atypical protein kinase C
To see where aPKC is expressed during embryonic development, the mRNA distribution was analyzed by RNA in situ hybridization. aPKC mRNA is already detectable in freshly laid eggs before the onset of zygotic transcription and thus must be deposited maternally during oogenesis. At the cellular blastoderm stage, aPKC mRNA is present in all cells except for the pole cells. During gastrulation, strong expression of aPKC is detectable in tissues that undergo morphogenetic movements; e.g., the invaginating mesoderm, the proctodeum, and the cephalic furrow. In embryos at the extended germ band stage, prominent aPKC expression is detectable in neuroblasts. In several epithelial tissues, in particular in the fore- and hind-gut and in the Malpighian tubules, aPKC mRNA is highly enriched in the apical cytocortex, reminiscent of the polarized localization of baz and crumbs (crb) mRNAs (Wodarz, 2000).
The distribution of aPKC protein was analyzed using anti-PKCzeta antibody C20. During cellularization of the embryo, aPKC becomes localized to the apical cytocortex of all cells except for the pole cells, which do not contain detectable amounts of aPKC. Already at this stage aPKC is enriched at apico-lateral cell borders, giving rise to a honeycomb pattern in en face views of the blastoderm. After completion of cellularization, DaPKC is highly concentrated in the apico-lateral cortex and shows little overlap with the basolateral marker Nrt. Apical localization is maintained throughout embryonic development in most epithelia that are derived from the ectoderm (e. g. epidermis, fore-, and hind-gut), Malpighian tubules, and the tracheal system. The only ectodermal epithelium devoid of DaPKC expression is the amnioserosa (Wodarz, 2000).
Strong expression of DaPKC is also detected in neuroblasts, the stem cells of the embryonic CNS. During delamination of neuroblasts, aPKC is localized in the apical stalk that is wedged between adjacent cells of the neuroectodermal epithelium. In pro- and meta-phase, aPKC forms apical cortical crescents. In anaphase, aPKC staining is strongly diminished and expands over a broader region of the neuroblast cortex, but is clearly excluded from the budding ganglion mother cell. Thus, from delamination through pro- and meta-phase, localization of DaPKC in neuroblasts is very similar to that of Baz and Insc. Simultaneously with aPKC, cell outlines of epithelial cells and neuroblasts were visualized with the Nrt antibody. Similar to epithelial cells, Nrt expression is clearly polarized in neuroblasts. Strong staining for Nrt is detectable in the basal and lateral membrane of neuroblasts, whereas staining is strongly reduced in apical regions where DaPKC is expressed (Wodarz, 2000).
Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib). Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. A computer model of apicobasal polarity was build that suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarization. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt-aPKC-Par6-cdc42, aPKC phosphorylation of Crb, and recruitment of Expanded (Ex) and Kibra (Kib) to prevent endocytic removal of Crb from the plasma membrane. Lgl antagonizes the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. The results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells (Fletcher, 2012).
These above results define an apical positive feedback loop that centers on endocytic regulation of Crb. If such a positive feedback loop exists, it must be antagonized by the basolateral determinants to prevent spreading of apical determinants into the basolateral domain. In the computer model, ectopic spreading of apical determinants caused by simulated inhibition of endocytosis (strongly reducing the rate at which apical determinants are removed from the plasma membrane) can be counteracted simply raising the number of basolateral determinants by 5-fold. In follicle cells, inhibiting endocytosis with RNAi against the AP2/clathrin component AP50 leads to ectopic spreading of apical determinants into the basolateral domain, as in the model. Overexpression of Lgl-GFP was sufficient to restore normal polarity even in the presence of AP50 RNAi, again similar to the simulations. Furthermore, expression of Lgl-GFP also rescued the spreading of apical determinants caused by Rab5 RNAi or overexpression of Crb. These results suggest that Lgl may be a rate-limiting basolateral determinant and that it acts to inhibit positive feedback among apical determinants and thereby promote endocytic removal of Crb from the basolateral membrane (Fletcher, 2012).
Once Crb has been endocytosed by the AP2/clathrin machinery, it could be either degraded in the lysosome or recycled. Recent evidence indicates that Crb avoids the lysosome due to the action of the retromer machinery. The recycling endosome protein Rab11 is essential for Crb to remain at the plasma membrane in embryos. By costaining for Crb and Rab11 in follicle cells, it was possible to detect many endosomes that are positive for both proteins. Furthermore, when Rab11 is knocked down by RNAi in follicle cells, a loss of Crb from the plasma membrane was detected and an accumulation in enlarged endosomes. In contrast, RNAi of Rab5 causes accumulation of Crb at the plasma membrane. Accordingly, the Rab11 RNAi phenotype -- unlike that of Rab5 -- cannot be suppressed by coexpression of Lgl-GFP. These results confirm that Crb undergoes Rab11-mediated recycling to maintain its polarized plasma membrane localization (Fletcher, 2012).
One difference between the computer model and in vivo data is that inactivation of apical determinants in the model leads to complete loss of apical determinants from the membrane. However, in follicle cells, mutation of crb does not cause complete loss of apical aPKC from the plasma membrane. This residual aPKC is due to the Bazooka protein (Baz/Par3), which-like Crb-is able to bind to aPKC-Par6 and normally localizes to adherens junctions but can also occupy the apical membrane in the absence of Crb. Whether the Baz system operates by the same positive feedback principle as the Crb system remains to be explored (Fletcher, 2012).
These findings indicate that polarization of Crb in the follicle cell epithelium depends on the combination of two principles: positive feedback and mutual antagonism. The apical domain forms where Crb can recruit additional Crb molecules via Crb-Crb interactions, recruitment of Sdt and aPKC-Par6-cdc42, aPKC phosphorylation of the Crb FERM-binding domain, and recruitment of the FERM-domain protein Ex and its binding partner Kib. Although direct binding between these factors in follicle cells was not shown, work in other model systems indicates that they do bind directly. Disruption of any element of this feedback loop results in endocytosis of Crb from the plasma membrane. In contrast, ectopic activation of various components of this feedback loop-by overexpression of Crb, cdc42V12, or aPKCdeltaN-stabilizes Crb and the other apical determinants at the plasma membrane. The basolateral domain forms where Crb is endocytosed from the plasma membrane because Lgl (which can bind to aPKC-Par6 and inhibit aPKC kinase activity), presumably prevents Crb from engaging in a productive interaction with the other apical determinants, thereby disrupting Crb self-recruitment (Fletcher, 2012).
In conclusion, the model explains how epithelial polarity is a property of a complex system that can emerge spontaneously from the nature of the interactions between apical and basolateral polarity determinants. The principle of combined positive feedback and mutual antagonism outlined in this study in Drosophila follicle cells may prove to be widely used in the generation of polarity in many different cellular contexts (Fletcher, 2012).
Cell polarity is critical for epithelial structure and function. Adherens
junctions (AJs) often direct this polarity, but it has been found that Bazooka
(Baz) acts upstream of AJs as epithelial polarity is first established in
Drosophila. This prompted an investigation into how Baz is positioned and how downstream polarity is elaborated. Surprisingly, it was found that Baz localizes to an apical domain below (basally to) its typical binding partners atypical protein kinase C (aPKC) and
partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In
fact, Baz positioning is independent of aPKC and PAR-6 relying instead on
cytoskeletal cues, including an apical scaffold and dynein-mediated
basal-to-apical transport. AJ assembly is closely coupled to Baz positioning,
whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical
domain with Baz and AJs localizing basally to aPKC and PAR-6, and
specific mechanisms were identified that keep these proteins apart. These results reveal key
steps in the assembly of the apical domain in Drosophila (Harris, 2005).
These results frame a model of apical domain assembly during epithelial
polarity establishment in Drosophila. During
cellularization, Baz acts as a primary polarity landmark that positions AJs and
aPKC. Baz, itself, is positioned by two cues (an apical scaffold and
dynein-mediated transport). Baz recruits and colocalizes with AJ proteins in a
subapical region while helping direct aPKC to the extreme apical region.
During gastrulation, a third cue becomes important for
Baz and AJ positioning. At this stage, aPKC becomes required for maintaining Baz
and AJs. PAR-6 is also recruited to the extreme apical region and maintains Baz
and AJs. Although Baz can interact
with aPKC and PAR-6 at this stage, Crb blocks these interactions. It is proposed
that this interaction network establishes a
robust, stratified apical domain from the earliest stages of epithelial
development (Harris, 2005).
AJs are often key polarity landmarks.
However, Baz positioning is AJ independent at the time that epithelial polarity is first
established in Drosophila.
Here, Baz appears to act as a primary polarity landmark, but what cues position
Baz?
The data indicate that Baz is initially positioned by cytoskeletal cues that
support an apical Baz-binding scaffold and mediate basal-to-apical Baz
transport. The apical scaffold is saturable. Its function requires actin; Baz
becomes basally mislocalized after actin disruption. However, since Baz
overlaps only the basal reaches of the apical actin network, it is unlikely that Baz
simply binds actin. Interestingly, Baz remains largely membrane associated when
actin is disrupted. One caveat is that there is some residual actin. However,
the same treatment dissociates APC2 from the cortex.
Actin is also required for PAR-3
cortical association in C. elegans one-cell embryos. During Drosophila
cellularization, it is speculated that Baz may have other cortical anchors and that actin
may control their distribution -- of course, actin is critical for many cellular
processes and could play other roles in positioning Baz. It will be important to
identify the apical scaffold for Baz (Harris, 2005).
Baz positioning also requires the minus-end-directed MT motor dynein.
Live imaging of BazGFP revealed basal-to-apical translocation of BazGFP puncta
during cellularization. Baz-GFP that diffuses to ectopic basal positions appears
to engage a preexisting, dynein-based, basal-to-apical transport system. Such a
system transports Golgi vesicles apically during
cellularization. Baz-dynein
associations appear to cease once dynein brings Baz to the apical region, where
Baz presumably docks with its apical scaffold. Although BazGFP puncta move
slower than in vitro dynein velocity measurements,
dynein-mediated lipid droplet movements have
similar speeds during Drosophila cellularization.
In vivo, BazGFP puncta may be slowed because they form large
cortical complexes. Indeed, DE-Cad, aPKC, and PAR-6 associate with these puncta
and Baz oligomerization may promote complex assembly.
Further supporting a role for dynein, endogenous Baz is
positioned near MT minus ends in WT embryos, but mislocalizes basally in
dhc64Cm/z mutants. dhc64C mutations also enhance the
baz mutant embryonic phenotype. This is the first report
of dynein positioning Baz or its homologues (Harris, 2005).
Analysis of dynein mutants also revealed a third mechanism that can
reposition Baz apically during gastrulation. Perhaps the apical Baz-binding
scaffold is strengthened during this stage. Alternatively, a distinct polarizing
mechanism may be activated, or aPKC and PAR-6 may be
involved. Having three Baz positioning mechanisms may ensure proper Baz
localization for regulating downstream polarity (Harris, 2005).
Baz acts upstream of AJs as epithelial polarity is first established in
Drosophila. The following model is proposed
in which AJ assembly may be coupled to Baz positioning. During
cellularization, AJ proteins accumulate in both apical and basal junctions.
Basal junctions form transiently near the base of each invaginating furrow. Baz
is not required for basal junctions, but is required for recruiting AJ proteins
into apical junctions. Apical Baz
may provide a landmark for apical AJ assembly (Harris, 2005).
The data also suggest that Baz may be involved in ferrying DE-Cad to the apical
domain via dynein-mediated transport. Dynein is required for correct apical
positioning of both Baz and DE-Cad, and their colocalization in ectopic basal
complexes in dhc64Cm/z mutants suggests they may normally be
transported to the apical domain together. Indeed, Baz can form complexes with
DE-Cad and Arm. Although most endogenous Baz is apical during WT cellularization, its
basal mislocalization in dhc64Cm/z mutants suggests that some
Baz may normally move basally. In fact, excess BazGFP displaced from the apical
domain preferentially accumulates at basal junctions. It is hypothesized that some
Baz may normally interact transiently with basal junctions. From there, it may
help ferry AJ proteins apically via dynein-mediated transport. MT motors have
been implicated in AJ assembly. For example, dynein interacts with
ß-catenin and may tether MTs to AJs assembling between PtK2 cells.
Kinesin transports AJ proteins to nascent
AJs in cell culture, and the mitotic kinesin-like protein 1 is
required for apical targeting of AJs and other cues in C. elegans
epithelia. It will be important
to see if these targeting mechanisms have commonalities with AJ positioning in
Drosophila, and if Baz homologues are involved (Harris, 2005).
Finally, it is hypothesized that the third Baz-AJ positioning mechanism
revealed in dhc64Cm/z mutants might be related to the normal
maturation/stabilization of AJs at gastrulation. At this stage, precursory spot
AJs fuse into continuous belt junctions around the top of each cell.
In mammalian cell culture, aPKC is
required for such AJ maturation.
Similarly, aPKC is required for proper AJ and Baz positioning
during Drosophila gastrulation, as has been shown for PAR-6.
Considering aPKC and PAR-6 are
positioned apically as dhc64Cm/z mutants gastrulate, they
might recruit Baz and AJs apically in this context as well (Harris, 2005).
Based on their shared roles in polarity in C. elegans, characterized
physical interactions, and colocalization in mammalian cells, Baz, aPKC, and
PAR-6 are thought to function, at least in some cases, as an obligate tripartite
complex. The data suggest that the bulk of cortical Baz and aPKC/PAR-6 do not form
obligate complexes during epithelial development in Drosophila. Instead,
aPKC and PAR-6 localize to an apical region above Baz and AJs, and are
positioned there by distinct mechanisms. Baz/PAR-3 also segregates from aPKC and
PAR-6 in other cell types. In C. elegans one-cell embryos, PAR-3, aPKC,
and PAR-6 each localize in clusters on the anterior cortex, but these different
clusters have limited colocalization (60%-85% fail to colocalize.
aPKC and PAR-6 colocalize without PAR-3 at the leading edge of
migrating mammalian astrocytes.
In Drosophila photoreceptors, Baz colocalizes with AJs below
aPKC, PAR-6, and Crb. Even in
polarized MDCK cells, aPKC and PAR-6 show some segregation above PAR-3, and
although they mainly colocalize at tight junctions,
mammalian PAR-3 can regulate tight junction assembly
independently of aPKC and PAR-6.
Thus, in many contexts interactions between Baz/PAR-3, aPKC, and PAR-6 are
dynamic and/or regulated (Harris, 2005).
Baz (PAR-3), aPKC, and PAR-6 often recruit each other to the cortex, but the
assembly pathways vary. In C. elegans, one-cell embryos, PAR-3, aPKC, and
PAR-6 are mutually dependent for their cortical recruitment.
However, in Drosophila neuroblasts, Baz
can be positioned without aPKC and PAR-6.
Similarly, apical Baz is positioned without aPKC and PAR-6 during
Drosophila cellularization. In contrast, apical aPKC recruitment requires
Baz, whereas PAR-6 is largely nonpolarized at this stage. Given the lack of
extensive colocalization of Baz and aPKC in WT embryos, Baz may control aPKC
positioning indirectly, perhaps regulating binding to a separate apical
scaffold. Alternately, cortical recruitment might involve cytoplasmic
Baz-aPKC complexes. Apical PAR-6 accumulates at gastrulation, and this
appears partially Baz independent. Indeed, cdc42 recruits PAR-6 at this stage,
and at the same time aPKC and PAR-6 become required for maintaining apical Baz.
Thus, although Baz is first positioned
independently of aPKC and PAR-6, these cues soon develop complex
interdependencies (Harris, 2005).
Although Baz can directly bind both aPKC and PAR-6, at least two
mechanisms keep them apart. During cellularization, Baz colocalizes with aPKC
and PAR-6 when overexpressed, but normally it localizes with AJs below aPKC and
PAR-6. This normal segregation may thus involve competition with other binding
partners. After cellularization, Crb also becomes important for segregating Baz
and AJs from aPKC and PAR-6. These segregation mechanisms help form a stratified
apical domain from the earliest stages of epithelial development (Harris, 2005).
A stratified apical domain may strengthen the boundary between the apical and
basolateral domains. This boundary forms via reciprocal antagonism between
polarity cues. For example, aPKC phosphorylates and excludes Lethal giant larvae
(Lgl) from the apical domain in Drosophila epithelia and Lgl appears to
repel PAR-6 from the basolateral domain.
The Crb and Dlg complexes also have mutual antagonism. It is proposed
that the subapical Baz-AJ region may insulate the
apical and basolateral domains. For example, it may inhibit active aPKC from
moving basally. Indeed, PAR-3 binding can block mammalian aPKC kinase activity.
The Baz-AJ subapical region could
also block basolateral cues, since AJs are required to segregate Dlg. In this way, the Baz-AJ
subapical region could help define a distinct apical-basolateral boundary (Harris, 2005).
To conclude, Baz appears to be a primary epithelial polarity landmark in
Drosophila. It is positioned by multiple mechanisms, including an apical
scaffold and dynein-mediated transport, and organizes a stratified apical
domain, in which it colocalizes with AJs below its typical partners aPKC and
PAR-6 (Harris, 2005).
Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, 'budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g., cortical mark) or an extrinsic cue (e.g., cell-cell signal). Using live imaging and in vitro culture, and using the distributions of Baz and aPKC as markers, it was found that neuroblasts in contact with epithelial cells always 'bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remain anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. It is concluded that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity (Siegrist, 2006).
This study shows that embryonic neuroblasts require an extrinsic signal from
the overlying epithelium to anchor their centrosome(s) at the apical side of
the cell, induce Par cortical polarity at prophase, and position Par cortical
crescents at the apical cortex. How does the extrinsic cue stabilize centrosome position throughout multiple rounds of cell division? It is likely to stabilize centrosome-cortex interactions, perhaps by regulating association of microtubule plus-ends with the apical neuroblast cortex. During mitosis, the apical cortex is enriched with several proteins with the potential to interact with microtubules
directly and indirectly, such as Pins, Gαi, Dlg and Insc, but it
remains unknown whether one or more of these are involved in transducing the
extrinsic cue that promotes centrosome anchoring. During interphase, none of
these proteins shows apical enrichment, although several have uniform cortical
localization (e.g., Dlg, Galphai) and could help stabilize the neuroblast
centrosome following the completion of telophase (Siegrist, 2006).
The epithelial extrinsic signal is also required for the timing and
position of Par cortical polarity in embryonic neuroblasts. In the presence of
the extrinsic cue, Par polarity, as evidenced by the distribution of Baz and aPKC, is established around the G2/prophase
transition; without the extrinsic cue, Par polarization is delayed until
prometaphase/metaphase. Because adjacent neuroblasts divide asynchronously, it
is likely that the epithelial cue is always present, but the neuroblast only
becomes competent to form the Par crescent at the G2/prophase transition. The
best candidates would be mitotic kinases or phosphatases that change levels at
the G2/prophase transition (Siegrist, 2006).
The position of the Par cortical crescent is also determined by the
epithelial cue. In isolated neuroblasts, the Par cortical crescent forms at
random positions during subsequent cell cycles, correlating with randomization
of the cell division axis. It is not known how Par protein crescents are
formed in wild-type embryonic neuroblasts exposed to the epithelial cue or in
isolated neuroblasts that lack extrinsic signals. In wild-type neuroblasts,
the initial events in Par protein polarization are likely to involve
polarization of Baz or Insc, the two most upstream components in the Par
cortical polarity pathway. In isolated neuroblasts, Par crescents form over one pole
of a randomly oriented mitotic spindle, raising the possibility that astral
microtubules may induce Par crescents, similar to their ability to trigger
Pins/Galphai/Dlg crescents. Although Par crescents can still form in the absence
of both microtubules and extrinsic cues (such as in Colcemid-treated isolated
neuroblasts), astral microtubules may be necessary to direct
the position of Par crescents in isolated neuroblasts (Siegrist, 2006).
In the future, it will be important to determine the relationship between
centrosome position and position of cortical polarized Par proteins. Both
require an extrinsic signal from the overlying epithelium, but they could be
independently regulated by two different signals, independently regulated by
the same signal, or they could act in a linear pathway. For example, a single
extrinsic cue could anchor the G2 centrosome pair, and then the centrosome
pair could induce apical cortical polarity at the G2/prophase transition,
similar to centrosome-induced cortical polarity in the C. elegans
zygote (Siegrist, 2006).
One of the best candidate pathways for regulating orientation of the
neuroblast division axis by extrinsic cues is the non-canonical Wnt signaling
pathway, because it is known to orient cell divisions in Danio rerio, C.
elegans and Drosophila. This pathway uses the Frizzled (Fz) receptor and the cytoplasmic Disheveled (Dsh) and Gsk3 proteins from the Wnt pathway, but does not use a Wnt ligand. In addition,
these three components are joined by the two transmembrane proteins Strabismus
(Stm) and Flamingo (Fmi) during planar cell polarity signaling in
Drosophila. However, no evidence was found to support a role for
this pathway in orienting embryonic neuroblast divisions. RNAi of each of the
four Drosophila Fz receptors, individually and in combination, had
little effect on neuroblast spindle orientation or cortical polarity. Nor were spindle orientation defects observed following expression of
a dominant-negative Fz1 lacking the cytoplasmic domain, expression of the Wnt
pathway antagonist Axin, or in dsh maternal zygotic mutants,
fmi zygotic mutants, stm maternal zygotic mutants or fz1
fz2 double mutants. The non-canonical Wnt pathway may
still be involved in the ectodermal signal that regulates neuroblast
orientation, but its role may be masked by genetic redundancy (Siegrist, 2006).
A second candidate pathway for regulating epithelial-to-neuroblast
signaling is an extracellular matrix (ECM)-integrin pathway. ECM is
deposited by the basal surface of epithelia, which is where neuroblasts
contact the overlying embryonic epithelia. However, no major
integrin ligand, Laminin, is detected at the basal surface of the embryonic ectoderm
during stages 9-11, nor was the core ß-integrin protein detected in
neuroblasts. In addition, maternal zygotic mys mutants lacking
ß-integrin show normal embryonic neuroblast spindle orientation. It is unlikely that the ECM-integrin signaling regulates embryonic
neuroblast spindle orientation (Siegrist, 2006).
Interestingly, neuroblasts located in the procephalic neural ectoderm are
reported to undergo asymmetric cell divisions within the plane of the
epithelium and reproducibly orient along the apicobasal embryonic axis to bud
GMCs towards the interior of the embryo. Similarly, during adult PNS
development, the pIIb cell lies within the imaginal disc epithelium yet
divides along the apicobasal axis. In both cases, the reproducibly apicobasal
spatial pattern of cell divisions occurs independent of an overlaying
polarized epithelium. It remains unknown whether the oriented pattern of these
cell divisions is regulated by intrinsic cues or extrinsic cues (e.g., more
internal cells). Unlike ventral cord embryonic neuroblasts, neuroblasts in the
brain and in the PNS contain several cell-cell junctions, including
cadherin-containing adherence junctions and septate junctions. These signaling
rich sites could provide spatial information for spindle orientation as seen
in other cell types (Siegrist, 2006).
Although the nature of the cue required to orient embryonic neuroblasts is
not clear, there are several approaches to identify potential genes required
for this process. As extrinsic cues are required for early localization of Par
proteins and because baz and insc mutants have mis-oriented
spindles relative to the epithelium, identifying binding partners for either
Insc or Baz could be informative. In addition, a small
genetic deficiency has been identified that, when homozygous, results in embryonic neuroblast
spindle orientation defects relative to the overlying ectoderm without
affecting epithelial morphology; one or more genes within this genetic
interval would be excellent candidates for components of the extrinsic
signaling pathway (Siegrist, 2006).
Finally, does neuroblast cell behavior in culture accurately reflect
neuroblast behavior in vivo? It has previously been shown that in vivo
embryonic neuroblasts establish apicobasal spindle orientation through one of
two behaviors. Either the mitotic spindle first forms parallel to the
overlaying epithelium and then rotates 90° to align orthogonal to the
overlaying epithelium or the spindle forms as it rotates into its proper
orientation. Centrosome separation and rotation behavior were not
described. Both behaviors were also observed in cultured neuroblasts, however,
with several differences: (1) rotations of fully formed
spindles were observed at a very low frequency and this behavior usually correlated with an
unhealthy culture; (2) if both centrosomes moved basally or away from the
epithelial contact site after separation, an initial
spindle formation coinciding with rotation into a position orthogonal to
epithelial cells is frequently observed, similar to some of the reported in vivo cases. One
additional difference in the analysis between these two systems involves the
Drosophila stocks used for live imaging. Following
microtubule behavior from cells relied on expressing endogenous levels of a
microtubule-associated protein fused in frame to GFP, rather than upon
overexpression of a tau:GFP fusion protein. This difference alone could
account for the observed differences between the two studies (Siegrist, 2006).
An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. The Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. It is concluded that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation (Lee, 2006a).
Some brat mutant neuroblasts show expanded aPKC cortical crescents, in some cases reaching the basal cortex. This phenotype appears specific for aPKC, because other apical cortical proteins (e.g., Baz, Pins) are unaffected. Brat might repress aPKC translation, leading to increased aPKC protein levels in brat mutants. Alternatively, the absence of Prospero or other basal cortical proteins may indirectly affect aPKC localization (Lee, 2006a).
What is the cellular origin of the brat mutant phenotype? brat mutant GMCs are compromised in three ways: they lack Brat translational repression activity, lack Prospero, and some may have ectopic aPKC. Loss of Brat translational repression activity could well play a role in the ectopic neuroblast self-renewal phenotype, because all brat mutants disrupting the NHL translational repression domain exhibit a brain tumor phenotype, and Brat has been previously shown to negatively regulate cell growth. Loss of Prospero also plays a role in the brat phenotype: prospero mutant GMCs have a failure to downregulate neuroblast gene expression and a failure in neuronal differentiation, similar to brat mutants. prospero null mutant embryos also show a slight delay in neuronal differentiation, although they appear to undergo normal neuroblast self-renewal. Finally, ectopic aPKC can also mimic aspects of the brat phenotype, including formation of supernumerary large Dpn+ neuroblasts. Interestingly, the mammalian paralogs of Drosophila aPKC (aPKCλ/ζ) are expressed in neural progenitors of the ventricular zone, and the mammalian Prospero ortholog Prox1 is expressed in differentiating neurons of the subventricular zone. Thus, identifying Prospero transcriptional targets and aPKC phosphorylation targets may provide further insight into the molecular mechanism of neural stem cell self-renewal in both Drosophila and mammals (Lee, 2006a).
Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. Atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, a balance of microtubule and actin interactions was found to control AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost (Harris, 2007).
This study reveals the roles of aPKC during polarity establishment and elaboration in Drosophila embryos. In contrast to C. elegans, aPKC is not critical during initial polarity establishment; Baz and AJs are initially localized correctly and the embryonic epithelium can undergo initial morphogenesis. However, aPKC plays an early and striking role in maintaining the symmetrical organization of AJs, via effects on MT organization, and also plays an important later role in the elaboration of polarity (Harris, 2007).
aPKC's later role in polarity elaboration may reflect effects on multiple targets. aPKC is critical for the cortical localization of its normal binding partner PAR-6 and the apical determinant Crb. This latter effect is consistent with the fact that aPKC can phosphorylate Crb, and disruption of aPKC phosphorylation sites in Crb destabilizes Crb in the apical domain. Since Crb stabilizes AJs after gastrulation, this likely contributes to the eventual AJ breakdown in apkcm/z mutants. Crb may act in concert with PAR-6 or in parallel. aPKC can also phosphorylate and exclude the basolateral cue Lgl from the apical domain, and consequenct failure to exclude Dlg from the apical domain in apkcm/z mutants. Thus, apical invasion of basolateral cues may also contribute to the eventual loss of epithelial polarity in apkcm/z mutants (Harris, 2007).
However, it is unlikely that these global changes in apical-basal cell polarity are responsible for the early clustering of AJs into planar-polarized puncta in apkcm/z mutants. Indeed, most of these other polarity players affect polarity after gastrulation. crb mutants have normal spot junctions during gastrulation and early germband extension. Lgl and Dlg are not normally excluded from the apical domain until after gastrulation. Similarly, while mammalian aPKC can restrict PAR-1 to the basolateral domain of epithelial cells, Drosophila PAR-1 is not normally excluded from the apical domain at gastrulation. Thus, effects on Crb, Lgl/Dlg, and PAR-1 cannot easily account for the focusing of AJs and Baz into discrete planar-polarized puncta as apkcm/z mutants gastrulate (Harris, 2007).
Instead, the data suggest that aPKC regulates AJ symmetry by regulating MTs. MT regulation may be a common aPKC function. For example, Drosophila aPKC promotes MT stability at synaptic boutons of neuromuscular junctions, where aPKC forms a complex with Futsch (a MAP1B-like protein) and tubulin, recruiting Futsch to boutons to stabilize MTs. aPKC also regulates MT orientation as mammalian astrocytes and fibroblasts undergo directed migration during wound healing, while in MDCK cells, aPKC helps organize the MT cytoskeleton during ciliogenesis (Harris, 2007).
MT organization and reorganization play important roles in epithelial morphogenesis, and the data demonstrate that loss of aPKC disrupts these events. During Drosophila cellularization, strong MT nucleation from apical centrosomes is likely necessary for assembling lateral MTs that support the apical transport of lipids and proteins to form cell membranes. These MTs also help direct the initial apical positioning of AJs and Baz. During later development, the analysis of apkcm/z mutants indicates that centrosomal MTs can affect the symmetric positioning of AJs around the apical domain. Without aPKC activity, the centrosomes become abnormally dominant, bipolar cues, directing AJ clustering and thus disrupting AJ symmetry. Although this abnormal MT organization differs from changes to MT organization observed in AJ mutants, the possibility cannot be ruled out that there is feedback between MTs and AJs during epithelial morphogenesis and that aPKC may regulate these interactions. Indeed, such feedback is very likely and it will be critical to define MT-AJ cross talk mechanisms in future studies (Harris, 2007).
In apkcm/z mutants, MT-associated AJ/Baz puncta assemble at the dorsal and ventral sides of the cells. This suggests that MTs may normally function in AJ assembly at these newly formed cell-cell contacts. However, these polarized AJ assembly events must also be counterbalanced to maintain AJ symmetry and proper epithelial structure. Cytoskeletal inhibitor studies suggest that AJ symmetry may normally be regulated by a balance of MT-AJ and actin-AJ interactions at this stage -- actin appears to counteract MT-based AJ assembly at dorsal and ventral cell contacts. Actin as been shown to be enriched at anterior and posterior cell contacts, suggesting that it may be an early planar polarity cue at this stage. Perhaps this planar-polarized actin stabilizes a pool of AJs at anterior and posterior cell contacts, thereby counterbalancing MT-based AJ assembly at dorsal and ventral contacts. Alternatively, lower levels of actin at dorsal and ventral cell contacts could directly counteract MT-based AJ assembly at these sites. Distinguishing these possibilities requires further study. Nonetheless, the apkcm/z mutant phenotype appears to arise from a gain-of-function effect in which MTs become overactive and the proper balance between MT-AJ and actin-AJ interactions is lost. As a result, there is a break in AJ symmetry in apkcm/z mutants, MT-associated AJ puncta eventually become randomly positioned, and the epithelium dissociates (Harris, 2007).
The data suggest a speculative mechanistic model by which aPKC could normally regulate MT-AJ interactions. This study shows that MT association is responsible for the abnormal AJ asymmetry seen in apkcm/z mutants, and that Dynein accumulates at these abnormal AJ/Baz puncta. Since Dynein plays a role in apical transport of AJ/Baz proteins during cellularization, it is proposed that aPKC may normally regulate release of AJ/Baz complexes from Dynein, allowing a complete transport cycle. In the absence of this release, AJ/Baz complexes could maintain an abnormal association with MTs, and localized Dynein activity may pull the centrosomes and spot junctions together into the abnormal puncta seen in apkcm/z mutants. This abnormal cortical Dynein activity might also stabilize MTs emanating from the centrosomes, resulting in the persistent centrosomal MTs in the mutants. Alternatively, aPKC may function at the centrosomes to decrease MT nucleation or increase MT severing. Future experiments will illuminate these mechanisms and the generality of aPKC's role in controlling MT organization and AJ positioning (Harris, 2007).
Apical constriction is a major mechanism underlying tissue internalization during development. This cell constriction typically requires actomyosin contractility. Thus, understanding apical constriction requires characterization of the mechanics and regulation of actomyosin assemblies. This study analyzed the relationship between myosin and the polarity regulators Par-6, aPKC and Bazooka (Par-3) (the PAR complex) during amnioserosa apical constriction at Drosophila dorsal closure. The PAR complex and myosin accumulate at the apical surface domain of amnioserosa cells at dorsal closure, the PAR complex forming a patch of puncta and myosin forming an associated network. Genetic interactions indicate that the PAR complex supports myosin activity during dorsal closure, as well as during other steps of embryogenesis. It was found that actomyosin contractility in amnioserosa cells is based on the repeated assembly and disassembly of apical actomyosin networks, with each assembly event driving constriction of the apical domain. As the networks assemble they translocate across the apical patch of PAR proteins, which persist at the apical domain. Loss- and gain-of-function studies show that different PAR complex components regulate distinct phases of the actomyosin assembly/disassembly cycle: Bazooka promotes the duration of actomyosin pulses and Par-6/aPKC promotes the lull time between pulses. These results identify the mechanics of actomyosin contractility that drive amnioserosa apical constriction and how specific steps of the contractile mechanism are regulated by the PAR complex (David, 2010).
The repeated assembly and disassembly of apical actomyosin networks is an integral part of amnioserosa tissue morphogenesis during DC. Restricting myosin to the amnioserosa alone is sufficient for amnioserosa apical constriction and overall DC. Dynamic apical myosin has been described in the amnioserosa. This study defined these dynamics as repeated assembly and disassembly cycles of actomyosin networks. Moreover, assembly and disassembly are linked to apical constriction and relaxation, respectively. This is consistent with laser ablation studies showing that the apical surfaces of amnioserosa cells maintain tension across the tissue. Moreover, AJ live imaging has revealed general pulsing of amnioserosa cells from germband retraction through DC. The pulsing actomyosin networks arise with this same developmental timing. A 230±76 second periodicity of cortical pulsing has been described at DC, similar to that of the pulsing actomyosin networks. This study found that increased network durations and decreased lull times with amnioserosa-targeted Baz overexpression coincide with faster DC, as compared with amnioserosa-targeted Par-6 plus aPKC-CAAX overexpression, which increases lull times. It is concluded that the pulsing actomyosin networks mediate the constriction of individual amnioserosa cells and that this contributes to DC (David, 2010).
Remarkably, a single amnioserosa apical constriction event is followed by an almost equal relaxation. However, over many constrictions the cells progressively reduce their apical surface area. This suggests that ratcheting mechanisms incrementally harness the constrictions for overall tissue change. Intracellular and extracellular ratchets are possible. Cells of the Drosophila ventral furrow also display pulsed contractions of apical actomyosin networks as they apically constrict. However, there is minimal relaxation after each constriction. Instead, residual myosin filaments are retained between pulses, and may act as intracellular ratchets to harness the pulsed contractions. By contrast, residual myosin filaments were rarely observed between actomyosin pulses in amnioserosa cells, possibly explaining their relaxation after each cell constriction. It has been proposed that the leading edge actomyosin cable of the surrounding epidermis acts as an extracellular ratchet to harness amnioserosa contractility. However, the ability of myosin expression in the amnioserosa alone to drive DC suggests that other mechanisms contribute. Indeed, DC is a robust process with redundant contributions from both amnioserosa and epidermis. At later stages, filopodia-based epidermal zippering at the canthi could provide another extracellular ratchet. In addition, each amnioserosa cell has a persistent circumferential actin belt that might act as an intracellular ratchet, and other uncharacterized processes, such as membrane trafficking or basal activities, could also contribute (David, 2010).
Actomyosin activity also appears to be linked between cells. The networks display preferential D-V movement, and a network in one cell appears to promote network formation in neighbors. Overall amnioserosa cell shape changes are also coordinated between neighbors. Moreover, myosin activity in isolated amnioserosa cells can elicit cortical myosin accumulation in neighboring epidermal cells. It is speculated that feedback from epidermal cells might orient the D-V movement of amnioserosa actomyosin networks. Interestingly, amnioserosa cells also preferentially contract along the D-V axis. Although the actomyosin networks move in this direction, it is unlikely that they are solely responsible for the directional cell shape changes -- the networks affect the cell circumference both along the axis of their trajectory and perpendicular to it, and, as discussed, both effects are transient. Thus, forces from the epidermis might be needed for the biased D-V amnioserosa cell contraction, and they might also direct the D-V movement of amnioserosa actomyosin networks to facilitate DC (David, 2010).
As the actomyosin networks assemble and disassemble, they translocate across a persistent PAR protein patch. These transient associations and lack of specific colocalization between the actomyosin networks and the PAR proteins argue against PAR proteins being integral parts of the actomyosin networks. However, the current results show that the PAR proteins regulate the networks. Genetic interaction tests indicate that Baz, Par-6 and aPKC support myosin activity for proper DC. Strikingly, the live imaging revealed that Baz and Par-6/aPKC regulate distinct phases of the myosin assembly/disassembly cycle. Together, the loss-of-function and gain-of-function studies show that Baz promotes network durations, whereas Par-6 and aPKC promote lull times between pulses. Baz overexpression also decreased lull times, which could result indirectly from increased network durations or from more direct inhibition of the lull phase. Importantly, overexpression experiments indicate that the effects occur specifically in amnioserosa cells, and analyses of cell polarity and AJs indicate that the PAR proteins have relatively direct effects on the actomyosin networks. However, it remains possible that the PAR proteins have additional functions in the amnioserosa (David, 2010).
A number of molecular interactions must control PAR protein activity in the apical domain of amnioserosa cells. The PAR proteins often, but not exclusively, colocalize in amnioserosa cells, suggesting a dynamic relationship consistent with separate Baz and Par-6/aPKC functions. They also show colocalization with Crb, an apical transmembrane protein at the core of the Crb polarity complex. Interestingly, Crb is known to regulate DC, and Par-6 and aPKC can bind Crb complex components. Thus, Crb might be one anchor for PAR proteins at the apical surface of amnioserosa cells (David, 2010).
Molecular mechanisms connecting PAR proteins to myosin and actin have been implicated in a number of studies. For example, aPKC phosphorylates and inhibits mammalian myosin IIB, although these sites are not present in Drosophila Myosin II (Zipper). Par-6/aPKC also inhibits Rho by activating the ubiquitin ligase Smurf1 in mammalian cells. Additionally, Baz and aPKC immunoprecipitate with Sqh from Drosophila egg chambers. Analogous to amnioserosa morphogenesis, mammalian Par-3 and Par-6/aPKC regulate distinct aspects of cell shape change through different cytoskeletal regulators during dendritic spine morphogenesis: Par-3 inhibits cell protrusions by inhibiting Rac through sequestering the RacGEF Tiam1, whereas Par-6/aPKC promotes protrusions by inhibiting Rho via p190 RhoGAP (David, 2010 and references therein).
Amnioserosa cell apical constriction has similarities to endoderm precursor cell apical constriction during C. elegans gastrulation. Here, myosin activity drives cell ingression. Similar to in amnioserosa cells, the PAR complex and myosin accumulate at the center of the apical surface of these cells and of earlier cells as well. However, these C. elegans actomyosin networks do not appear to undergo full assembly/disassembly cycles and instead progressively accumulate or display continual network flows. Interestingly, apical myosin enrichment requires PAR-3 in C. elegans endodermal precursor cells. Apical myosin enrichment also requires Baz, Par-6 and aPKC in Drosophila egg chamber follicle cells. These results suggest that the PAR complex initiates actomyosin network assembly, contrasting with the amnioserosa, in which networks can assemble without detectable Baz and are inhibited by Par-6/aPKC. Perhaps, actomyosin networks with full assembly/disassembly cycles are regulated distinctly. In the one-cell C. elegans embryo, PAR protein puncta move with a multifaceted cortical myosin network to the embryo anterior. Each facet of the network assembles and disassembles with durations similar to those of the amnioserosa actomyosin networks. The network can also form without the PAR proteins, but the overall flow of the network fails with loss of PAR-3, PAR-6 or aPKC. It would be interesting to test whether PAR-3, PAR-6 and aPKC have distinct effects on the individual facets of these networks (David, 2010).
What triggers actomyosin network assembly in amnioserosa cells? It appears to be independent of Baz, and must overcome Par-6/aPKC inhibition. The Rho pathway triggers actomyosin contractility in many contexts. However, amnioserosa-targeted expression of dominant-negative Rho does not appear to block DC. Alternatively, actin assembly might trigger the networks. Actin networks appear larger and last longer than myosin networks as both start forming during germband retraction. This suggests that actin might organize these networks during germband retraction and possibly DC. Intriguingly, Rac inhibition disrupts DC and reduces amnioserosa actin levels. The trigger might also involve intercellular forces from networks in neighboring cells (David, 2010).
How is the actomyosin assembly/disassembly periodicity regulated? Since more than one network per cell is rarely observed, network assembly might require disassembly of the existing network. Disassembly might begin a cascade that ultimately triggers formation of the next network. For cycling, assembly might likewise elicit disassembly. The data indicate that the PAR proteins are important elements of the regulatory network that is involved. Once a network is triggered, Baz prolongs it, but as the network persists, trigger and maintenance signals must be overcome for network disassembly. With disassembly, Par-6/aPKC activity appears to inhibit new assembly, promoting lull times. With time, this Par-6/aPKC activity must diminish and/or be overwhelmed by the trigger mechanism for new network assembly to occur. Identifying trigger and feedback mechanisms within this cycle will be key for understanding how pulsed actomyosin contractions are regulated in the amnioserosa (David, 2010).
To study the consequences of aPKC loss-of-function on epithelial polarity and asymmetric division of neuroblasts, the phenotype of mutants in the aPKC locus was analyzed. BLAST searches with the genomic sequence of the aPKC gene revealed that the P-element insertion l(2)k06403 is located in the third intron of the aPKC gene. This insertion line is homozygous lethal, contains a single PlacW P-element, and does not complement Df(2R)Jp1, which removes the cytological interval 51C3;52F5-9. Staining intensity with anti-PKCzeta antibody C20 was reduced to background level in embryos homozygous mutant for l(2)k06403. The P-element was mobilized using the transposase source P[delta2-3] and several revertants lacking the w+ marker of the original P-element insertion were recovered. Of seven revertant lines tested, 3 were homozygous viable, demonstrating that insertion of the P-element into the aPKC locus was the cause of lethality and of the observed phenotype. It was thus conclude that l(2)k06403 is an allele of aPKC and it was renamed DaPKCk06403 (Wodarz, 2000).
Homozygous mutant DaPKCk06403 embryos do not produce any cuticle. The reason for this phenotype is early embryonic lethality before the onset of cuticle secretion. Transheterozygous DaPKCk06403/Df(2R)Jp1 embryos show exactly the same cuticle phenotype, suggesting that DaPKCk06403 is a strong hypomorphic or null allele. The analysis of the aPKC loss-of-function phenotype was complicated by the fact that aPKC mutants show maternal haploinsufficiency with incomplete penetrance. The evidence for this conclusion comes from the following observations: it was noticed that, in egg collections from the DaPKCk06403/CyO stock, significantly more than 50% of embryos (including CyO/CyO homozygotes) fail to hatch, which means that even embryos with a zygotic wild-type allele of aPKC frequently show developmental defects. This finding was confirmed in crosses of heterozygous DaPKCk06403/CyO females to wild-type males, where considerable embryonic lethality was observed. The reciprocal cross does not show an increase of embryonic lethality compared with wild-type controls. It is therefore concluded that the decreased viability of the progeny from DaPKCk06403/CyO mothers is caused by the reduction of the maternal DaPKC level during oogenesis (Wodarz, 2000).
To produce embryos with the wild-type maternal contribution of aPKC, but lacking any zygotic aPKC expression, C(2)v females were crossed to DaPKCk06403/CyO males and the cuticle phenotype of their progeny was analyzed. One quarter of the embryos derived from that cross completely lacked zygotic expression of aPKC and produced cuticles with characteristic defects. In most cases, head structures were missing and the ventral cuticle either showed large holes or was missing altogether. These phenotypes are strikingly similar to those of baz mutants. baz null embryos derived from germ-line clones produce very little or no cuticle and resemble DaPKCk06403 mutant embryos derived from heterozygous mothers. baz mutant embryos lacking only the zygotic expression produce cuticles with characteristic head defects and ventral holes that are very similar to DaPKCk06403 mutant embryos derived from C(2)v mothers (Wodarz, 2000).
The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote,
subsequently have been shown to regulate cellular polarity in diverse
cell types by means of an evolutionarily conserved protein complex
including PAR-3, PAR-6, and atypical protein kinase C (aPKC).
The Drosophila homologs of par-1, par-3 (bazooka [baz]), par-6 (DmPar-6), and pkc-3
(Drosophila aPKC; DaPKC) each are known to play
conserved roles in the generation of cell polarity in the germ line as
well as in epithelial and neural precursor cells within the embryo. In
light of this functional conservation, the potential role
of baz and DaPKC in the regulation of
oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of
initial anterior-posterior polarity within germ-line cysts and
maintenance of oocyte cell fate. Germ-line clonal analyses indicate
both proteins are essential for two key aspects of oocyte
determination: the posterior translocation of oocyte specification
factors and the posterior establishment of the microtubule organizing
center within the presumptive oocyte. Baz and DaPKC
colocalize to belt-like structures between germarial cyst cells.
However, in contrast to their regulatory relationship in the
Drosophila and C. elegans embryos, these
proteins are not mutually dependent for their germ-line localization,
nor is either protein specifically required for PAR-1 localization to
the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).
To examine the potential oogenic function of baz and
DaPKC, protein null germ-line mutant clones were generated for
both baz and DaPKC. Germ-line clones, identified
by the absence of nuclear GFP expression, were counterstained with the
chromatin marker propidium iodide to examine the number and ploidy of
the germ-line nuclei. In contrast to wild-type egg chambers, which invariably contain 15 nurse cells and a single oocyte, baz mutant
germ-line clones, while containing the normal complement of germ-line
nuclei, fail to differentiate an oocyte, resulting in a 16-nurse cell
phenotype as revealed by the polyploid state of all 16 germ-line nuclei.
Similarly, DaPKCk06403 germ-line clones
also fail to differentiate an oocyte as indicated by the presence of 16 polyploid nurse cell nuclei in germ-line mutant egg chambers. These results reveal a germ-line autonomous requirement for DaPKC and confirm the
role of Baz in oocyte differentiation and/or maintenance (Cox, 2001).
These analyses further reveal that germ-line depletion of either
DaPKC or baz function from the follicle cells leads to their
multilayering, which disrupts the normal partitioning of germ-line
nuclei to successively mature egg chambers caused by mispositioning of
mutant follicle cells. These mispartitioned
baz+ nurse cell nuclei, into an otherwise
baz null germ-line clone, may provide the threshold of
germ-line Baz required to rescue the oocyte differentiation defect and
allow production of a mature egg. Alternatively, the Baz protein may
exhibit a long perdurance after mitotic clone induction, which depletes
over a period of days, resulting in the cessation of egg production in
these mosaic females. These results, however, in no way invalidate the
previous conclusions that maternally provided Baz masks the severity of the embryonic polarity phenotype in both epithelial cells and neuroblasts. Rather, the results indicate that these embryos are not likely to represent a complete maternal depletion for Baz (Cox, 2001).
Oocyte differentiation requires the polarized accumulation of oocyte
specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in
the localization of these factors, mutant germ-line clones
for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst
cells in region 2a, and then both molecules are targeted first to the
two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In
contrast, ORB fails to translocate from the anterior to a posterior
crescent in both baz and DaPKC null germ-line cysts in germarial
region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in
baz and DaPKC null germ-line clones in germarial
region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).
Furthermore, in contrast to wild-type germ-line cysts in which DHC64C
localizes to a single posterior cell, in DaPKC null germ-line clones DHC64C fails to localize to a single cell posteriorly, but rather accumulates in the two posterior-most presumptive
pro-oocytes of the mutant germ-line cyst.
Therefore, baz and DaPKC display essentially
identical phenotypes in germ-line mutant clones with regards to oocyte
differentiation and the establishment of initial A-P polarity within
the oocyte. The failure to maintain oocyte identity in either
baz or DaPKC mutant cysts can therefore be directly correlated with defects in the A-P translocation of oocyte specification factors within a single posterior cell of a germ-line cyst, suggesting oocyte differentiation depends on this early polarization event (Cox, 2001).
The posterior assembly of a functional MTOC has been directly
implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the
failure to translocate these factors to a posterior crescent in region
3 baz or DaPKC mutant cysts may result from a defect
in microtubule reorganization within these mutant cysts. In contrast to
wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of
oocyte specification factors in these mutants are likely caused, at
least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).
In addition to the microtubule network, both ring canals and the fusome
play critical roles in cyst polarization and oocyte differentiation. The formation and spatial distribution of ring canals as well as fusome morphogenesis was examined in both baz and
DaPKC mutant germ-line cysts. In baz mutant cysts, ring canal formation and spatial distribution are indistinguishable from wild-type germ-line cysts. Furthermore, the wild-type
spatial arrangement of ring canals in baz null germ-line
cysts suggests there is no apparent disruption in germ cell adhesion
within the cyst. Similarly, no defects were observed in ring canal
formation or spatial distribution in DaPKC mutant cysts (Cox, 2001).
These analyses indicate baz mutant cysts display relatively normal fusome morphology, although mutant fusome branches appear slightly thinner when compared with wild-type fusomes within the same germarium. Similarly, no apparent defect is observed in fusome morphology in DaPKC null germ-line cysts, indicating DaPKC
is dispensable in the germ line for proper fusome morphogenesis (Cox, 2001).
To investigate the mechanism by which Baz and DaPKC exert their effects
on oocyte differentiation, the localization of these
proteins was analyzed in the germ line and soma during oogenesis. The specificity of the Baz and DaPKC
antibodies was verified by using mosaic ovaries containing
baz or DaPKC null mutant clones. Baz is first detected in the germarium as a belt-like specialization on germ cell membranes at sites of germ cell interconnection. These belt structures are
reminiscent of ring canals with respect to their position between
germ-line cyst cells; however, in contrast to ring canals these "Baz
belts" are approximately 2-fold greater in diameter. Germaria
double-labeled for Baz and rhodamine phalloidin reveal that the Baz belts
localize adjacent to ring canals and further reveal an approximate 1:1 ratio between the two structures within germ-line cysts. The microtubule cytoskeleton as well as the fusome were observed projecting through individual cystocytes coincident with
the site of Baz belt expression on the germ cell membrane. Later in
oogenesis, Baz is transiently enriched in the oocyte cytoplasm at
stages 5-6 before the onset of vitellogenesis and is subsequently undetectable in the germ line of
vitellogenic egg chambers. As with Baz localization to the apical
junctional zone in the embryonic epithelium, tight apical localization of Baz is also observed in follicular epithelia (Cox, 2001).
DaPKC localizes to the Baz belts in the germarium, whereas in follicle cells DaPKC is apically constricted consistent with Baz localization in these cells (Cox, 2001).
In embryonic epithelia, Baz, DmPAR-6, and DaPKC apically colocalize and
partially overlap with the apico-laterally enriched Arm and DE-cadherin
proteins in the region of the apical zonula adherens. Furthermore,
these proteins are required to maintain epithelial cell polarity and
are mutually dependent for their proper localization. DE-cadherin is localized to belt-like structures adjacent to ring canals in region 2 germarial cysts reminiscent of Baz belt localization, suggesting that these adherens junction components may similarly colocalize in the germ line. To further investigate the molecular and functional nature of Baz belt expression in the
germarium, germaria were labeled with anti-Baz, anti-Arm, and
anti-DE-cadherin antibodies and their potential colocalization
within the germarium was examined. These
analyses reveal Baz colocalizes with both DE-cadherin and Arm to the Baz belts within the
germarium. Furthermore, consistent with the colocalization of Baz and
DaPKC to Baz belts within the germarium, partial
colocalization of DaPKC with DE-cadherin is observed in wild-type germarial cysts. To assay whether these proteins are mutually dependent for their germ-line localization, the localization of DE-cadherin and Arm was examined in baz null germ-line clones. In contrast to their mutual dependence in embryonic epithelia, these analyses indicate germ-line Baz function is dispensable for the localization of either DE-cadherin or Arm to the Baz belts in
the germarium. DaPKC is dispensable for the localization of either DE-cadherin or Arm
to these structures. These results
indicate Baz and DaPKC function are not required for the formation of
these structures because both Arm and DE-cadherin localization to these
belts in baz or DaPKC mutant cysts is indistinguishable from
that observed in wild-type cysts. Previous studies have revealed that germ-line clones of a strong allele of
shotgun (shgIG29), the gene
encoding DE-cadherin, disrupts the arrangement of germ cells in region
2b germarial cysts, suggesting DE-cadherin may mediate germ cell
adhesion. In contrast, these analyses of ring canal spatial distribution
in baz and DaPKC mosaic cysts strongly suggests
Baz and DaPKC function are dispensable for normal germ cell adhesion.
Furthermore, germ-line clonal analyses of either shg or
arm reveal neither gene is required for oocyte
differentiation, whereas both
baz and DaPKC are essential in oocyte determination. These results suggest the components of the Baz belts
likely mediate diverse cellular functions essential for germ-line cyst
development, which may include cell adhesion, cell signaling, and cyst
polarization (Cox, 2001).
In the C. elegans zygote, the PAR-3/PAR-6/PKC-3
complex is localized to the anterior where it is required for the
posterior localization of PAR-1. To investigate the potential regulatory relationship between baz and par-1 in the Drosophila germ line,
baz null germ-line clones were generated and PAR-1 expression and
localization was analyzed. In wild-type germ-line cysts, PAR-1 is localized to the spectrosome and fusome in germarial regions 1 and 2a and subsequently
is down-regulated on fusomes in regions 2b and 3. In
baz mutant germ-line cysts, PAR-1 localization to the
spectrosome is unaffected and is likewise present on fusomes although somewhat weaker localization is observed on fusome branches of baz mutant germ-line cysts when compared with wild-type germ-line cysts. Taken together, these results suggest germ-line baz is dispensable in the germarium for
normal PAR-1 expression and localization (Cox, 2001).
To determine whether par-1 may regulate Baz expression, par-1 null germ-line clones were generated and Baz
localization was examined. No defect was observed in Baz belt expression in
par-1 mutant germ-line cysts. Furthermore, in contrast to wild-type stage 5-6 egg
chambers in which Baz is transiently enriched in the oocyte cytoplasm, in par-1 mutant egg chambers Baz localization is abolished from the posterior presumably due to the
defect in oocyte differentiation observed in par-1 mutant egg chambers. These results indicate
germ-line par-1 is dispensable for normal Baz belt
expression and localization (Cox, 2001).
To assay the regulatory relationship between baz and
DaPKC the localization of DaPKC was analyzed in
baz mutant germ-line cysts, as well as
the localization of Baz in DaPKC mutant germ-line cysts. In contrast to their mutual dependence for
localization in the embryo, both Baz and DaPKC localization within the germ line is mutually independent. These results indicate that
despite the apparent functional conservation of Baz, DaPKC, and PAR-1
in generating oocyte polarity, the regulatory relationships among these
genes are not conserved in the germ line (Cox, 2001).
The colocalization of Baz, DaPKC, Arm, and DE-cadherin to
the Baz belt structures in the germarium strongly suggests the components of
the Baz belts are capable of mediating a multiplicity of functions in
germ-line cysts. In embryonic epithelia, these proteins function in the formation of the apical zonula adherens junction and are mutually dependent for their apical localization. The germ-line colocalization of these molecules to the Baz
belts suggests these structures may represent a potential polarity cue
on the germ cell plasma membrane. The restricted localization of Baz
belt components to germ cell membranes at points of cell-cell contact
represents an asymmetry on the plasma membrane of germ-line cyst cells
with regard to the A-P axis of the cyst and stage 1 oocyte. The
asymmetric localization of these molecules to one side of the germ cell
plasma membrane may act as a polarity cue in defining anterior versus
posterior within individual cystocytes of the 16-cell germ-line cyst
and thus contribute to the establishment of an initial A-P axis and to
subsequent germ-line cyst polarization. These results further suggest
that Baz and DaPKC likely function in a signaling capacity, rather than
a structural one, to mediate oocyte differentiation, whereas
DE-cadherin and Arm are more likely to function in maintaining germ
cell adhesion and cyst integrity (Cox, 2001).
Consistent with par gene function in other systems, these results indicate that Baz, DaPKC, and PAR-1 are required for the
establishment and maintenance of cellular polarity in the
Drosophila germ line; however, the regulatory relationships
observed between these genes in the germ line versus that observed in
embryonic blastomeres, epithelial cells, and neural precursor cells
indicates that, while these molecules are functionally conserved, the
mechanisms by which these genes act appear to be less well conserved.
In contrast to their mutual dependence for localization in the embryo,
Baz, DaPKC, and PAR-1 each are mutually independent for their
localization in the germ line. Taken together, these results underscore
the functional utility of the par genes and their effectors
as a molecular module for generating cellular polarity in diverse cell
types. Furthermore, the independence of Baz, DaPKC, and PAR-1 in their germ-line localization provides a unique opportunity to probe new
mechanisms by which these highly conserved proteins function in
regulating diverse processes such as cellular polarity, asymmetric cell
division, or growth control (Cox, 2001).
Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype; reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes (Rolls, 2003).
aPKCk06403 zygotic mutant embryos die before gastrulation and all epithelial and neuroblast polarity is lost. A much later onset phenotype is found: survival of aPKCk06403 homozygotes to second larval instar with no significant embryonic phenotype. Although surprising, it is felt that these results reflect the authentic zygotic aPKCk06403 mutant phenotype for several reasons. (1) Recent work from the Wodarz lab confirms that aPKCk06403 homozygotes survive to at least late embryonic stages without significant epithelial or neuroblast defects (Wodarz, A., personal communication to Rolls, 2003), presumably due to the absence of deleterious second site mutations that were on the original aPKCk06403 chromosome. (2) PCR was used to verify the presence of the aPKCk06403 allele in all of of the stocks used in this study, and dominantly marked balancer chromosomes were used to independently confirm the identity of every aPKCk06403 homozygote analyzed. (3) aPKCk06403 homozygous second instar larvae were shown to have very low levels of aPKC mRNA and no detectable full-length protein; thus, a very strong or null aPKC mutant phenotype was examined. (4) It was shown that aPKCk06403/aPKCk06403 and aPKCk06403/Df(2R)JP1 larvae have indistinguishable lethal phases and quantitative neuroblast phenotypes; thus, aPKCk06403 behaves as a genetic null allele. (5) aPKCk06403 homozygous embryos have persistent maternal aPKC protein, explaining the lack of an embryonic phenotype in these mutants (Rolls, 2003).
One of the more unexpected findings is that aPKC mutant neuroblasts show normal Baz/Par3 apical localization. The Baz/Par3-Par6-aPKC complex has been suggested to form a functional unit that is interdependent for localization in C. elegans, mammals, and Drosophila. Baz/Par3 shows normal apical localization in aPKC mutant neuroblasts, showing that normal Baz/Par3 localization can occur without being part of the Par3-Par6-aPKC complex. In addition, neuroblasts lacking apical aPKC and Par6 still form a molecularly defined apical cortical domain containing Baz/Par3, Insc, Pins, and Dlg. These results lead to the proposal of a hierarchy for apical protein localization in neuroblasts: Baz/Par3-Insc-Pins-Dlg --> aPKC --> Par6-Lgl. This hierarchy is consistent with recent biochemical analyses in which a protein complex was isolated containing Par6-aPKC-Lgl, but not Baz/Par3. It is suggested that aPKC may be required to anchor the Par6-aPKC-Lgl complex at the apical cortex of the neuroblast (Rolls, 2003).
Both aPKC and Lgl are required for Miranda basal localization in neuroblasts, and all available data support a model in which Lgl is required for targeting Miranda to the neuroblast cortex, whereas aPKC blocks Lgl function on the apical side of the neuroblast: (1) lgl mutants have little or no Miranda at the cortex; (2) aPKC mutants show uniform cortical Miranda localization; (3) a weak lgl phenotype can be suppressed by reducing aPKC levels, showing that aPKC activity antagonizes Lgl activity; (4) aPKC and Lgl physically interact; (5) an overexpressed nonphosphorylatable Lgl protein is uniformly cortical and able to induce uniform cortical Miranda localization, whereas phospho-Lgl is preferentially released from the cell cortex. This has led to a model in which Lgl acts as an anchor for Miranda at the basal cortex, but is absent from the apical cortex due to aPKC-mediated phosphorylation. Although this simple model is attractive, it is noted that Lgl has never been observed colocalized with Miranda in a basal cortical crescent, and a role for cytoplasmic Lgl in Miranda localization has not been definitively ruled out (Rolls, 2003).
The Baz/Par3-Par6-aPKC complex has a well-characterized role in regulating neuroblast spindle orientation. Spindle orientation can be measured relative to extrinsic landmarks around the neuroblast (e.g., perpendicular to the overlying ectoderm) or relative to intrinsic cues within each neuroblast (e.g., perpendicular to the Baz/Par3-Par6-aPKC apical crescent). Mutations in baz or par6 genes randomize embryonic neuroblast spindle orientation relative to the overlying ectoderm, but it is not clear whether these phenotypes are due to disruption of the ectodermal layer or to a cell-autonomous defect in the neuroblast. The function of aPKC in embryonic neuroblast spindle orientation cannot be assayed due to high levels of maternal aPKC protein present in aPKC zygotic mutant embryos. However, aPKC is not required for intrinsic spindle orientation in larval neuroblasts; the mitotic spindle is always perpendicular to the Baz/Par3-Insc-Pins apical crescent (Rolls, 2003).
The Baz/Par3 and Par6 proteins are required to establish epithelial polarity in Drosophila, and aPKC is also shown to be required for normal apical/basal epithelial cell polarity. The similar phenotype in baz, par6, and aPKC mutants may indicate that these proteins function together as a complex to provide a single function in epithelia, despite the evidence that they have independent functions in neuroblasts. One primary function may be the inhibition of Lgl activity because it is found that the lgl epithelial polarity defects can be strongly suppressed by reducing aPKC levels. Lgl-Dlg-Scrib activity can also antagonize Baz/Par3-Par6-aPKC activity, and it is tempting to speculate that aPKC inactivates Lgl by phosphorylation, whereas Lgl can inactivate aPKC by sequestering it into an Lgl-Par6-aPKC complex and out of the Baz/Par3-Par6-aPKC complex (Rolls, 2003).
The role of aPKC in cell proliferation has not been previously investigated in Drosophila. There are three lines of evidence showing that aPKC promotes cell proliferation in neuroblasts and epithelia. The number of cells in aPKC mutant mushroom body neuroblast clones is significantly lower than the number in wild-type clones. There appears to be a normal number of early-born gamma neurons in these clones, followed by only a few later-born alpha neurons. The normal number of early-born gamma neurons suggests that loss of aPKC does not lead to cell death in this population; moreover, no decrease is seen in the number of neuroblasts per brain lobe in aPKC mutant larvae. It is concluded that cell death is not contributing to the reduction in neurons observed in the clones, but rather, that the neuroblast stops dividing near the time the neuroblast switches over to generating alpha and ß neurons. The neuroblast may become arrested at some point in the cell cycle, or it may undergo a terminal division to generate a pair of GMCs (perhaps due to both daughter cells inheriting Miranda and Prospero GMC determinants). A second indication that aPKC promotes cell proliferation is that far fewer epithelial cells are observed in aPKC mutant eye imaginal discs compared with the wild type, even with an additional day of growth as second instar larvae. Finally, a 50% reduction in aPKC levels (aPKC/+) can strongly suppress the epithelial and brain overproliferation phenotypes of lgl mutants. Together, these data show that aPKC positively regulates cell proliferation in epithelia and neuroblasts. Interestingly, reduction in the function of the mammalian atypical PKCzeta (using overexpression of a dominant-negative kinase) can suppress Rac1/cdc42-induced overproliferation. Thus, aPKC may have an evolutionarily conserved role in promoting cell proliferation, as well as in the establishment of cell polarity (Rolls, 2003).
Although aPKC and Lgl act antagonistically to regulate many aspects of epithelial and neuroblast cell polarity and cell proliferation, they may share a common positive function in regulating neuroblast apical cell size. lgl zygotic mutants have some embryonic telophase neuroblasts with an abnormally small apical cortical domain, apical spindle pole, and neuroblast size. These defects are not suppressed by reducing aPKC levels, and in fact may be enhanced. Thus, it is proposed that Lgl and aPKC both act positively to promote large apical cell and spindle pole size. It has been reported that Baz/Par3-Par6-aPKC and Pins-Galphai act in parallel pathways to promote large apical cell size and apical spindle size; Lgl could be acting as part of the Pins-Galphai pathway, or as a third input promoting apical cell and spindle size (Rolls, 2003).
Par-3/Baz, Par-6, and aPKC are evolutionarily conserved regulators of cell polarity, and overexpression experiments implicate them as axon determinants in vertebrate hippocampal neurons. Their mutant and overexpression phenotypes were examined in Drosophila melanogaster. Mutants neurons have normal axon and dendrite morphology and remodel axons correctly in metamorphosis, and overexpression does not affect axon or dendrite specification. Baz/Par-6/aPKC are therefore not essential for axon specification in Drosophila (Rolls, 2004).
Therefore, Drosophila Baz, Par-6 and aPKC are not required for axon specification in vivo, and their overexpression has no effect on axon specification or outgrowth. In contrast, overexpression of Par-3 or Par-6 in cultured mammalian hippocampal neurons results in multiple axon-like processes, leading to the hypothesis that these proteins are axon determinants. How can these apparently paradoxical results be reconciled? One possibility is that vertebrate neurons require Par-complex proteins for axon specification, whereas Drosophila neurons do not. If this is the case, it would be interesting to learn how different molecular pathways in mammals and flies generate the same functional subcellular domain (the axon). Another possibility is that neither fly nor vertebrate neurons use Par proteins to specify axon identity in vivo; cultured hippocampal neurons are separated from normal external polarity cues and may use a different mechanism for axon specification. Polarity cues from surrounding cells may also inhibit neurons in vivo from changing polarity in response to extra Par-3 or Par-6, explaining the different effects of overexpressing these proteins in Drosophila and in hippocampal neurons. A third possibility is that the overexpression experiments, where proteins are present at higher-than-normal levels, do not reflect the in vivo functions of the proteins. Loss-of-function and overexpression experiments that examine vertebrate neurons in vivo or in slice preparations will be crucial for fully understanding the role of Par complex proteins in vertebrate axon specification (Rolls, 2004).
The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. At Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1B-related protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, Baz and Par-6 are also expressed at synapses and their synaptic localization depends on aPKC activity. These findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory (Ruiz-Canada, 2004).
During expansion of the NMJ, parent boutons located at the distal end of a branch give rise to new synaptic boutons by budding. New buds separate from parent boutons by the formation of a neck, and NMJ branches extend by neck elongation and bouton enlargement. Throughout this process, the postsynaptic membrane and underlying cytoskeleton impose a barrier to presynaptic extension, since synaptic boutons and their buds are completely surrounded by the muscle cell membrane and underlying cytoskeleton. During branch elongation, a presynaptic signal may induce the retraction of the postsynaptic cytoskeleton barrier. It is proposed that changes in both the pre- and postsynaptic cytoskeleton during branch elongation mediate these events and that these processes are regulated by aPKC with the collaboration of Baz and Par-6 in both locales (Ruiz-Canada, 2004).
The localization of aPKC to MT-rich domains at the NMJ and the marked reduction in new synaptic bouton formation observed in dapkc mutants prompted an investigation of the cytoskeletal changes during branch elongation. At the presynaptic arbor of wild-type larvae, NMJ expansion is accompanied by the presence of unbundled MTs at the distal tip of NMJ branches and by little if any colocalization between MTs and the MAP1B-related protein Futsch. In vertebrates, MAP1B protects MTs from depolymerization . Similarly in Drosophila, MT integrity in axons and at the NMJ is preserved by Futsch; severe mutations in futsch result in MT fragmentation. This Futsch-dependent protection of MT integrity at presynaptic arbors has an important role during NMJ expansion, since MT fragmentation in futsch and dapkc mutants results in marked reduction in bouton number (Ruiz-Canada, 2004).
In epithelial cells and growth cones, an increase in aPKC activity enhances MT lifetime, although the involvement of MT-associated proteins in this process has not been investigated. The results suggest that aPKC activity enhances MT stability in a process that depends on Futsch. (1) Coimmunoprecipitation experiments indicate that aPKC, Tubulin, and Futsch exist in the same biochemical complex and that the interaction between aPKC and MTs is likely to be through Futsch. (2) Loss of dapkc function results in fragmented MTs at terminal boutons, and an increase in aPKC activity by pre-PKM (PKM is a persistantly active kinase) results in longer than normal MTs and an increased colocalization between MTs and Futsch. (3) Presynaptic expression of PKM in a futsch mutant background results in MT fragmentation similar to futsch mutants alone, suggesting that PKM acts through Futsch to stabilize MTs (Ruiz-Canada, 2004).
The results also show that changes in aPKC activity affect both postsynaptic MT and Actin domains. Based on the lack of aPKC within the Actin domain and the enrichment of aPKC at the MT domain, a primary action of aPKC in muscle cells might be through MTs that surround the peribouton area. Alternatively, the effect of aPKC activity on muscle MTs may arise as a consequence of changes in Baz and Par-6 in the Actin-rich peribouton area, which is spatially segregated from postsynaptic MTs (Ruiz-Canada, 2004).
An interesting finding is that both an increase and decrease in aPKC activity, either pre- or post-synaptically, result in reduction of NMJ expansion. This may reflect the possibility that the pre- and post-synaptic cytoskeleton antagonize one another during NMJ expansion and that an asymmetric perturbation of the cytoskeleton in each cell prevents normal synaptic growth. An alternative or additional possibility is that aPKC is asymmetrically regulated at the pre- and postsynaptic cell, being activated in one cell and inhibited in the other. In this regard, it was noteworthy that while increasing aPKC activity increases the stability of presynaptic microtubules, increasing aPKC postsynaptically results in microtubules that appeared to retract from the junctional area (Ruiz-Canada, 2004).
These studies indicate that Baz and Par-6 are colocalized with aPKC, although this colocalization is only partial. Further, a decrease in Baz or Par-6 gene dosage has been shown to alter NMJ growth and the genes interact genetically with aPKC. That all three proteins coimmunoprecipitate supports the notion that they exist in a tripartite complex. However, it is also likely that at different regions of the NMJ, the composition of the complex is reduced to aPKC-Par-6 or Baz-Par-6. This is suggested by the colocalization studies showing that only Par-6 and aPKC are concentrated at the MT bundle and that only Par-6 and Baz are concentrated at the peribouton area (Ruiz-Canada, 2004).
Baz and Par-6 are localized to the Actin/Spectrin peribouton area, and loss of Baz in dapkc mutants or baz4/+ mutants decreases peribouton Spectrin localization, suggesting that Baz regulates the Actin/Spectrin network. In epithelial cells, Baz is required for the maintenance of the zonula adherens, an Actin belt that encircles the cell just below its apical face. At the NMJ, Baz may similarly contribute to the maintenance of the Actin-rich domain (Ruiz-Canada, 2004).
The composition of the Baz/Par-6/aPKC complex is likely to be regulated by the kinase activity of aPKC; expressing PKM increased the amount of Baz associated to the complex. Mammalian Par-6 is known to bind to both aPKC and Baz at distinct sites, and Par-6 activates aPKC when bound to activated Cdc42 and Rac1. Mammalian Baz/Par-3 is also known to bind to both aPKC and Par-6 at distinct sites, but in contrast to Par-6, Baz inhibits aPKC activity. This inhibition can be suppressed by aPKC-dependent Baz phosphorylation at a highly conserved protein region, and this phosphorylation promotes the dissociation of Baz and aPKC. At the NMJ, it was found that increasing PKM, which lacks the Par-6 binding site, increases the binding between Par-6 and Baz, suggesting that Baz phosphorylation may promote the association between Baz and Par-6. A potential scenario is that Baz and aPKC may exist as an inactive complex at the muscle cortex. Phosphorylation of Baz dissociates the complex and phosphorylated Baz may accumulate at the peribouton region. In agreement with this model, it was found that overexpressing PKM postsynaptically results in an expansion of the peribouton area and increased accumulation of Baz at this area (Ruiz-Canada, 2004).
Electrophysiological studies show that aPKC activity also influences synaptic efficacy. This may result from cytoskeletal changes, which may alter the localization of synaptic proteins, such as GluRs. Indeed, changes in aPKC activity were found to affect both GluR levels or distribution and mEJP amplitude. Many synaptic receptors are anchored to the Actin submembrane matrix. For example, the scaffolding protein DLG, which is responsible for the clustering of Shaker K+ channels and the cell adhesion molecule FasII at the peribouton area, depends on normal Spectrin levels for proper localization at this area. Similarly, in mammals, the DLG homolog SAP97 binds to band 4.1, which is anchored at the Actin/Spectrin network, and NMDA receptors bind alpha-Actinin, an Actin binding protein. Therefore, the changes in GluR levels and distribution found in dapkc mutants may result from alterations in the postsynaptic Actin network (Ruiz-Canada, 2004).
Despite the changes in mEJP amplitude, synaptic junction efficacy (represented by quantal content) was decreased in both aPKC gain- and loss-of-function mutants. This is in contrast to other mutants that affect synaptic transmission in which quantal content is maintained despite changes in postsynaptic sensitivity. For example, reduction of GluR at the postsynapse results in an increase in the amount of neurotransmitter release at Drosophila NMJs. The results raise the possibility that aPKC may be affecting the mechanism that controls retrograde regulation of neurotransmitter release (Ruiz-Canada, 2004).
In addition to changes in quantal content and mEJP amplitude, a reduction was also observed in mEJP frequency. Changes in the frequency of mEJP may arise from a decrease in the probability of release or in the number of release sites. At the NMJ, the reduction in mEJP frequency may reflect the reduction in bouton number observed in dapkc mutants (Ruiz-Canada, 2004).
In the mammalian hippocampus, atypical PKMzeta is necessary and sufficient for LTP maintenance. In flies, overexpression of PKMzeta enhances memory in a Pavlovian olfactory learning paradigm. Moreover, aPKC inhibition using a kinase dead dominant-negative or chelerythrine treatment, which specifically inhibits the catalytic domain of aPKC, diminishes memory without affecting learning. Although these studies suggest that aPKC is involved in functional plasticity of synapses, the cellular mechanism for this effect is unknown (Ruiz-Canada, 2004).
Recent studies suggest that morphological modifications of dendritic spines accompany synapse plasticity, and therefore, changes in spine structure might be at the core of learning and adaptive mechanisms. Spines are particularly enriched in Actin, and interfering with the Actin cytoskeleton inhibits spine motility. Further, many members of the postsynaptic complex, including NMDA receptors, CaMKII, PSD-95, SPAR, and Shank associate with F-Actin through Actin binding proteins. MTs, in contrast, localize to dendritic shafts and are believed to constitute a more stable component. This partitioning between MT and microfilament domains, however, is reminiscent of these domains in growth cones, where Actin and MT dynamics are highly interdependent and ultimately responsible for growth cone dynamics. Similarly, in these studies it has been shown that interfering with normal MT dynamics though modifications in aPKC activity has important consequences for the arrangement of the Actin-rich peribouton area and the normal localization of GluRs. Therefore, although the influence of MTs in spine structure has received less attention, it may be the case that spine architecture is ultimately defined by an interplay between Actin- and MT-rich domains (Ruiz-Canada, 2004).
These studies demonstrate that changes in MT organization are an essential aspect of synapse development and that the aPKC/Baz/Par-6 complex plays an important role in their regulation. In addition, the results show that at the postsynaptic cell, changes in aPKC activity result in dramatic changes in both the MT and Actin networks. Commensurate with the behavioral and electrophysiological studies in which increasing aPKC activity enhanced LTP and memory maintenance, it was found that increases and decreases in aPKC activity inversely regulated the synaptic cytoskeleton. These observations raise the attractive possibility that aPKC regulates synapse plasticity, at least in part, by affecting the organization of the synaptic cytoskeleton (Ruiz-Canada, 2004).
How a cell chooses to proliferate or to differentiate is an important issue in
stem cell and cancer biology. Drosophila neuroblasts undergo self-renewal
with every cell division, producing another neuroblast and a differentiating
daughter cell, but the mechanisms controlling the self-renewal/differentiation
decision are poorly understood. This study tested whether cell polarity genes,
known to regulate embryonic neuroblast asymmetric cell division,
also regulate neuroblast self-renewal. Clonal analysis
in larval brains shows that pins mutant neuroblasts rapidly fail to
self-renew, whereas lethal giant larvae (lgl) mutant neuroblasts
generate multiple neuroblasts. Notably, lgl pins double mutant
neuroblasts all divide symmetrically to self-renew, filling the brain with
neuroblasts at the expense of neurons. The lgl pins neuroblasts show
ectopic cortical localization of atypical protein kinase C (aPKC), and a
decrease in aPKC expression reduces neuroblast numbers, suggesting that aPKC
promotes neuroblast self-renewal. In support of this hypothesis,
neuroblast-specific overexpression of membrane-targeted aPKC, but not a
kinase-dead version, induces ectopic neuroblast self-renewal. It is concluded that
cortical aPKC kinase activity is a potent inducer of neuroblast
self-renewal (Lee, 2005).
Drosophila neuroblasts are an
excellent model system in which to investigate the molecular control of
self-renewal versus differentiation. Larval neuroblasts repeatedly divide
asymmetrically to self-renew a neuroblast and to produce a smaller daughter
cell, called a ganglion mother cell (GMC), that typically makes two postmitotic
neurons; this process enables a single neuroblast to generate many hundreds of
neurons. Self-renewal is defined as the capacity of a
neuroblast to maintain all attributes of its cell type (molecular markers and
proliferation potential). In this regard, a neuroblast is very similar to a
germline stem cell: both maintain their stem cell identity while generating
differentiating progeny. About 100 neuroblasts per
brain lobe are formed during embryogenesis, where they proliferate briefly
before entering quiescence. Brain neuroblasts
re-enter the cell cycle between 10 and 72 h after larval hatching
(ALH), and then a stable population of ~100 mitotic, self-renewing neuroblasts is
maintained. This invariant neuroblast
number was used to screen for mutants altering self-renewal versus differentiation:
mutants in which a neuroblast makes two neuroblast progeny (ectopic
self-renewal) will have >100 neuroblasts, whereas mutants in which a
neuroblast makes two GMC progeny (failure in self-renewal) will have <100
neuroblasts. This assay was used to test known cell polarity mutants
for a role in neuroblast self-renewal (Lee, 2005).
Two classes of cell
polarity regulators were assayed for an effect on larval neuroblast self-renewal.
lgl and discs large (dlg) zygotic mutants were examined, because
these mutants form brain tumours and promote basal
protein targeting in embryonic and larval neuroblasts. Lgl
and Dlg have several protein interaction motifs and are localized around the
neuroblast cortex. In addition, pins
and Galphai zygotic mutants were examined; these genes regulate cell
polarity in embryonic neuroblasts, but have not
been well characterized in larval neuroblasts. Pins and Galphai are
colocalized with Inscuteable and the evolutionarily conserved
Bazooka-Par6- aPKC proteins at the apical cortex of mitotic
neuroblasts, and all of these proteins are partitioned into the neuroblast
during cytokinesis (Lee, 2005).
In wild-type larvae, a population of ~100 neuroblasts
could be identified by the markers Worniu,
Deadpan and Miranda, and by labelling with a pulse of 5-bromodeoxyuridine
(BrdU); by contrast, the thousands of differentiating GMCs and neurons rapidly
downregulate neuroblast markers and express nuclear Prospero and/or Elav.
A clear increase in neuroblast number is observed in
lgl and dlg mutants; there are supernumerary neuroblasts at all
stages examined; all extra neuroblasts expressed Deadpan and Miranda and
are proliferative on the basis of their ability to incorporate BrdU. Galphai
zygotic mutants have a complex phenotype that will be described
in a later publication; however, pins zygotic mutants show a marked decrease in
neuroblast number. Notably, this phenotype is not due to a subset of
neuroblasts remaining quiescent, because neuroblast numbers peak and then
decline over time, and it is not due to neuroblast
cell death. The relatively late onset of the pins phenotype
is probably due to the gradual depletion of maternal pins gene product
in these larvae (Lee, 2005).
To determine whether the pins and lgl larval brain phenotypes are
due to defects in neuroblast self-renewal, positively marked genetic
clones were induced in single neuroblasts to trace their
progeny. Clone induction
parameters were adjusted to ensure that each clone was derived from a single neuroblast (1.2
clones per lobe). In wild-type brains, neuroblast clones always
contained a single Worniu+ Miranda+
nuclear-Prospero- neuroblast and numerous smaller Worniu-
Miranda- nuclear-Prospero+ progeny,
confirming that wild-type neuroblasts always
divide to self-renew and to generate a smaller differentiating GMC.
By contrast, lgl mutant brains had an average of
2.3 neuroblasts per clone, with up to six neuroblasts per clone, showing that
lgl mutant neuroblasts can divide symmetrically to yield two neuroblasts.
The opposite phenotype was seen in pins
mutant brains: 72.8% of the clones had no neuroblast and the remainder had a
single neuroblast.
The neuroblasts did not die in the pins mutants as evidenced by the following: the cell death marker
caspase-3 was not upregulated, neuroblast-specific expression of the p35 cell death inhibitor
did not rescue the missing neuroblasts, and one
clone was observed in which the largest cell coexpressed neuroblast and GMC markers,
consistent with an intermediate stage in neuroblast-to-GMC differentiation. It is concluded
that wild-type neuroblasts exclusively
generate neuroblast/GMC siblings; lgl mutant neuroblasts occasionally
undergo ectopic self-renewal to generate neuroblast/neuroblast siblings; and
pins mutant neuroblasts occasionally fail to self-renew, resulting in
GMC/GMC siblings and termination of the lineage (Lee, 2005).
Next to be examined was whether lgl pins double mutants had fewer neuroblasts (like
pins mutants) or extra neuroblasts (like lgl mutants).
Unexpectedly, a phenotype was detected in which the larval brain was full of
cells expressing the neuroblast markers Worniu, Miranda and Deadpan and lacking
expression of the neuronal marker Elav. Additional markers that distinguish neuroblasts and
GMCs were examined to determine whether these cells were neuroblasts or a hybrid
neuroblast/GMC identity. Both wild-type neuroblasts and lgl
pins cells actively transcribed the worniu, deadpan,
miranda and prospero genes, maintained proliferation, did not
express the Elav neuronal differentiation marker, and did not extend axons.
The only potential GMC attribute found in lgl pins
neuroblasts was nuclear Prospero protein but, because wild-type
neuroblasts and GMCs both contain Prospero protein, which can accumulate in
neuroblast nuclei if not properly localized, this protein is not a definitive
marker for the GMC cell type. Thus, lgl pins brains contain large numbers
of ectopic, proliferating, self-renewing neuroblasts. Combining these
lgl, pins and lgl pins mutant data leads to the conclusion
that Lgl inhibits self-renewal, whereas Pins has dual functions in promoting and
inhibiting self-renewal (Lee, 2005).
To understand how Lgl and Pins
regulate neuroblast self-renewal at the cellular level, cortical
polarity marker localization was examined in mitotic larval neuroblasts. In wild-type larval
neuroblasts, the Par complex (Bazooka-Par6-aPKC) and
Pins-Galphai proteins forms an apical crescent at metaphase
and are partitioned into the self-renewing neuroblast at telophase, whereas the
Miranda and Prospero proteins form a basal crescent at metaphase and are
partitioned into the differentiating GMC at telophase. In lgl
pins double mutants, in which all neuroblasts divide symmetrically to
generate self-renewing neuroblast/neuroblast siblings, most mitotic neuroblasts
show uniform cortical aPKC, cytoplasmic Bazooka and Par6, and uniform cortical
Miranda at metaphase and telophase.
Thus, only aPKC maintained its correct subcellular localization and correlated
with neuroblast self-renewal (Lee, 2005).
aPKC localization was examined in lgl and pins single mutants, in which symmetric
divisions occurred at lower frequency. In lgl mutants, aPKC showed weak
ectopic cortical localization in about half the metaphase neuroblasts, whereas
Miranda was delocalized from the cortex; by telophase, however, both proteins
appeared to be localized normally. Ectopic cortical
aPKC was also observed in dlg mutant larval neuroblasts.
A role for Lgl in restricting aPKC to the apical cortex of neuroblasts has not
been reported but
would be consistent with the observation that basolateral Lgl restricts aPKC to
the apical surface of Drosophila and vertebrate epithelia and
Xenopus blastomeres. In pins mutants, aPKC and cytoplasmic Miranda
showed weak uniform cortical distribution in metaphase neuroblasts, but were properly
localized in most telophase neuroblasts
Thus, both Lgl and Pins are required to restrict aPKC to the apical cortex in
metaphase neuroblasts (Lee, 2005).
Whether aPKC is required for neuroblast self-renewal was examined.
aPKC mutant clones in larval
mushroom body neuroblasts showed premature lineage termination,
consistent with aPKC being required for neuroblast
self-renewal. In addition, aPKC null mutants died as second instar larvae
with reduced neuroblast numbers. Because this was a
relatively mild phenotype and there was no detectable aPKC protein at this
stage, it is likely that there are additional pathways for stimulating
neuroblast self-renewal. Next, whether aPKC is required for ectopic
neuroblast self-renewal in the lgl mutants was tested. lgl aPKC double
mutants had normal numbers of neuroblasts, showing
that aPKC is required for the ectopic neuroblast self-renewal seen in lgl
mutants. aPKC mutants also suppressed ectopic neuroblast self-renewal in
several independently isolated lgl mutations, further supporting a role
for aPKC in self-renewal. In addition, it was found that aPKC is fully epistatic to
lgl in regulating Miranda localization. Thus, aPKC is required for the ectopic
neuroblast self-renewal and Miranda delocalization phenotypes seen in lgl
mutants (Lee, 2005).
These data are most consistent with a model in which
Lgl negatively regulates aPKC, and aPKC directly promotes self-renewal. This
model is based on the observations that Lgl restricts aPKC localization to the
apical cortex of neuroblasts and that a reduction in aPKC blocks the lgl
self-renewal phenotype. To test this model,
worniu-Gal4 line was used to drive neuroblast-specific expression of
constitutively active aPKC or Lgl proteins, and an increase or
decrease in neuroblast numbers was assayed. Neuroblast-specific expression of aPKC targeted
to the plasma membrane with a CAAX prenylation motif
(UAS-aPKCCAAXWT)
resulted in ectopic cortical aPKC localization, loss of
cortical Miranda, and a large increase in the number
of neuroblasts. These effects were not observed after overexpression of wild-type
aPKC or a membrane-targeted kinase-dead aPKC
(UAS-aPKCCAAXKD).
Expression of a constitutively active aPKC
(UAS-aPKCDeltaN) that was predominantly
cytoplasmic gave only a slight increase in
neuroblast number, showing that cortical
localization of aPKC is essential to generate ectopic neuroblasts. By contrast,
neuroblast-specific expression of a constitutively active Lgl protein (Lgl3A)
resulted in the expected uniform cortical localization of Miranda, but no change in
neuroblast numbers. Combined overexpression of both
Lgl3A and aPKCCAAXWT, however, resulted in strong suppression of the
aPKCCAAXWT ectopic neuroblast phenotype,
even though Lgl3A alone had no effect on neuroblast
numbers, consistent with Lgl inhibiting aPKC function either directly or
through its downstream effectors. Thus, neuroblast-specific overexpression of
aPKC can expand the neuroblast population (most probably by promoting symmetric
neuroblast/neuroblast cell divisions) without eliminating the ability of these
neuroblasts to undergo asymmetric neuroblast/GMC divisions to generate
differentiating progeny. It is concluded that aPKC is sufficient to promote
neuroblast self-renewal, Lgl can inhibit aPKC function, and membrane-targeting
and kinase activity are essential for aPKC function (Lee, 2005).
This study has established
Drosophila larval neuroblasts as a model system for studying self-renewal
versus differentiation. A simple model is proposed in which Pins anchors
aPKC apically and Lgl inhibits aPKC localization basally, thereby restricting
aPKC to the apical cortex where it promotes neuroblast self-renewal.
In addition, aPKC can phosphorylate and directly
inhibit Lgl function, which together with the current
data provides evidence for mutual inhibition between Lgl and aPKC in
neuroblasts, similar to the mutual inhibition seen between these two proteins in
epithelia. Mutual inhibition between aPKC and Lgl would result in stabilization of apical
aPKC localization and more reliable partitioning of aPKC into the neuroblast
during mitosis. In pins mutants, aPKC is delocalized and nonfunctional
owing to Lgl activity, thereby reducing self-renewal; in lgl mutants,
aPKC shows weak ectopic cortical localization that increases self-renewal, and
in lgl pins double mutants, aPKC is both delocalized and fully active:
thus all neuroblasts undergo symmetric self-renewal.
Although the targets of aPKC involved in self-renewal are unknown, aPKC
may directly phosphorylate and inactivate GMC determinants,
and/or phosphorylate and activate neuroblast-specific
proteins. Notably, lgl1 mutant mice have neural progenitor
hypertrophy and knockdown of a pins
mammalian homologue (AGS3) leads to depletion of neural progenitors:
phenotypes that are very similar to those described
in this study. In the future, it will be important to determine the role of aPKC in mammalian neural progenitor self-renewal and to identify the aPKC-regulated
phosphoproteins that regulate neuroblast self-renewal in Drosophila (Lee, 2005).
By acting as a dynamic link between adjacent cells in a monolayer, adherens junctions (AJs) maintain the integrity of epithelial tissues while allowing for neighbor exchange. Although it is not currently understood how this combination of AJ stability and plasticity is achieved, junctionally associated actin filaments are likely to play a role, because actin-based structures have been implicated in AJ organization and in the regulation of junctional turnover. Through exploring the role of actin cytoskeletal regulators in the developing Drosophila notum, this study has identified a critical role for Cdc42-aPKC-Par6 in the maintenance of AJ organization. In this system, the loss or inhibition of Cdc42-aPKC-Par6 leads to junctional discontinuities, the formation of ectopic junctional structures, and defects in apical actin cytoskeletal organization. Affected cells also undergo progressive apical constriction and, frequently, delamination. Surprisingly, this Cdc42-aPKC-Par6-dependent regulation of junctional stability was found to be independent of several well-known targets of Cdc42-aPKC-Par6: Baz, Lgl, Rac, and SCAR. However, similar AJ defects are observed in wasp, arp2/3, and dynamin mutant cells, suggesting a requirement for actin-mediated endocytosis in the maintenance of junctional stability downstream of Cdc42. This was confirmed in endocytosis assays, which revealed a requirement for Cdc42, Arp2/3, and Dynamin for normal rates of E-cadherin internalization. In conclusion, by focusing on the molecular mechanisms required to maintain an epithelium, this analysis reveals a novel role for the epithelial polarity machinery, Cdc42-Par6-aPKC, in local AJ remodeling through the control of Arp2/3-dependent endocytosis (Georgiou, 2008).
This analysis of Cdc42 in Drosophila epithelial cells reveals a novel role for Cdc42, both in the regulation of the actin cytoskeleton and in AJ maintenance and stability. In this capacity, Cdc42 appears to function together with junctional Par6 and aPKC. It may appear surprising that Cdc42, aPKC, and Par6 act relatively independently of Baz (Par3) in the notum, given that Baz has been shown to act as a landmark to define the future site of E-cad localization and AJ formation in the embryo and to define neuroblast polarity upstream of Cdc42, aPKC, and Par6. However, the localization and function of Baz are distinct from those of Par6 and aPKC in many tissues. Moreover, the differences go further, because in the context of the notum, Cdc42, Par6, and aPKC are not required to maintain a polarized epithelium, and do not appear to function together with Lgl. A possible explanation for the reduced dependency of epithelial architecture in the notum on these molecules is the structural support gained from overlying apical cuticle and the basal lamina during its relatively slow development. Because of this, junctional defects in cdc42 mutant clones in this tissue are less likely to be an indirect consequence of a primary defect in apical-basal polarity. Moreover, the inherent stability of this epithelium made it possible to identify a novel role for the Cdc42-Par6-aPKC complex in the communication of polarity information across cell-cell junctions within the plane of the epithelium, something that may have been obscured by apical-basal polarity defects in studies in less stable epithelia (Georgiou, 2008).
Several lines of evidence suggest that the AJ defects seen in cdc42, aPKC, or par6 mutant cells result from defects in the internalization of junctional material, rather than from defects in the delivery of E-cadherin to the plasma membrane. First, junctional E-cadherin levels remain high in cdc42 mutant clones, enabling these cells to pull on surrounding cells. Second, in cdc42 clones, tubules and tubule-derived puncta containing E-cadherin, alpha-Catenin, and beta-Catenin remain continuous with the cell surface—as visualized with a probe for extracellular E-cadherin. This is the case, even though the vast majority of the rare, large E-cadherin puncta seen by light microscopy in the wild-type are found associated with an internalized fluid phase marker. Third, similar structures accumulate after a transient block in endocytic vesicle scission, resulting from the inhibition of Dynamin function. These data suggest that the primary defect induced by loss of Cdc42 is a defect in the endocytosis-mediated turnover of junctional material. Nevertheless, a detailed analysis of the molecular dynamics of E-cadherin-GFP at wild-type and mutant AJs is needed to quantify the contribution of Cdc42-mediated endocytosis to the normal rate of E-cadherin turnover (Georgiou, 2008).
How then do Cdc42, aPKC, and Par6 activate endocytosis to drive normal junctional turnover? Having identified a role for Cdc42 (aPKC and Par6) in apical actin organization, attention was focused on actin cytoskeletal regulators known to act downstream of Cdc42 in answering this question. Significantly, both WASp and the Arp2/3 complex (but not Rac or SCAR) were found to be required for the maintenance of a normal apical actin cytoskeleton and AJs. Because WASp is a well-established Cdc42 target, this suggested that the effect of Cdc42 on junctional endocytosis is mediated directly through WASp. The junctional phenotypes observed in WASp, however, appear weaker than those seen after the loss of Arp2/3, Cdc42, or Dynamin. This may be the result of protein perdurance. Alternatively, this observation may point to the existence of other proteins that act in parallel with WASp to stimulate Arp2/3-mediated vesicle scission downstream of Cdc42. In fact, a number of Cdc42 targets have been shown to affect actin nucleation and membrane tubulation. Nevertheless, the striking similarities between the AJ phenotypes induced by loss of Arp2/3, Dynamin, and Cdc42, together with recent data implicating Cdc42, Par6, and aPKC in the regulation of vesicle trafficking and endocytosis, provide strong evidence that the primary defect in each case is a block in actin-mediated endocytosis. On the basis of this analysis, it is suggested that the cdc42, par6, and apkc phenotype arises in the following way. First, a reduction in the activity of Cdc42-aPKC-Par6 on one side of an AJ translates into a reduction of Cdc42-aPKC-Par6 activity on the opposing side, resulting in a concomitant reduction in the activity of WASp and the Arp2/3 complex, leading to defects in Dynamin-dependent endocytosis along the entire cell-cell interface. The resulting failure to remove excess material from the ends of the AJ causes junctional spreading, as observed in electron micrographs. This leads to the formation of the discontinuous junctions, junctional extensions, and punctate surface structures visible in confocal images. Over time, this has the effect of destabilizing AJs, leading to the loss of apical material and, eventually, to cell delamination. In this view, Cdc42-Par6-aPKC regulate local Arp2/3-mediated endocytosis to maintain AJs in a state of dynamic equilibrium. Internalized junctional material can then be recycled back to the cell surface to engage in cell-cell adhesion in a well-regulated fashion. Importantly, this model predicts that the stability of AJs is intimately linked to their turnover -- a feature that makes AJs inherently plastic (Georgiou, 2008).
Polarity of many cell types is controlled by a protein complex consisting of Bazooka/PAR-3 (Baz), PAR-6 and atypical protein kinase C (aPKC). In Drosophila, the Baz-PAR-6-aPKC complex is required for the control of cell polarity in the follicular epithelium, in ectodermal epithelia and neuroblasts. aPKC is the main signaling component of this complex that functions by phosphorylating downstream targets, while the PDZ domain proteins Baz and PAR-6 control the subcellular localization and kinase activity of aPKC. The mutant phenotypes of an aPKC null allele were compared with those of four novel aPKC alleles harboring point mutations that abolish the kinase activity or the binding of aPKC to PAR-6. These point alleles retain full functionality in the control of follicle cell polarity, but produce strong loss-of-function phenotypes in embryonic epithelia and neuroblasts. These data, combined with molecular dynamics simulations, show that the kinase activity of aPKC and its ability to bind PAR-6 are required only for a subset of its functions during development, revealing tissue-specific differences in the way that aPKC controls cell polarity (Kim, 2009).
The Drosophila anterior-posterior (AP) axis is determined by the polarisation of the stage 9 oocyte and the subsequent localisation of bicoid and oskar mRNAs to opposite poles of the cell. Oocyte polarity has been proposed to depend on the same PAR proteins that generate AP polarity in C. elegans, with a complex of Bazooka (Baz; Par-3), Par-6 and aPKC marking the anterior and lateral cortex, and Par-1 defining the posterior. The function of the Baz complex in oocyte polarity has remained unclear, however, because although baz-null mutants block oocyte determination, egg chambers that escape this early arrest usually develop normal polarity at stage 9. This study characterised a baz allele that produces a penetrant polarity phenotype at stage 9 without affecting oocyte determination, demonstrating that Baz is essential for axis formation. The dynamics of Baz, Par-6 and Par-1 localisation in the oocyte indicate that the axis is not polarised by a cortical contraction as in C. elegans, and instead suggest that repolarisation of the oocyte is triggered by posterior inactivation of aPKC or activation of Par-1. This initial asymmetry is then reinforced by mutual inhibition between the anterior Baz complex and posterior Par-1 and Lgl. Finally, it was shown that mutation of the aPKC phosphorylation site in Par-1 results in the uniform cortical localisation of Par-1 and the loss of cortical microtubules. Since non-phosphorylatable Par-1 is epistatic to uninhibitable Baz, Par-1 seems to function downstream of the other PAR proteins to polarize the oocyte microtubule cytoskeleton (Doerflinger, 2010).
The baz358-12 allele causes a fully penetrant defect in the localisation of bcd and osk mRNAs and in the positioning of the oocyte nucleus and gurken mRNA, providing the first demonstration that Baz is required for the polarisation of the Drosophila AP and dorsal-ventral axes. This raises the question of why baz-null mutant germline clones that escape the block in early oogenesis sometimes develop into eggs with normal polarity. Although it is formally possible that Baz is not absolutely essential for oocyte polarity and that the baz358-12 allele has a dominant-negative effect, this seems very unlikely. First, baz358-12 behaves like a typical hypomorphic mutation as it is recessive and fails to complement the lethality of baz-null alleles. Second, nearly half of the escapers from baz-null germline clones show similar polarity defects to baz358-12 at stage 9, indicating that this is a loss-of-function phenotype. Thus, it seems more likely that whatever allows a few of the null germline clones to escape the early-arrest phenotype also allows some of them to escape the polarity defect at stage 9. For example, other polarity pathways might be activated in baz-null mutant germaria that can partially compensate for the loss of Baz in both oocyte determination and axis formation (Doerflinger, 2010).
The observation that baz358-12 does not cause any defects in the initial polarisation of the oocyte, although it is essential for the AP polarisation at stage 9, indicates that there must be some differences in the functions of Baz at each stage. During early oogenesis, Baz localises in a ring around each ring canal at the anterior of the oocyte and shows perfect colocalisation with DE-cadherin (Shotgun - FlyBase) and Armadillo. Since the PDZ domains of Baz have been shown to interact with Armadillo, it might be recruited to the anterior rings through this interaction, which should still occur normally in the baz358-12 mutant. By contrast, the truncated Baz protein does not localise to the cortex of the oocyte at stages 7-9, indicating that the C-terminal region is necessary for its cortical recruitment at this stage. The only identified domain in this region is CR3, which binds to the kinase domain of aPKC. However, a point mutation in CR3 that disrupts its interaction with aPKC has no effect on the cortical localisation of Baz at stage 9. There must therefore be another domain in the C-terminal region of Baz that is required for its recruitment to the oocyte cortex (Doerflinger, 2010).
Another important difference between the initial polarisation of the oocyte and the repolarisation at mid-oogenesis is the relationship between the PAR proteins. During early oogenesis, the localisation of Baz is unchanged by loss of Par-1 and vice versa. By contrast, Baz and Par-1 show mutually exclusive localisations at stage 9, with Par-1 spreading around the lateral cortex in baz mutants, and Baz and Par-6 localising to the posterior in par-1 mutants. Baz is required to recruit Par-6 to the cortex in mid-oogenesis, as Par-6 disappears from the anterior cortex in baz358-12 clones and localises to the posterior with BazS151A S1085A-GFP. Thus, Baz, Par-6 and presumably also aPKC form a complex in the stage 9 oocyte, making the arrangement of PAR proteins much more similar to that in the C. elegans zygote, with Baz (PAR-3), Par-6 and aPKC defining the anterior and lateral cortex and Par-1 the posterior. As in C. elegans, these complementary localisations are also maintained by mutual antagonism between the anterior and posterior PAR proteins. It has been shown that Par-1 phosphorylates Baz to exclude it from the posterior. This study shows that mutation of the conserved aPKC site in the Par-1 linker region leads to the mislocalisation of Par-1 around the anterior and lateral cortex, strongly suggesting that aPKC phosphorylates this site to restrict Par-1 to the posterior (Doerflinger, 2010).
Although the final pattern of PAR proteins in the stage 9 Drosophila oocyte is similar to that in the C. elegans zygote, this pattern develops over a much longer period of time and in a different way. Baz-GFP is enriched at the posterior of the oocyte at the beginning of stage 7 and gradually spreads anteriorly during the succeeding 12 hours, before finally disappearing from the posterior at stage 9. Since Par-1 appears at the posterior early in stage 7, Baz and Par-1 overlap at the posterior for some considerable time. By contrast, Par-6-Cherry starts to disappear from the posterior during stage 7, and already shows a complementary pattern to Par-1 at stage 8. This raises the question of why Par-6, which is recruited to the cortex by Baz, disappears more rapidly from the posterior. Although this might mean that they are excluded by different mechanisms, both Par-6 and Baz localise to the posterior in par-1 mutants and in BazS151A S1085A-GFP-expressing oocytes, indicating that their exclusion depends on the phosphorylation of Baz by Par-1. Thus, Par-1 phosphorylation might first release Par-6 from Baz, and then more gradually displace Baz from the cortex. The phosphorylation of serine 1085 of Baz by Par-1 disrupts the interaction of Baz with aPKC and this might be sufficient to release the Par-6-aPKC complex. However, Par-6 also binds directly to the PDZ domains of Baz, and the phosphorylation of serine 1085 alone would not be expected to interfere with this interaction. Thus, Par-1 might also act in some other way to release Par-6, perhaps by promoting the posterior recruitment of Lgl, as the latter is known to inhibit the interaction of Par-6-aPKC with Baz in neuroblasts (Doerflinger, 2010).
The gradual evolution of PAR protein localisation during stages 7-9 argues against the idea that the oocyte is polarised by a cortical contraction, as in C. elegans, and no any evidence has been observed for cortical movements of the actin cytoskeleton. This raises the question of how this asymmetry arises. Two possible scenarios for how the polarising signal from the posterior follicle cells triggers PAR protein asymmetry can be invisioned. First, the initial cue could remove or inactivate aPKC and Par-6 at the posterior, which would then allow Par-1 to localise there because aPKC is no longer present or able to exclude it. Although aPKC can be inhibited at the posterior by Lgl, this seems unlikely to provide the cue because Lgl localises to the posterior after Par-1 and is not essential for oocyte polarity. Alternatively, the initial asymmetry could be generated by the posterior recruitment and activation of Par-1. Work in mammals has shown that LKB1 (STK11) phosphorylates the activation loop of PAR-1 (MARK2) to turn on its kinase activity, and this is likely to be case in Drosophila as well, as lkb1 mutants exhibit a very similar phenotype to par-1 mutants. LKB1 activity is regulated by protein kinase A (PKA), which is required for the transduction of the polarising follicle cell signal in the oocyte. Thus, it is possible that the initial asymmetry is generated by a kinase cascade at the posterior of the oocyte, consisting of PKA, which activates LKB1, which activates Par-1 (Doerflinger, 2010).
Once the PAR polarity has been established, it must somehow polarise the oocyte microtubule cytoskeleton to direct the localisation of bcd and osk mRNAs. The epistasis experiment suggests that Par-1 provides the primary output from the PAR system, as uniformly distributed Par-1 makes the whole cortex behave like the posterior cortex regardless of whether Baz is also uniformly distributed or not. Based on the par-1 loss- and gain-of-function phenotypes in the oocyte and follicle cells, Par-1 might act to stabilise microtubule plus ends at the cortex and to inhibit the nucleation or anchoring of microtubule minus ends (Doerflinger, 2010).
One key remaining question is the identity of the Par-1 substrates that mediate its effect on microtubule organisation. In addition to Baz, Par-1 has also been shown to phosphorylate Exuperantia and Ensconsin in the oocyte to regulate bcd mRNA localisation and the activity of Kinesin. However, neither of these targets can account for the dramatic effects of Par-1 on microtubule organisation. It has recently been claimed that Par-1 regulates the oocyte microtubule cytoskeleton by phosphorylating the microtubule-stabilising protein Tau, thereby destabilising the microtubules at the posterior of the oocyte. This conclusion was based on the observation that germline clones of tauDf(3R)MR22 produce a partially penetrant defect in the anchoring of the oocyte nucleus. However, the tauDf(3R)MR22 mutation is a 65 kb deletion that removes eight other genes as well as tau, and the phenotype could therefore be due to the loss of one of these other loci. More importantly, tau can be specifically removed without deleting any other genes by generating heterozygotes for two overlapping deficiencies, and these tau-null flies are homozygous viable and fertile and develop normally polarised oocytes. Thus, it seems highly unlikely that Tau is a relevant substrate for Par-1 in the polarisation of the oocyte. A full understanding of oocyte polarity will therefore depend on the identification of the Par-1 targets that control microtubule nucleation, anchoring and stability (Doerflinger, 2010).
Epithelial cells mostly orient the spindle along the plane of the epithelium (planar orientation) for mitosis to produce two identical daughter cells. The correct orientation of the spindle relies on the interaction between cortical polarity components and astral microtubules. Recent studies in mammalian tissue culture cells suggest that the apically localised atypical protein kinase C (aPKC) is important for the planar orientation of the mitotic spindle in dividing epithelial cells. Yet, in chicken neuroepithelial cells, aPKC is not required in vivo for spindle orientation, and it has been proposed that the polarization cues vary between different epithelial cell types and/or developmental processes. In order to investigate whether Drosophila aPKC is required for spindle orientation during symmetric division of epithelial cells, advantage was taken of a previously isolated temperature-sensitive allele of aPKC. This study showed that Drosophila aPKC is required in vivo for spindle planar orientation and apical exclusion of Pins (Raps). This suggests that the cortical cues necessary for spindle orientation are not only conserved between Drosophila and mammalian cells, but are also similar to those required for spindle apicobasal orientation during asymmetric cell division (Guilgur, 2012).
This work characterised a novel hypomorphic allele of aPKC (apkcts). This allele contains a point mutation at a highly conserved phenylalanine within the protein kinase domain and shows reduced levels of in vitro kinase activity without any significant reduction of aPKC protein levels or any detectable change in its apical localisation. At restrictive temperature, larval wing discs mutant for apkcts showed significant levels of cell extrusion and apoptosis, yet their epithelial architecture was normal. Since loss of aPKC activity was commonly associated with a complete collapse of apicobasal polarity, abnormal AJs, apical constriction and cell extrusion, it was hypothesized that the apkcts allele specifically failed to complement a function of aPKC that was not directly related to apicobasal polarity and/or the formation and maintenance of junctional components. Previously, it was suggested that different epithelial tissues can have differential requirements for aPKC activity during Drosophila development (Kim, 2009). The current data expands this hypothesis and suggests that, even within the same epithelial cell, aPKC is likely to have distinct thresholds of activity to correctly regulate different cellular processes (Guilgur, 2012).
Despite the fact that zygotic mutants of apkcts showed significant temperature-sensitive phenotypes, no decrease in aPKCts in vitro kinase activity was detected at the restrictive temperature when compared with the permissive temperature. It was hypothesized that the observed phenotypes were possibly due to an overhaul property of the epithelial cell whereby, at restrictive temperature, there were higher requirements for aPKC kinase activity. Alternatively, it is also possible that in vivo the aPKCts mutant protein behaved as a temperature-sensitive kinase, and in vitro kinase assay failed to detect such behaviour (Guilgur, 2012).
Consistent with the mammalian tissue culture work (Durgan, 2011; Hao, 2010; Qin, 2010), this analysis of apkcts Drosophila wing discs revealed a requirement of aPKC for spindle orientation during symmetric mitosis. Complete randomization of spindle positioning would potentially lead to spindle misorientation in 50% of epithelial cell divisions. In apkcts wing discs, there was a significant decrease in the proportion of dividing epithelial cells with the correct planar orientation of the spindle, which suggested an increase in the randomization of spindle orientation. Although it is unclear when a departure from planarity should be considered functionally significant, an increase in the randomization of spindle orientation is likely to be associated with an increase in the probability of cell extrusion and apoptosis, as misorientation of the mitotic spindle along the apicobasal axis could potentially cause rotation of the mother cell cleavage plane, a reduction of the apical and sub-apical domains of the basally localised daughter cell, and its subsequent extrusion and apoptosis. It was hypothesized that spindle misorientation was likely to be one of the main causes for the cell extrusion and apoptosis phenotypes observed in apkcts wing discs at restrictive temperature. Consistently, both the apoptosis and spindle orientation defects observed in the apkcts wing discs were similarly temperature sensitive (Guilgur, 2012).
The highly conserved Pins/LGN protein is important for spindle orientation as it links the cell cortex of the dividing cell with the astral microtubules of the mitotic spindle. In mammalian epithelial tissue culture cells, LGN needs to be correctly excluded from the apical cortex of the dividing cells to ensure planar orientation of the spindle. It has been proposed that phosphorylation of LGN/Pins by apical aPKC inhibits its binding to apically anchored Gai, resulting in its exclusion from the apical cortical region of the dividing cell (Hao, 2010). Consistent with this model, this study observed that at restrictive temperature larval wing discs mutant for apkcts showed significant mislocalisation of Pins in a subset of mitotic cells, which suggested that Drosophila aPKC is also required in vivo to exclude Pins from the apical domain of dividing epithelial cells (Guilgur, 2012).
Further supporting the hypothesis that apical mislocalisation of Pins is one of the main causes of the phenotypes observed in apkcts mutants, it was observed that a strong hypomorphic allele of pins (pins193) could behave as a dominant suppressor of apkcts wing disc apoptosis at the semi-permissive temperature. Moreover, and suggesting that the aPKC-dependent regulation of Pins is not restricted to the wing disc epithelia, it was also observed that pins193 behaved as a dominant suppressor of the abdomen dorsal closure midline defects observed in adult flies mutant for apkcts (Guilgur, 2012).
Altogether, these data suggest that aPKC is an important in vivo regulator of spindle orientation during the symmetric division of epithelial cells, with the aPKC-dependent phosphorylation of Pins resulting in its exclusion from the apical domain of dividing cells, a role that is most likely conserved between Drosophila and mammalian cells. Yet, and similarly to chicken neuroepithelial cells (Peyre, 2011), it is nevertheless possible that other Drosophila epithelial tissues use distinct (or possibly redundant) polarization cues [e.g. integrin signalling to orient the spindle during symmetric mitosis (Guilgur, 2012).
The observations indicate that identical aPKC-dependent cortical cues are likely to be used to orient the mitotic spindle during symmetric and asymmetric mitosis. Similarly, Pins is required for both types of cell division, which suggests that modulation of the aPKC-dependent apical exclusion of Pins is likely to play a key role in the means by which similar cortical cues can be differentially interpreted in epithelial cells (spindle planar orientation) and neuroblasts (spindle apicobasal orientation) (Guilgur, 2012).
Polarity of the Drosophila oocyte is essential for correct development of the egg and future embryo. The Par proteins Par-6, aPKC and Bazooka are needed to maintain oocyte polarity and localize to specific domains early in oocyte development. To date, no upstream regulator or mechanism for localization of the Par proteins in the oocyte has been identified. This study analyzed the role of the small GTPase Cdc42 in oocyte polarity. Cdc42 was shown to be required to maintain oocyte fate, which it achieves by mediating localization of Par proteins at distinct sites within this cell. Cdc42 localization itself is polarized to the anterolateral cortex of the oocyte, and Cdc42 is needed for maintenance of oocyte polarity throughout oogenesis. The data show that Cdc42 ensures the integrity of the oocyte actin network and that disruption of this network with Latrunculin A phenocopies loss of Cdc42 or Par protein function in early stages of oogenesis. Finally, it was showm that Cdc42 and Par proteins, as well as Cdc42/Par and Arp3, interact in the context of oocyte polarity, and that loss of Par proteins reciprocally affects Cdc42 localization and the actin network. These results reveal a mutual dependence between Par proteins and Cdc42 for their localization, regulation of the actin cytoskeleton and, consequently, for the establishment of oocyte polarity. This most likely allows for the robustness in symmetry breaking in the cell (Leibfried, 2013).
The findings show that Cdc42 is required for oocyte polarity throughout oogenesis. The following findings were made: (1) Cdc42 localizes to the anterolateral cortex of the young oocyte; (2) Cdc42 interacts with Par proteins in the germline in vivo; (3) mutants for Cdc42, aPKC or Baz display a disrupted actin cytoskeleton at the anterolateral cortex; and (4) disrupting the actin cytoskeleton with Latrunculin A results in loss of anterior-to-posterior movement of the oocyte-specific protein Orb, phenocopying loss of Cdc42 or the Par proteins. Thus, the cortical actin cytoskeleton is crucial for the establishment of oocyte polarity (Leibfried, 2013).
This is in line with previous observations linking the actin cytoskeleton and Par proteins in the generation of cell polarity. Loss of Baz results in an increase in actin protrusions in Drosophila epithelia and a decrease in actin at synapses. In C. elegans, active CDC-42 localizes to the anterior during the polarity maintenance phase, when it is important for PAR-6 localization, and the anterior actin cap is depleted in par-3 mutants during polarity establishment. Similar to the current observations, actin depolymerization does not affect Par protein localization in C. elegans. By contrast, drug-induced actin depolymerization has been shown to disrupt Baz apical localization during cellularization and to interfere with its cortical association during gastrulation in Drosophila (Leibfried, 2013).
Although the molecular relationship between Par proteins and actin has not been clearly delineated, in mammals Par-3 (Pard3) associates with actin regulators, including the RacGEF Tiam1 and LIM kinase 2. In the current study, it was shown that Cdc42 localization depends on the Par complex and that Cdc42, aPKC, Baz and Par-6 interact in vivo in biochemical or genetic assays. This interaction is required for oocyte polarity. Par-6 interacts biochemically with Cdc42 and Baz via its semi-CRIB and PDZ domains and via its PB1 domain with the PB1 domain of aPKC. Baz interacts biochemically with the kinase domain of aPKC. Indeed, a quaternary complex of Myc-Cdc42, HA-Par-6b, PKCτ/λ and Par-3 can be isolated from transfected COS-7 cells. In Drosophila, a Baz mutant lacking its aPKC-interaction domain supports early oogenesis and an aPKC mutant that cannot bind Par-6 also develops late egg chambers. Together, these results and the current data indicate that interaction of Cdc42, Par-6, aPKC and Baz is required for their correct function in the germline, and that the binding of aPKC to either Par-6 or to Baz is sufficient to ensure this interaction, highlighting the role of all three Par proteins in actin regulation via their interaction with Cdc42. This quaternary relationship seems important for the regulation of polarity establishment, whereas studies in mature epithelial cells have delineated separate functions of Par-6/aPKC/Cdc42 and Baz for polarity maintenance (Leibfried, 2013).
Early oocyte polarity and its maintenance were previously linked to the microtubule network. Microtubules play an important role in early oogenesis, as their disruption with Colchicine leads to a 16-nurse-cell phenotype. Indeed, oocyte specification depends on the accumulation of the oocyte-specific protein BicD, which is a component of the microtubule-related dynactin complex (Leibfried, 2013).
The results point to a sequential involvement of actin and microtubules in polarizing the oocyte: in the early stages, after oocyte specification, the Par proteins together with Cdc42 establish cortical domains and a pronounced cortical actin cytoskeleton. The interdependence of these proteins for their localization persists during oogenesis, allowing for robustness of symmetry breaking. At later stages, knockdown of Cdc42 results in reduced amounts of Baz and Par-1 at the anterior and posterior of the oocyte, respectively. As Par-1 is required for microtubule organization, this most likely leads to the observed mislocalization of axis determinants. Similarly, disrupting the actin cytoskeleton with drugs or by knockdown of actin-binding proteins has been shown to result in bundling of microtubules and premature ooplasmic streaming, leading to loss of oocyte polarity. Hence, microtubules act in oocyte specification and late polarity events, whereas Cdc42 and actin dominate in the establishment and maintenance of polarity in the developing oocyte (Leibfried, 2013).
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date revised: 20 December 2020
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