chickadee
The ability of neuronal growth cones to be guided by extracellular cues requires intimate communication between signal
transduction systems and the dynamic actin-based cytoskeleton at the leading edge. Profilin (Chickadee in Drosophila), a small, actin-binding protein,
has been proposed to be a regulator of the cell motility machinery at leading edge membranes. However, any requirement it may have in
the developing nervous system has been unknown. Profilin associates with members of the Enabled family of proteins,
suggesting that Profilin might link Abl function to the cytoskeleton.
In a genetic screen in Drosophila to identify genes required for the correct navigation and
outgrowth of motoneuron growth cones two alleles of a
stranded (sand) mutation were recovered in which motor growth cones arrest before reaching their final targets. The molecular genetic analysis reveals that stranded alleles are zygotic lethal mutations in chickadee. In vitro experiments
confirm that axon extension is impaired in Profilin mutants. Moreover, phenotypic comparisons and genetic interactions
between chic and abl mutants support the notion that Profilin and Abl cooperate to promote axon
extension. Genetic analysis in Drosophila has been used to
demonstrate that mutations in Profilin (chickadee) and Abl (abl) display an identical growth cone arrest phenotype for axons
of intersegmental nerve b (ISNb). Moreover, the phenotype of a double mutant suggests that these components function
together to control axonal outgrowth (Wills, 1999).
Although axon outgrowth defects are observed in all motor pathways in chic alleles, supporting a general role for Profilin
in all aspects of axon outgrowth, these defects arise only late in development, when motor growth cones must navigate
certain key choice points. Since Profilin is expressed abundantly during oogenesis, it is
possible that a maternal supply of Profilin protein can function in the absence of zygotic Profilin for the initial stages of
embryogenesis, thus masking a more general role for Profilin. To test whether maternally supplied Profilin function might
modulate the zygotic phenotype of chicsand mutants, the phenotypes of embryos were compared from mothers with one or
two copies of the wild-type Profilin gene. Comparing embryos with the same zygotic genotype (chicsand/Df), it was found
that those derived from mothers carrying a duplication at the Profilin locus show substantial rescue of the
stranded axonal phenotype. Thus, doubling the supply of maternally expressed Profilin rescues the chic mutant zygote through
embryonic stage 17. The failure of motor growth cones at particular locations in the periphery of chicsand
embryos is likely to reflect regions with the highest requirement for Profilin function (Wills, 1999).
The choice point regions where chic mutant growth cones frequently arrest correspond to locations where growth cones
typically slow down, become more complex in shape, and probe their environment. Although it is thought that the failure of these mutant growth cones
reflects an intrinsic deficit in forward locomotion, it is also possible that chic mutant growth cones are unable to respond to
specific extrinsic guidance cues. Therefore, the context dependence of chic mutant axon outgrowth was assessed by
measuring outgrowth from dissected nerve cords in vitro. In these experiments, mutant and wild-type nerve cords were
removed from embryos at embryonic stage 16 and cultured on poly-L-lysine-coated coverslips. Under these in vitro
conditions, regenerating axons begin extending from transected motor nerve roots and central nervous system (CNS)
longitudinal connectives after 3 hr and continue to extend for the next 8-9 hr, long after growth cones would have arrested in
mutant embryos. In this assay system, homozygous chicsand nerve cords produce Fasciclin II- (Fas
II-) positive neurite fascicles, but the mutant axons do not extend as far as wild-type controls. Neurite fascicles extend 50
µm on average from wild-type nerve cords, while chic mutant fascicles extended 32 µm on
average, only 64% of wild-type growth. These results support the conclusion
that Profilin plays a role in general axonal extension, regardless of the environment, and suggest that the axonal defects in
chic embryos result from loss of Profilin function in the mutant axons and not in the surrounding tissue (i.e., a cell
autonomous function for Profilin in motoneurons) (Wills, 1999).
The discovery that Profilin interacts biochemically with members of the Enabled family suggests that Profilin might provide a link between the Abl tyrosine kinase pathway and the actin
cytoskeleton. Although axonal phenotypes have been observed in ena mutants, previous studies employing general axon markers have identified defects only when abl mutations were
combined with mutations in other axon guidance genes. However, using the mAb 1D4 antibody to examine
motor pathways during late embryonic development (stage 17), a previously unappreciated growth cone arrest
phenotype was observed in the ISNb projection of abl homozygous mutant embryos that is essentially identical to the ISNb phenotype of
chic mutants. In abl mutants, ISNb axons frequently stop at contact with muscle 13 and/or the adjacent muscle
30, failing to reach the distal target muscle 12. Less frequently, abl mutant ISNb axons stop earlier
at contacts with muscles 14 or 28; such defects are rare in wild-type controls but less penetrant in abl mutants than in strong
chic backgrounds. Other peripheral motor axon pathways appear normal in abl mutants, as
assessed with mAb 1D4. Abl was shown to require an active kinase domain to function normally in ISNb
development (Wills, 1999).
In addition to the requirement for Profilin and Abl in ISNb development, both components are also necessary for the
accurate formation of axon pathways within the CNS. Staining of chic or abl mutant embryos with mAb 1D4 reveals
similar disorganization in the parallel longitudinal fascicles of Fas II-positive axons on either side of the CNS midline.
Although the prevalent phenotype observed in both single mutant genotypes is mild, a range of defects can be seen, from mild, to intermediate, to extreme. The prevalent defects are not likely to be a product of alterations in CNS cell fates, since patterning in
strong chicsand mutants has been assessed with several different antibody probes. In
mildly effected embryos, longitudinal pathways are often diverted, causing fusions and/or breaks in these fascicles; occasionally,
inappropriate midline crossing can be seen. In embryos with intermediate effects, Fas II-positive axons often cross the midline barrier, in
addition to the collapse of longitudinal fascicles. In embryos with extreme effects, axonal connections between segments along the
anterio-posterior axis are often absent, consistent with a major failure in axonal extension; this extreme phenotype is very
rare in chic or abl single mutants. In such extreme cases, defects are also observed in muscle patterning; such
defects have been found in abl mutants (Wills, 1999 and references).
The similarity between the chic and abl phenotypes, both in the CNS and periphery, raised the question of whether these
genes cooperate in axonal development. To determine if the function of Profilin is sensitive to the amount of Abl, as
expected for components in the same pathway, chic homozygous embryos that lack one allele of abl were tested. Two-fold reduction of Abl function in the chic background results in a dramatic shift
in the distribution of CNS axon phenotypes; in these embryos, the extreme phenotype increases 10-fold
in comparison to chic mutants alone. This dose-sensitive genetic interaction suggests that Profilin and Abl
cooperate in the same overall process (Wills, 1999).
A dynamic actin cytoskeleton is essential for the remodeling of cell shape during development, but the specific roles of many actin partners remain unclear. A novel actin binding protein, Ciboulot (Cib), corresponding to CG4944, plays a major role in axonal growth during Drosophila brain metamorphosis. Loss of Cib function
leads to axonal growth defects in the central brain, while overexpression of the gene during development leads to overgrown projections. The Cib protein displays
strong sequence similarity to beta-thymosins but has biochemical properties like profilin: the Cib-actin complex participates in actin filament assembly
exclusively at the barbed end, and Cib enhances actin-based motility in vitro. Genetic experiments show that Cib and the Drosophila profilin protein Chickadee
(Chic) cooperate in central brain metamorphosis (Boquet, 2000).
In the adult brain protocerebrum, two central structures have been the focus of particular attention, the mushroom bodies (MB) and the central complex (CX). In the
adult, the MB send their axons into the peduncle to terminate in one of three sets of lobes. MB are already present in a miniature state in the late embryo and are
remodeled through larval and pupal development in a sequential manner. The
CX, in contrast, is entirely built up during metamorphosis from immature neurons of the brain commissure. The mechanisms involved in the remodeling of the MB and CX during metamorphosis remain largely unknown (Boquet, 2000).
The CX is located at the adult interhemispheric junction and consists of a neuropilic structure composed of ~20,000 neurons divided into four substructures,
organized rostrally to caudally: the ellipsoid body (EB), the fan-shaped body (FB) with its pair of nodulli, and the protocerebral bridge. In
cib mutants, the ellipsoid body is ventrally opened or even more severely disorganized. The central brain cib defect was further characterized using EB-specific enhancer trap lines as structural markers. The gal1625 line displays an expression pattern
that is restricted to some ring neurons in the adult EB. At the onset of normal pupal formation, the EB precursor appears as a flat neuropilic structure,
consisting of dorsal fibers of the future EB. During metamorphosis, the putative EB neuropil elongates downward to form the characteristic ring-like structure. In a cib context, the EB shows axonal growth arrest with variable expressivity (Boquet, 2000).
The sequence of Cib exhibits three similar domains: D1, D2, and D3. D1 and D2 display 47% sequence identity; D1 and D3, 50%, and D2 and D3,
67%. D3 contains a 10 amino acid sequence, KLKHTETNEK, which represents an actin binding motif very similar to that found in beta-thymosins, a family of small (5 kDa) actin binding proteins present in vertebrates and sea urchins. A motif similar to
KLKHTETNEK is also found in other actin binding proteins, including actobindin, a 9 kDa actin binding protein found in Acanthamoeba castellanii, as well as verprolin in Saccharomyces cerevisiae,
its homolog WIP (WASP-interacting protein), and proteins of the WASP family in mammalian cells. A C. elegans Cib homolog was
identified that exhibits four similar domains. The D2 and D3 Cib domains display an overall sequence identity with rat thymosin beta4 (Tbeta4) of 54% and 65%, respectively. All beta-thymosins bind G-actin specifically in a 1:1 molar ratio with an affinity in the 106 M-1 range and behave
as passive G-actin sequestering proteins that create a pool of unassembled monomeric actin. The
strong sequence identity between Cib and thymosin beta4 suggests that it may interact with actin (Boquet, 2000).
The Cib protein was detected in wild-type embryos but not in cib mutant embryos laid by mutant females, indicating that
the embryonic product corresponds exclusively to maternal protein accumulated during oogenesis. The temporal expression pattern of Cib thus confirms the conclusions of the genetic analysis and supports the idea that cib is required for correct central nervous
system development during metamorphosis. In situ hybridization reveals the presence of CIB mRNA in the brain of third instar larvae and
of 60-hr-old pupae. Strong expression is detected in the pars intercerebrallis and in four clusters of cell bodies in each hemisphere, which most likely
belong to the four clusters of MB Kenyon cells (Boquet, 2000).
The functional homology between Cib and profilin was tested in vivo by examining the genetic interaction between cib and chic. In chic221/+ flies, which presumably have only half of the profilin found in normal flies, there are no structural brain abnormalities. Similarly, heterozygote cibP/+ females display no abnormal brain phenotype in paraffin section. However, cibP/+; chic221/+ doubly heterozygote females display a cib-like phenotype, with a partially split fan-shaped body. Thus, the interaction between cib and chic is not additive but synergistic. Reducing by half the Chic activity in cib mutants exacerbates the phenotype normally associated with cib: cibP/Y; chic221/+ males display a fan-shaped body split in the middle, an ellipsoid body strongly disturbed, similar to that observed in trio mutants, and disorganized MB medial lobes that rarely reached their normal position. These results show that chic is involved in adult brain formation and, moreover, support the view that Cib acts synergistically with Chic in vivo. Furthermore, cibP/Y; chic221/+ males display other phenotypes that can be related to impaired actin-dependent developmental processes, including sterility and a thoracic bristle defect with shorter and forked ends, possibly resulting from the defective formation of the peripheral nervous system. Chic rather than Cib is the principal source of profilin function during gametogenesis since chic but not cib mutants are sterile. However, the reduced profilin in chic221/+ flies is partially compensated by Cib+, allowing fertility (in other words, chic221 is recessive in an otherwise wild-type background but semidominant in a cib background) (Boquet, 2000).
chic and the tyrosine kinase encoding gene Abl interact genetically. cibP/Y;Abl04674/+ individuals display a stronger phenotype than their cibP/Y;TM3/+ siblings. Thus, cib and chic interact similarly with Abl, which supports the assumption that they both are active in the same pathway (Boquet, 2000).
As a decreased axonal growth is observed in the absence of Cib, it was of interest to see whether overexpressing the Cib, an actin binding protein, would lead to increased axonal growth. An inducible hs-cib+ transgene was made that allowed overexpression of Cib during development. The effect was assessed at the level of the MB, since they consist of parallel neuron bundles, the ends of which can be easily traced. MB beta lobes overgrow and cross the midline when Cib is overexpressed. Identical results were obtained when Cib expression was driven specifically in the MB using a UAS-cib+ construction and MB-expressed PGal4 lines (Boquet, 2000).
This work demonstrates the importance of the regulation of actin assembly in neuronal outgrowth during brain formation, linking molecular events to changes at the
cellular and the organ level. It is anticipated that Cib will cooperate in vivo not only with Chic but also with other putative Drosophila components of the motile
machinery, such as the G-actin sequestering protein Act-up and the signaling pathway toward actin cytoskeleton (Boquet, 2000).
In the course of a genetic mosaic screen for new mutations affecting early eye development, alleles of a conserved gene were isolated and the gene was called capulet or act up (acu) for its effect on actin. acu encodes a homolog of yeast cyclase-associated protein (CAP) that sequesters
monomeric actin; acu is required to prevent actin filament polymerization in the eye disc. acu is required for cells to change shape in the
morphogenetic furrow, and it also prevents premature neuronal differentiation in the eye disc, probably by restricting the movement of Hedgehog (Benlali, 2000).
The actin cytoskeleton has been implicated in mediating cell shape changes in many systems. Filamentous (F) actin is assembled
from monomeric (G) actin subunits, with kinetics that favor addition of subunits to the barbed ends of the filaments and dissociation of subunits from the pointed ends.
This process is regulated by a variety of proteins. Profilin (Drosophila's Chickadeebinds to actin monomers and was originally thought to sequester them, preventing polymerization. However, profilin has now been shown to acts as a nucleotide exchange factor for actin and promotes the elongation of actin filaments at their barbed ends. ADF/cofilin stimulates pointed end depolymerization, increasing the G actin pool and promoting filament turnover.
Capping and severing proteins also limit the length of filaments. GTPases of the Rho family appear to regulate actin filament dynamics in
response to extracellular signals (Benlali, 2000 and references therein).
In yeast, overexpression of profilin can rescue the defects caused by mutation of the C-terminal domain of CAP, suggesting that profilin may act to sequester actin monomers in yeast. However, in other systems, profilin appears to stimulate actin polymerization. The phenotype of chic clones were examined in the eye disc. Unlike acu clones, chic clones show greatly reduce phalloidin staining. Profilin and CAP thus appear to have opposite effects on actin polymerization in the eye disc. Clones of cells doubly mutant for acu and chic predominantly show the chic phenotype of lack of phalloidin staining, although some cells near the edges of the clone showed high levels of staining. Thus, profilin is required for normal actin polymerization even in the absence of monomer sequestration by CAP (Benlali, 2000).
Since premature photoreceptor differentiation was observed both in acu mutant clones, in which actin filament levels are increased, and in chic clones, in which actin filament levels are decreased, an effect on cell shape might be common to both mutations. Using an antibody to the membrane-associated protein Armadillo (Arm), it was observed that acu mutant cells in the region of the morphogenetic furrow do not undergo the normal shape changes, and instead retain large apical profiles. The same phenotype was observed in chic mutant clones. This suggests both that coordinated alterations in actin filaments are required for apical constriction of cells in the morphogenetic furrow, and that this cell shape change is important in controlling the pattern and timing of differentiation (Benlali, 2000).
Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance
receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively
active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and
ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout
enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho
suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by
heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase
(ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho
or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin
activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).
Thus, when
expressed alone, only ctDrac and ctDcdc42 cause midline
crossing errors. However, the mutant GTPases interact
genetically with mutations in robo, Sos, and chic and with
overexpression of ctMLCK. The interactions are surprisingly
specific. Midline crossing errors caused by expression
of ctDrac or ctDcdc42 are suppressed by heterozygous loss
of Profilin and enhanced by expression of ctMLCK. These
results indicate that Drac1 and Dcdc42 encourage axons to
cross the midline by regulating actin polymerization and/or
myosin activity. CtRho and dnRho interact strongly with
expression of ctMLCK or heterozygous loss of Robo, which
suggests that regulation of myosin activity by Rho is crucial
for midline repulsion. This work demonstrates that Rho,
Drac1, and Dcdc42 are involved in dictating which axon
may cross the midline, presumably by aiding in the transduction
of attractive and/or repulsive cues operating at the
midline. By using mutations in signaling molecules known
to prevent pCC/MP2 axons from crossing the midline, this
analysis concentrates on how Rho, Drac1, and Dcdc42 may
regulate cytoskeletal dynamics in response to midline repulsive
cues (Fritz, 2002).
Rho family GTPases activate a number of effectors that
may affect axon outgrowth by regulating adhesion, myosin
force generation, and/or actin polymerization. The ctDrac- and
ctDcdc42-induced midline crossing errors are suppressed
by heterozygous loss of Profilin, an actin-binding
protein, which stimulates actin polymerization. Since reducing
actin polymerization partially rescues the ctDrac
and ctDcdc42 phenotypes as well as errors caused by
heterozygous loss of Robo, it is likely that the midline
crossing errors are caused by excessive actin polymerization.
Increased actin polymerization may produce more
filopodia to explore the midline, which leads to midline
crossing. There are several pathways through which Drac1
and Dcdc42 might affect actin polymerization. The Cdc42/
Rac effector p21-activated kinase (PAK) activates LIM kinase
to phosphorylate cofilin, an actin-depolymerizing factor
required for neurite outgrowth. Cdc42 also activates actin polymerization through WASP,
which stimulates polymerization by binding to the Arp2/3
complex. The activation of WASP by Cdc42 is enhanced by
Profilin, which may explain why the suppression of the
ctDcdc42 phenotype is stronger than that of the ctDrac-induced
errors. However, actin polymerization
may not be the only process regulated by Rho family
GTPases to increase outgrowth (Fritz, 2002).
The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Genetics and cell biology have been combined to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. Profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, Capulet (Capt), the Drosophila homologue of Adenylyl Cyclase Associated Proteins, functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. It is therefore proposed that Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila (Baum, 2001).
In vitro, Capt has been shown to inhibit actin polymerization by sequestering actin monomers, mirroring its role in vivo, where it limits actin-filament formation. Profilin, encoded by the chicadee (chic) gene, is another well-characterized monomeric actin-binding protein that can serve to promote or to inhibit actin polymerization. To determine which of these biochemical activities dominate in follicle cells, the phenotype of chic-null mutant clones was examined. In chic mutant columnar follicle cells, levels of F-actin seem markedly decreased at all cortices, although actin filaments are lost preferentially from the basal cortex. Thus, profilin promotes actin polymerization in follicle cells as it does in the Drosophila germline and in imaginal discs. To test whether profilin is also required for the formation of apical actin aggregates in capt mutant cells, actin filaments were examined in chic capt double-mutant cell. In clones of the double mutant, ectopic actin filaments are not formed. Thus, profilin is required for actin-filament formation at the cortex of wild-type follicle cells and for the synthesis of apical F-actin aggregates in the capt mutant. Interestingly, chic capt double-mutant clones exhibit an additional morphological phenotype not seen in clones of either single mutant. Double-mutant cells lose their columnar morphology and collapse, forming a thin squamous-like layer of cells. The loss of Capt therefore perturbs cell architecture in the chic mutant, even though a corresponding change in the level of cortical F-actin is not observed. Moreover, the chic capt double-mutant phenotype shows that an ordered actin cytoskeleton is likely to be crucial for the proper morphogenesis of the columnar epithelium (Baum, 2001).
As a test of Ena function, follicular ena clones were generated. Cells homozygous for hypomorphic ena alleles (ena210, ena23) lose cortical actin filaments from apical, basal and lateral sites. However, whereas chic clones preferentially lose basal actin filaments, ena mutant cells also exhibit a marked decrease in the amounts of apical F-actin. This might reflect the fact that Ena is concentrated at apical junctions in the wild type, whereas profilin has a broader cellular distribution. It is concluded that Ena facilitates actin-filament formation in Drosophila, much as it does in mammalian cells (Baum, 2001).
Having found genetic evidence to suggest that Ena and Abl cooperate with Capt in the control of epithelial F-actin, the distribution was examined of Ena and Abl proteins in capt mutant follicle cells. In the capt mutant, Ena's distribution is altered so that the majority of the protein becomes localized with apical actin filaments. Thus Ena is found tightly associated with apical F-actin both in the wild type and in capt mutant cells. In contrast, significant amounts of Ena are not observed at the apical surface of capt chic double-mutant cells, in which apical F-actin aggregates are not formed. Therefore, both in the wild type and in various mutants, the amount of Ena present at adherens junctions closely parallels the level of apical F-actin. The localization of Abl was also examined in the wild type and in capt mutant tissue. Abl, like Capt, seems to have a diffuse staining pattern within wild-type follicle cells. However, in capt clones, Abl protein becomes concentrated at the apical cell surface, partly localizing with Ena. Thus, a loss of Capt leads to a change in localization of both Ena and Abl. Because these proteins act together with Capt to control the spatial organization of the follicular actin cytoskeleton, their altered distribution is likely to contribute to the generation of the marked capt mutant phenotype (Baum, 2001).
This study has used Drosophila genetics and the follicular epithelium to characterize how various actin-binding proteins act to regulate the spatial organization of F-actin. The results show that actin dynamics are regulated by distinct mechanisms within different domains of a polarized epithelial cell. Capt, Ena and Abl seem to modulate apical actin-filament formation, whereas cofilin and profilin seem to have a more global function, regulating cortical actin-filament dynamics throughout the cell. Moreover, the accumulation of F-actin at apical, basal and lateral sites in tsr mutant follicle cells and the loss of cortical actin filaments in chic mutant cells indicates that cortical actin filaments are turned over continuously throughout the cell. This being so, it is striking that F-actin becomes so highly polarized in the capt mutant (Baum, 2001).
A model is envisaged in an effort to explain the aetiology of the pronounced capt mutant phenotype. Normally, in the columnar epithelium, Ena protein is concentrated at adherens junctions, where it promotes local F-actin synthesis. This activity of Ena is counterbalanced by Capt, which limits the amount of apical F-actin. Excess F-actin therefore forms at apical junctions in capt mutant cells. This newly formed apical F-actin is able to recruit additional molecules of Ena from the cytoplasm, because Ena binds microfilaments, which leads to further actin-filament formation. This explains why, in the absence of apical F-actin aggregates, Ena does not become concentrated at the apical cortex of cells in the capt chic mutant. Thus, the loss of Capt initiates an explosive cycle of local actin polymerization and Ena recruitment at adherens junctions, culminating in the striking polar actin aggregates observed in capt mutant cells. It is speculated that within wild-type epithelial cells, controlled autocatalytic cycles of actin-filament formation of this type might help to limit the accumulation of actin filaments to a single site within a cell. For instance, during the formation of a Drosophila wing hair, a similar process might be required to generate a single bundle of actin filaments at the apical cortex of the epithelium (Baum, 2001).
The correct localization of myosin II to the equatorial cortex is crucial for
proper cell division. A collection of genes was examined that causes defects
in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin
II localization. Three genes in the rho1 signaling pathway, pebble (a Rho
guanidine nucleotide exchange factor), rho1, and rho kinase, are
required for the initial recruitment of myosin II to the equatorial cortex. This
initial localization mechanism does not require F-actin or the two components of
the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and
racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin
(diaphanous), and profilin (chickadee) are required to stably
maintain myosin II at the furrow. In the absence of these latter genes, myosin
II delocalizes from the equatorial cortex and undergoes highly dynamic
appearances and disappearances around the entire cell cortex, sometimes
associated with abnormal contractions or blebbing. These findings support a model
in which a rho kinase-dependent event, possibly myosin II regulatory light chain
phosphorylation, is required for the initial recruitment to the furrow, whereas
the assembly of parallel, unbranched actin filaments, generated by
formin-mediated actin nucleation, is required for maintaining myosin II
exclusively at the equatorial cortex (Dean, 2005).
This study has discovered three steps in the myosin II
localization/activation process that involve distinct groups of genes:
(1) an initial recruitment of myosin II to the equatorial cortex that is
independent of F-actin and centralspindlin but requires rho1 signaling;
(2) a secondary stabilization of myosin II at the midzone that requires
F-actin and a second set of genes that are likely involved in building a
specific type of actin network, and (3) the activation of furrowing
once myosin II is localized that depends on centralspindlin (Dean, 2005).
Rho1, its
activating guanidine nucleotide exchange factor pebble, and rho kinase are each
required for the initial recruitment of myosin II to the equatorial cortex. Rho1
has been implicated in two pathways that are important for cytokinesis.
In the first pathway, rho1 signals to F-actin
through the formin diaphanous. However, proteins on this F-actin
pathway, including F-actin itself, are not essential for the initial myosin II
recruitment to the equatorial cortex. However, rho kinase, another
downstream target of rho1, is essential. Because rho kinase phosphorylates the
myosin II RLC, it is possible that
phosphorylation of the RLC is essential for myosin II recruitment to the furrow.
This hypothesis could not be directly tested, because the myosin II heavy
chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).
Phosphorylation of the RLC both activates the motor domain and, in some myosins,
increases bipolar thick filament formation. Because
F-actin is not required for myosin II recruitment, activation of the motor is
unlikely to be the mechanism by which phosphorylation of the RLC would cause
recruitment of myosin II to the equatorial cortex. It is quite possible,
however, that the rho kinase-mediated myosin II phosphorylation leads to thick
filament assembly and that this assembly is important for localization of myosin
to the equatorial cortex. Indeed, in Dictyostelium, it is clear that
bipolar thick filament formation is sufficient for myosin II localization to the
midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains
unknown (Dean, 2005).
In contrast to the lack
of F-actin involvement in the early recruitment of myosin II to the equatorial
cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain
myosin II in the equatorial region. Interestingly, the downstream rho1 effectors
diaphanous/formin and chickadee/profilin are also necessary for myosin II
maintenance at the equatorial midzone. Although the loss of these genes could
deplete F-actin, phalloidin staining has shown that F-actin is still present in all
of the RNAi-treated cells. In addition, these RNAi-treated
cells still contract, unlike when F-actin is completely disrupted with LatA.
Thus, myosin II appears to be interacting with F-actin in the cortex as it
disperses in dynamic patches throughout the cortex of these diaphanous- or
chickadee-depleted cells (Dean, 2005).
It is suggested that the role of diaphanous/formin and chickadee/profilin in
maintaining the myosin II contractile ring is through the creation of specific
F-actin structures. In particular, formin- and profilin-mediated nucleation
results in unbranched actin filaments because profilin
promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has
shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled
filaments. These parallel
filaments contrast with Arp2/3-mediated nucleation, which creates a highly
branched actin filament network. Indeed, Arp2/3, although essential for
lamellipodia formation, is not required for cytokinesis in Drosophila
cells. The hypothesis here
is that once myosin II is recruited to the equatorial cortex of
the cell by a rho kinase-dependent mechanism, possibly localized activation of
RLC phosphorylation, it is retained there because of its higher affinity for
parallel, unbranched actin filaments than to branched actin networks. Consistent
with this hypothesis, myosin II is depleted from the lamellipodia in migrating
cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are
more likely to be aligned in parallel bundles. Thus,
it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow
maintains a higher concentration of parallel actin filaments in this region
compared with the rest of the cortex, and these parallel filaments serve to
selectively retain myosin II at the equator to form a stable contractile ring.
In the absence of these parallel actin filaments, myosin II can bind branched
F-actin throughout the cortex, perhaps occasionally organizing them into
parallel bundles that cause increased myosin recruitment corresponding to the
flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).
Live-cell imaging shows that when pavarotti or racGAP50c
are depleted, the cells do not display significant contractions despite
recruiting myosin II to the equatorial cortex. Although there is some modest
membrane contractile activity in these cells, it is clear that significant
contraction or furrowing requires both components of the centralspindlin
complex. It is surprising that only these proteins were found to be necessary
for cortical contraction at sites of myosin II localization. Data from fixed
cells, as well as earlier studies, indicated that Drosophila cells do not
undergo equatorial contractions during mitosis when Diaphanous or Chickadee is
depleted. However, live-cell imaging shows that
when either of these two genes is depleted in S2 cells, not only is myosin II
transiently localized to the equatorial cortex before dispersing, but cells do
indeed display transient equatorial contraction. It is difficult to recognize
these events in fixed cells because of their transient nature and the somewhat
irregular shapes of cells depleted of these proteins. This work highlights the
importance of live-cell imaging in the study of dynamic processes such as
cytokinesis (Dean, 2005).
In addition to the suppression of furrowing, depletion of centralspindlin also
leads to an inability to retain F-actin exclusively at the equatorial cortex
during cytokinesis. This similar phenotype of the centralspindlin complex and
the F-actin affecting proteins suggests that centralspindlin may be an upstream
regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti
have been shown to accumulate at the spindle poles and are associated with an
abnormal accumulation of F-actin near the centrosomes.
Centralspindlin may be acting indirectly by helping to localize an important
actin-affecting protein at the central spindle, or it may act more directly on
the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been
hypothesized that centralspindlin affects the F-actin cortex through rho1
signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does
not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion,
and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of
centralspindlin may itself be important for signaling to the F-actin cortex.
Finally, centralspindlin cannot be the major actomyosin ring positioning signal
because myosin II is properly recruited in its absence (Dean, 2005).
The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. This study report a careful analysis of the oocyte cytoskeleton. Thick actin bundles are identified at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, the data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. Actin bundle formation requires Profilin but not Capu and Spire. Thus, the results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, the data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. The release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent (Wang, 2008).
This study reports the existence of actin bundles at the cortex of the oocyte that are involved in the cortical localisation of γTubulin. γTubulin is part of the γTubulin ring complex that is stabilising the minus ends of MTs. The presence of γTubulin alone does not allow to distinguish whether the protein is part of a microtubule organising centre (MTOC) that nucleates MTs or whether it only protects existing MTs from depolymerisation. This study used γTubulin solely as a maker for the MT minus ends; these are embedded within the cortical actin bundles before stage 10b (Wang, 2008).
The cytoskeletal rearrangements at stage 10b include the disassembly of the cortical actin bundles, the redistribution of the MT minus ends from the cortex to subcortical regions and the formation of MT arrays parallel to the oocyte cortex. Concomitantly with these cytoskeletal changes, the transition from slow to fast cytoplasmic streaming is triggered. What is the causal relationship between these events? The finding that interfering with actin bundle formation by drug treatment and GFPactin5c overexpression results in MT minus ends redistribution, MT array formation and premature fast streaming indicates that actin bundling acts upstream of MT reorganisation and streaming. The analysis of Khc mutants allows to further dissect the subsequent steps reorganising the MT cytoskeleton. In the absence of streaming, caused by the loss of Khc function, the redistribution of MT minus ends occurs normally, while the formation of MT arrays is abolished. Thus, minus end redistribution is upstream of streaming, and array formation is downstream. It is therefore proposed that streaming is initiated by the disassembly of the cortical actin bundles resulting in loss of cortical MT minus end anchoring. It is further proposed that the redistribution of the minus ends to subcortical regions is important for the reorganisation of the MT cytoskeleton into arrays that run parallel to the oocyte cortex. At this step a previously suggested self amplifying loop could be initiated, in which MT array formation and Kinesin movement enhance each other. In this loop the Kinesin driven streaming helps to sweep MTs into parallel arrays, which in turn allow more robust currents in the cytoplasm (Wang, 2008).
How do the actin binding proteins Capu, Spire and Profilin act on the oocyte cytoskeleton to prevent premature cytoplasmic streaming? chic/Profilin mutants and latrunculin A treatment both interfere with bundle formation. Latrunculin A treatment inhibits actin polymerisation by binding to and sequestering actin monomers. Profilin is involved in actin polymerisation by delivering actin monomers to the growing ends of actin filaments. Thus, latrunculin A and Profilin mutants appear to interfere with bundling by limiting the pool of monomers that can be added to growing actin filaments. In contrast, capu and spire mutants are not required for the formation of actin bundles. It is proposed that Capu and Spire anchor the MT minus ends in a scaffold provided by the cortical actin bundles. The lack of Capu and Spire activity in the mutants prevents cortical MT anchoring and allows streaming in the presence of actin bundles. This model is supported by the work that has shown that Capu and Spire proteins are able to crosslink F-actin and MTs, and that both proteins localise to the oocyte cortex (Wang, 2008).
The regulation of fast ooplasmic streaming could be controlled at the level of the cortical localisation of Capu and Spire. The displacement of the two proteins from the cortex at stage 10b might result in loss of MT minus end anchoring, and thereby induce fast streaming. To test this the localisation of GFP-Capu and GFP-Spire was analysed in cross sections of oocytes. However, no difference was detected in the localisation of the two proteins before and after onset of fast streaming. In addition, no displacement of GFP-Capu and GFP-Spire was detected after induction of premature streaming by latrunculin A treatment. Thus, Capu and Spire activities are not regulated at the level of their localisation (Wang, 2008).
A different mode of Capu and Spire regulation is suggested by their genetic and biochemical interaction with Rho1. This interaction has led to a model in which Rho1 initiates fast streaming by regulating the crosslinking activities of Capu and Spire. This study shows that the prevention of streaming requires not only capu and spire but also the presence of actin bundles. The formation of these bundles occurs, however, independently of capu and spire. This suggests that the onset of fast streaming is not only controlled by regulating Capu and Spire activities, but also by disassembly of the actin bundles (Wang, 2008).
Genes that are involved in actin regulation in the oocyte were also tested but these do not induce premature streaming. For capulet, swallow and moesin mutants the formation of ectopic actin clumps has been reported reflecting defects in the organisation of the oocyte actin cytoskeleton. The presence of ectopic F-actin in the oocyte cytoplasm was confirmed in these mutants, but nevertheless the formation cortical actin bundles was detected. Thus, actin defects in the oocyte do not necessarily affect cortical actin bundling (Wang, 2008).
Generation and maintenance of proper lumen size is important for tubular organ function. This study reports on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 results in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restores proper distribution of F-actin and phosphorylated Moesin and rescues the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin (Chickadee) phenocopies the Rho1 lumen size defects to a large extent. Ribbon, a BTB domain-containing transcription factor, functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. These studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin (Xu, 2011).
Rho1 acts both in salivary gland cells and in the surrounding mesoderm to maintain apical polarity during gland invagination and to mediate cell shape change during gland migration. This study demonstrates a novel role for Rho1 in controlling salivary gland lumen size through regulation of actin polymerization and distribution and regulation of Moesin activity. By analyzing Rho1 alleles for which salivary gland cells invaginated and formed a gland, it was shown that zygotic loss of function of Rho1 results in shortening and widening of the gland lumen, which is accompanied by defects in cell shape change and cell rearrangement and failure of apical domains to elongate along the Pr-Di axis of the gland. These effects of Rho1 are mediated through Rok; inhibition of Rok completely phenocopies loss of Rho1 in these cellular events. Based on these studies, a model is proposed for Rho1 control of salivary gland lumen size, in particular lumen width, which is determined by cell rearrangement and apical domain elongation. Rho1 and Rok, through inhibition of cofilin, regulate cell rearrangement and apical domain elongation by promoting actin polymerization to localize F-actin at the basolateral membrane and by limiting the apical accumulation of F-actin. In parallel to its role in actin polymerization and distribution, Rho1 acts independently of Rok to limit apical p-Moe with Rib by an unknown mechanism and this function of Rho1 is specific for apical domain elongation. The data on cofilin (Twinstar) are consistent with those in cultured HeLa cells that showed that mammalian ROCK can inhibit cofilin activity indirectly through LIMK-mediated phosphorylation of cofilin (Xu, 2011).
Although manipulating Moe activity through gland-specific expression of MoeT559D was sufficient to completely phenocopy the Rho1 lumen defects, including cell rearrangement, it did so without disrupting actin polymerization or distribution. This is likely to be due to activated Moe strengthening the link between the actin cytoskeleton and the apical plasma membrane (without affecting levels of apical F-actin), which would increase apical membrane stiffness and remove the ability of gland cells to rearrange. Indeed, Moesin has been shown to control cortical rigidity during mitosis of cultured Drosophila S2R+ cells. Thus, Rho1 regulates cell rearrangement and apical domain elongation by controlling the actin cytoskeleton and Moesin activity through distinct mechanisms (Xu, 2011).
The observation that chic mutant glands phenocopy Rho1 mutant glands to a large extent, suggests that Rho1 control of salivary gland lumen size is mainly dependent on a requirement for Rho1 in actin polymerization. However, as the chic and Rho1 gland lumen phenotypes are not identical, with chic mutant glands lacking the apical accumulation of F-actin and p-Moe observed in Rho1 mutant glands, Rho1 probably has an additional function in limiting accumulation of F-actin and p-Moe at the apical membrane. This function of Rho1, at least for limiting apical F-actin, might partly involve Rab5- or Shi-mediated endosome trafficking, because inhibition of Rab5 alone or Shi alone led to accumulation of F-actin at the apical membrane. Although Rab5DN- or ShiDN-expressing salivary gland cells were enriched with apical F-actin, lumen size was not affected. This could be due to Rab5DN and ShiDN affecting a pool of apical F-actin distinct from that affected by Rho1 and/or because Rab5DN-expressing gland cells retain basolateral F-actin and the ratio of apical to basolateral F-actin is not altered sufficiently to cause lumen size defects. In Rho11B mutant gland cells, some early endosomes were not coated with F-actin. Actin is known to contribute to multiple steps of the endocytic pathway, including movement of endocytic vesicles through the cytoplasm and their transport to late endosomes and lysosomes. One possible mechanism by which Rho1 normally limits apical accumulation of F-actin is by promoting its removal from the apical membrane and accumulation on endocytic vesicles (Xu, 2011).
Currently, it is not know how Rho1 limits accumulation of apical p-Moe. Membrane localization and activity of Moesin can be regulated via a number of mechanisms, such as its phosphorylation on a conserved Threonine residue, binding to phosphatidylinositol-(4,5)bisphosphate [PtdIns(4,5)P2] and association with components of the sub-membrane cytoskeleton, such as Crb. Studies in cultured mammalian cells have demonstrated that Rho signaling activates Moe either through phosphorylation of Moe by ROCK or through ROCK-mediated inhibition of myosin phosphatase, which is known to dephosphorylate p-Moe. Although it is possible that Drosophila Rho1 positively regulates Moe activity by one or more of these mechanisms, this study shows that in the developing salivary glands Rho1 in fact negatively regulates Moe activity. In rib mutant embryos, in which p-Moe is enriched apically, salivary gland and tracheal cells showed decreased staining for Rab11 GTPase, which localizes to the apical recycling endosomes and to secretory vesicles destined for the apical membrane. Thus, Rho1, like Rib might limit apical p-Moe through its membrane transport (Xu, 2011).
In Drosophila imaginal disc epithelia, Moe negatively regulates Rho1 activity to maintain epithelial integrity and to promote cell survival. These studies demonstrating that in the developing salivary gland Rho1 antagonizes Moe activity by limiting its localization at the apical membrane, shed novel insight into the functional relationship between Rho1 and Moe. It is possible that in a dynamic epithelium, such as the developing salivary gland, Rho1 contributes to the precise spatial and temporal regulation of Moe activity to fine-tune selective changes in apical domain shape. By contrast, in the imaginal disc epithelium, Rho1 regulation of Moe might not be necessary and, instead, Moe regulation of Rho1 activity is required to maintain epithelial integrity and cell survival. Thus, Rho and Moe can antagonize each other's activities depending on the type of epithelia or cellular event (Xu, 2011).
Rescue studies with Rho1WT demonstrate that Rho1 functions predominantly in the salivary gland cells to control apical domain elongation and cell rearrangement. Interestingly, expression of Rho1WT in the mesoderm with twi-GAL4 has no effect on cell rearrangement and has little effect on apical domain elongation and lumen size, whereas it has been shown that Rho1WT expression in the mesoderm significantly rescues the gland migration defect of Rho11B mutant embryos. This suggests that gland migration and lumen size control are regulated by distinct mechanisms. In support of this conclusion, embryos mutant for multiple edematous wings, encoding the ?PS1 integrin subunit, which have defects in gland migration, show no defects in gland lumen width. Identifying the distinct and overlapping mechanisms by which salivary gland lumen width and length are controlled will help to elucidate the mechanisms by which lumen size is controlled in tubular organs (Xu, 2011).
F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. This study has focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, embryonic Drosophila primary neurons were as one consistent cellular model to study filopodia regulation. The data for loss-of-function of capping proteins, Enabled, different Arp2/3 complex components, the formin DAAM, and profilin, reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of the Drosophila model system. Using combinatorial genetics, this study demonstrated that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM, filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. These genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps (Goncalves-Pimentel, 2011).
chickadee Effects of mutation: back to part 1/2
A test was performed to see whether the defects induced by the lack of Cib could be rescued by increasing the supply of Chic profilin. A chic+ duplication was introduced into a cib mutant. Two duplications were available, Dp(2;2)C619 (26A;28E), which rescues embryonic chic axonal phenotype, and Dp(2;2)Cam2 (23D01-02;26C01-02). These two duplications overlap over 26A;26C01-02, a small region that spans the chic+ locus (26A09–26B01). The phenotype of cibE10/Y; Dp(2;2),chic+/+ was compared to that of cibE10/Y; CyO/+ siblings, the latter genotype being used as a control of cib phenotype in the appropriate genetic background. Interestingly, cibE10/Y; Dp(2;2),chic+/+ individuals display a normal brain, whereas cibE10/Y; CyO/+ adults display a strong cib phenotype. The full rescue of cib defects by increasing the ubiquitous Chic supply confirms the profilin-like function of Cib in vivo (Boquet, 2000).
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