lacking the first vinculin-homology region (VH1), was fused to either BazOD (BazOD::αCatΔVH1) or Baz (Baz::αCatΔVH1), which recruited αCatΔVH1 to adherens junctions. αCatΔVH1 does not support adherens junctions alone, but did so when fused to DEcad. BazOD::αCatΔVH1 and Baz::αCatΔVH1 showed weak biochemical interactions with Arm and DEcad and little rescue of adherens junctions. These findings suggest that the physical link between α-catenin and β-catenin not only recruits α-catenin to adherens junctions, but needs to persist for normal adherens junction function. Both the physical linkage and allosteric regulation models propose that α-catenin interacts with the actin cytoskeleton at adherens junctions; however, they differ on whether binding of α-catenin to β-catenin is required to physically link cadherin and the actin cytoskeleton. The data argue for persistent physical linkage as a core requirement for α-catenin function. It was also found that localization of αCatΔVH1 to adherens junctions through fusion to Ed did not support adherens junction integrity, in contrast to DEcad::αCatΔVH1, suggesting that cadherins have distinct properties that are important for α-catenin function (Desai, 2013).
Similar to C. elegans and D. discoideum α-catenin proteins, αN-catenin was found to be monomeric in solution. αN-catenin can functionally replace the Drosophila protein, which formed a large dimer fraction in solution similar to αE-catenin. These results indicate that monomeric α-catenin can support adherens junction function, and that the in vitro monomer/dimer ratio may not correlate with the in vivo function of α-catenins (Desai, 2013).
To address the relationship between α-catenin dimerization and adherens junction function, the effects were tested of enhanced α-catenin dimerization. α-Cat or αCatΔVH1 fusion to BazOD or Baz probably causes enhanced multimerization, including dimerization of α-Cat. Although these chimaeras localize to adherens junctions they show reduced interactions with Arm and perform poorly. Dimerization was also enhanced by removing the N-terminal 56 or 64 amino acids from αN-catenin and α-Cat, respectively. Similar to αEcatΔ57, αCatΔ64 interacted with β-catenin/Arm. However, these constructs performed poorly when compared with their respective full-length proteins, suggesting that α-catenin dimers are inactive in adhesion and may represent a cytoplasmic pool that forms a dynamic equilibrium with α-catenin monomers. Monomers are recruited to adherens junctions through their interaction with β-catenin/ Arm, which probably stabilizes monomeric α-catenin that links cadherin to the actin cytoskeleton (Desai, 2013).
αE-catenin operates as a tension sensor and mechanotransducer at adherens junctions, changing conformation in response to pulling forces exerted by actomyosin. To achieve this, α-catenin needs to be suspended between two anchor points, which could be cadherin- β-catenin and F-actin, consistent with the physical linkage model. However, αE-catenin homodimers contain two actin-binding domains and can bundle actin filaments, raising the possibility that actin filament sliding as a result of myosin activity could apply tension to α-catenin. As the allosteric regulation model poses that α-catenin homodimers form preferentially at adherens junctions, tension sensing could be restricted to adherens junctions even without α-catenin linking cadherin to actin. However, actomyosin-generated tension also applies to E-cadherin and depends on the presence of αE-catenin, suggesting that at least part of the tension exerted on αE-catenin occurs when it physically links cadherin to the actin cytoskeleton. These data are complementary to the analysis of Drosophila α-Cat. Although it has not been possible to document a quartenary complex of the cadherin-catenin complex with F-actin, the data presented in this study are consistent with α-catenin physically linking cadherin to the actin cytoskeleton as a core requirement of α-catenin and cadherin-catenin complex function (Desai, 2013).
The results on the function of different regions within Drosophila α-Cat are in line with data from tissue culture studies on αE-catenin. The Arm and actin-binding regions at the N and C termini of α-Cat, respectively, are essential for function. The central region of α-Cat enhances adherens junction stability but does not contribute to α-Cat recruitment to adherens junctions, which relies only on the VH1-dependent binding to Arm. The central region between the VH1 and VH3 domains includes multiple parts that make partly independent contributions to α-Cat function, most likely through interactions with other binding partners. If the central region is activated by actomyosin pulling forces, then the tension-mediated conformational change in α-Cat would be expected to facilitate multiple interactions (Desai, 2013).
Although α-catenins can bind directly to F-actin in vitro, whether this occurs in vivo remains unresolved. It is possible that interactions with F-actin are indirect and mediated through F-actin-binding proteins. α-catenin organizes a complex interface between cadherin and the actin cytoskeleton. Uncovering how the multiple interactions between α-catenin and actin-binding proteins such as vinculin, formin, afadin or EPLIN contribute to adherens junction regulation during morphogenesis remains a major challenge for future investigation (Desai, 2013).
E-cadherin-mediated cell-cell adhesion is fundamental for epithelial tissue morphogenesis, physiology and repair. E-cadherin is a core transmembrane constituent of the zonula adherens (ZA), a belt-like adherens junction located at the apicolateral border in epithelial cells. The anchorage of ZA components to cortical actin filaments strengthens cell-cell cohesion and allows for junction contractility, which shapes epithelial tissues during development. This study reports that the cytoskeletal adaptor protein Girdin physically and functionally interacts with components of the cadherin-catenin complex during Drosophila embryogenesis. Fly Girdin is broadly expressed throughout embryonic development and enriched at the ZA in epithelial tissues. Girdin associates with the cytoskeleton and co-precipitates with the cadherin-catenin complex protein alpha-Catenin (alpha-Cat). Girdin mutations strongly enhance adhesion defects associated with reduced DE-cadherin (DE-Cad) expression. Moreover, the fraction of DE-Cad molecules associated with the cytoskeleton decreases in the absence of Girdin, thereby identifying Girdin as a positive regulator of adherens junction function. Girdin mutant embryos display isolated epithelial cell cysts and rupture of the ventral midline, consistent with defects in cell-cell cohesion. In addition, loss of Girdin impairs the collective migration of epithelial cells, resulting in dorsal closure defects. It is proposed that Girdin stabilizes epithelial cell adhesion and promotes morphogenesis by regulating the linkage of the cadherin-catenin complex to the cytoskeleton (Houssin, 2015).
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