bazooka
PAR-3, a homolog of Drosophila Bazooka, required for asymmetric division in C. elegans Polarized asymmetric divisions play important roles in the development of plants and animals. The first two embryonic
cleavages of Caenorhabditis elegans provide an opportunity to study the mechanisms controlling polarized asymmetric
divisions. The first cleavage is unequal, producing daughters with different sizes and fates. The daughter blastomeres
divide with different orientations at the second cleavage; the anterior blastomere divides equally across the long axis of
the egg, whereas the posterior blastomere divides unequally along the long axis. The results of an
analysis of the genes par-2 and par-3 are reported with respect to their contribution to the polarity of these divisions. Strong
loss-of-function mutations in both genes lead to an equal first cleavage and an altered second cleavage. Interestingly,
the mutations exhibit striking gene-specific differences at the second cleavage. The par-2 mutations lead to transverse
spindle orientations in both blastomeres, whereas par-3 mutations lead to longitudinal spindle orientations in both
blastomeres. The spindle orientation defects correlate with defects in centrosome movements during both the first and
the second cell cycle. Temperature shift experiments with a par-2 temperature sensitive mutant indicate that the par-2(+) activity is not required
after the two-cell stage. Analysis of double mutants shows that par-3 is epistatic to par-2. A model is proposed wherein
par-2(+) and par-3(+) act in concert during the first cell cycle to affect asymmetric modification of the cytoskeleton.
This polar modification leads to different behaviors for the centrosomes in the anterior and posterior and leads ultimately to
blastomere-specific spindle orientations at the second cleavage (Cheng, 1995).
The par-3 gene is required for establishing polarity in early C. elegans embryos. Embryos from par-3 homozygous
mothers show defects in segregation of cytoplasmic determinants and in positioning of the early cleavage spindles. The PAR-3 protein is asymmetrically distributed at the periphery of the zygote and asymmetrically
dividing blastomeres of the germline lineage. The PAR-3 distribution is roughly the reciprocal of PAR-1, another
protein required for establishing embryonic polarity in C. elegans. Analysis of the distribution of PAR-3 and PAR-1 in
other par mutants reveals that par-2 activity is required for proper localization of PAR-3 and that PAR-3 is required for
proper localization of PAR-1. In addition, the distribution of the PAR-3 protein correlates with differences in cleavage
spindle orientation and suggests a mechanism by which PAR-3 contributes to control of cleavage pattern (Etemad-Moghadam, 1995).
The generation of asymmetry in the one-cell embryo of Caenorhabditis elegans is necessary to establish the
anterior-posterior axis and to ensure the proper identity of early blastomeres. Maternal-effect lethal mutations with a
partitioning defective phenotype (par) have identified several genes involved in this process. A new
gene, par-6 (see Drosophila par-6), has been identified that acts in conjunction with other par genes to properly localize cytoplasmic components in the early
embryo. The early phenotypes of par-6 embryos include the generation of equal-sized blastomeres, improper
localization of P granules and SKN-1 protein, and abnormal second division cleavage patterns. Overall, this phenotype
is very similar to that caused by mutations in a previously described gene, par-3. The probable basis for this similarity
is revealed by genetic and immunolocalization results; par-6 acts through par-3 by localizing or maintaining the
PAR-3 protein at the cell periphery. In addition, loss-of-function par-6 mutations are found to act as dominant bypass
suppressors of loss-of-function mutations in par-2 (Watts, 1996).
Daughter cells with distinct fates can arise through intrinsically asymmetrical divisions. Before such divisions, factors
crucial for determining cell fates become asymmetrically localized in the mother cell. In Caenorhabditis elegans, PAR
proteins are required for the early asymmetrical divisions that establish embryonic polarity, and are asymmetrically
localized in early blastomeres, although the mechanism of their distribution is not known. Nonmuscle myosin II heavy chain (designated NMY-2) has been identified in C. elegans by means of its interaction with
the PAR-1 protein, a putative Ser/Thr protein kinase. Injections of nmy-2 antisense RNA into ovaries of
adult worms causes embryonic partitioning defects and leads to mislocalization of PAR proteins. It is therefore concluded
the NMY-2 is required for establishing cellular polarity in C. elegans embryos (Guo, 1996).
The par genes participate in the process of establishing cellular asymmetries during the first cell cycle of Caenorhabditis
elegans development. The par-2 gene is required for the unequal first cleavage and for asymmetries in cell cycle length
and spindle orientation in the two resulting daughter cells. The PAR-2 protein is present in adult
gonads and early embryos. In gonads, the protein is uniformly distributed at the cell cortex, and this subcellular
localization depends on microfilaments. In the one-cell embryo, PAR-2 is localized to the posterior cortex and is
partitioned into the posterior daughter, P1, at the first cleavage. PAR-2 exhibits a similar asymmetric cortical
localization in P1, P2, and P3, the asymmetrically dividing blastomeres of germ line lineage. This distribution in
embryos is very similar to that of PAR-1 protein. By analyzing the distribution of the PAR-2 protein in various par
mutant backgrounds, proper asymmetric distribution of PAR-2 depends on par-3 activity but not upon
par-1 or par-4 (see Drosophila Lkb1). par-2 activity is required for proper cortical localization of PAR-1 and this effect requires wild-type
par-3 gene activity. Although par-2 activity is not required for posterior localization of P granules at
the one-cell stage, it is required for proper cortical association of P granules in P1 (Boyd, 1996).
The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the
posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm,
while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from
blastomere-specific rotations of the nuclear-centrosome complex at prophase. Mutations in the
maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these
divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant
embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior
cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form
transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage.
The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early
let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of
actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is
abnormal but is not reversed in let-99 mutant embryos. Based on these data, it has been concluded that let-99(+) is required for
the proper orientation of spindles after the establishment of polarity, and it is postulated that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton (Rose, 1998).
Asymmetric cell divisions, critically important to specify cell types in the development of multicellular organisms, require polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus. In Caenorhabditis elegans, the maternally expressed protein, PAR-3, is localized to one pole of asymmetrically dividing blastomeres and is required for these asymmetric divisions. An atypical protein kinase C (PKC-3) is essential for proper asymmetric cell divisions and co-localizes with PAR-3. The predicted amino acid sequence of PKC-3 shows extensive similarity to atypical mammalian PKC subfamily members PKCzeta and PKClambda. The amino terminal half contains one cysteine-zinc finger motif and lacks a potential Ca 2+ binding domain conserved in the conventional PKC family members. These structural features characterize atypical PKCs, which are dependent on neither Ca 2+ nor diacylglycerol for their activation. The carboxy-terminal half of the predicted PKC-3 protein exhibits about 70% similarity to the kinase domain of atypical PKCs. A separate study (Wu, 1998) shows that purified PKC-3 protein requires phosphatidylserine but is independent of Ca 2+ and diacylglycerol for its activation, two characteristics of aPKCs (Tabuse, 1998).
The par genes are required to establish polarity in the Caenorhabditis elegans embryo. Mutations in two of these genes,
par-3 and par-6, exhibit similar phenotypes. A third gene, pkc-3, gives a similar phenotype when the protein is
depleted by RNA interference. PAR-3 and PKC-3 protein are colocalized to the anterior periphery of asymmetrically
dividing cells of the germline lineage. The peripheral localizations of both proteins depend on the activity of
par-6. The molecular cloning of par-6 is reported and the immunolocalization of PAR-6 protein. par-6 encodes a PDZ-domain-containing protein and has homologues in mammals and flies. Moreover, PAR-6 colocalizes with PAR-3 and par-3 and pkc-3 activity are required for the peripheral localization of
PAR-6. The localization of both PAR-3 and PAR-6 proteins is affected identically by mutations in the par-2, par-4 and
par-5 genes. The co-dependence of PAR-3, PAR-6 and PKC-3 for peripheral localization and the overlap in their
distributions lead to a proposal that they act in a protein complex (Hung, 1999).
Polarization of the one-cell C. elegans embryo establishes the animal's anterior-posterior (a-p) axis. Reduction-of-function anaphase-promoting complex (APC) mutations have been identified that eliminate a-p polarity. The APC activator cdc20 (Drosophila homolog: Fizzy) is required for polarity. The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there. The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome. Depletion of the protease separin (Drosophila homolog: Separin), a downstream target of the APC, causes similar pronuclear and a-p polarity defects. It is proposed that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation (Rappleye, 2001).
To better understand how factors such as PAR proteins that are strictly involved in establishing polarity interact with the basic cellular machinery, pod, or polarity and osmotic defective, genes have been identified that are required both for a-p polarity and more general cellular functions. The first gene in this new class, pod-1, encodes an actin binding protein asymmetrically localized at the anterior cortex of P0. The second, pod-2, functions in the same pathway as pod-1 and was identified in a screen for cold-sensitive mutants. Mutation of either pod-1 or pod-2 causes loss of a-p polarity in ~50% of one-cell embryos. Mutations in
pod-1 and pod-2 also give rise to osmotically sensitive embryos, suggesting that they affect more general cell functions (e.g., membrane trafficking) required both for the production of the secreted eggshell that protects the embryo and for a-p axis formation (Rappleye, 2001).
Five additional pod loci have been identified and characterized. In these new pod mutants, complete disruption of a-p polarity, as judged by symmetric cleavage and mislocalization of polarized proteins, occurs in nearly all one-cell embryos. These Pod mutant alleles represent partial
loss-of-function mutations in five components of the anaphase-promoting complex; the APC functions around the time of meiosis to establish polarity. The APC activator, cdc20, and the downstream protease separin are required for a-p polarity. The loss of APC and separin leads to a failure of the paternal pronucleus/centrosome to associate with the actin-rich cortex and, therefore, a failure in the transduction of the presumed polarity signal from the centrosome to the cortex. It is concluded that the APC can function to regulate metazoan axis formation (Rappleye, 2001).
In embryos mutant for the Pod alleles of the APC, PAR-3 is uniformly distributed around the cortex and PAR-2 is present only as cytoplasmic foci. In contrast, in embryos mutant for par-3 alone, PAR-2 (there is no Drosophila homolog) is still found at the cortex, albeit uniformly. Given that par-2 and par-3 are thought to act antagonistically to each other, whether cortical PAR-3 is excluding PAR-2 from the cortex in Pod/APC mutant embryos was tested. After depleting PAR-3 in Pod/APC mutant embryos, PAR-2 returns to the cortex in a uniform, nonpolarized distribution. Thus, in an APC mutant embryo, uniform PAR-3 excludes PAR-2 from the cortex. Since in wild-type PAR-3 is initially symmetric around the cortex but becomes asymmetric by meiosis II, it is concluded that the APC normally functions to restrict PAR-3 to the anterior, allowing cortical association of PAR-2 at the posterior (Rappleye, 2001).
How might the APC limit PAR-3 to the anterior? Microtubule interactions with the cortex and a functional centrosome appear to play important roles in dictating the localization of PAR-2 at the cortex. The centrosome is donated by the sperm and is attached to the paternal pronucleus during pronuclear stage one-cell embryos. The behavior of the paternal pronucleus/centrosome complex has been characterized. It becomes discernible toward the completion of meiosis II, sometimes in the very posterior or sometimes along a lateral edge near the posterior. Before the end of meiosis, the paternal pronucleus/centrosome becomes tightly associated with the cortex such that no cytoplasmic granules are seen between it and the embryonic cortex. Regardless of its initial position, the paternal pronucleus and its centrosome move to the very posterior cortex just before or within 21 s after the end of meiosis. The pronucleus remains attached at the cortex for 4 min 23 ± 52 s (relative to meiosis completion), after which it dissociates and meets the maternal pronucleus in the posterior cytoplasm. Subsequently, the mitotic spindle forms and becomes posteriorly displaced, leading to asymmetric cleavage (Rappleye, 2001).
The behavior of the paternal pronucleus/centrosome complex is markedly altered in pod-3(or319) mutant embryos. After appearing, the paternal pronucleus neither tightly associates with the cortex nor moves to the very posterior. Following pronuclear migration, it meets the maternal pronucleus in the center of the embryo, after which the embryo divides symmetrically. Thus, in embryos reduced for APC function, the characteristic close contact between the paternal pronucleus/centrosome and the embryonic cortex is absent during the early stages of polarity establishment. It is speculated that a close association of the sperm pronucleus and its centrosome with the cortex is required for specifying a posterior pole and thus required for excluding PAR-3. This model predicts that PAR-3, though affected by the centrosome-cortical interaction, should not be required for it. Indeed, in par-3 mutant embryos the paternal pronucleus behaves as in wild-type. It is tightly associated with the cortex on average 60 ± 48 s before the maternal pronucleus reformed, and full posteriorization occurred before or within 35 s of the completion of meiosis II. The paternal pronucleus remains there on average 4 min 21 ± 28 s prior to dissociating and meeting the maternal pronucleus. These par-3 mutant embryos subsequently divided symmetrically (Rappleye, 2001).
How does APCcdc20 function in the generation of polarity? The following model has been suggested: during meiosis II, perhaps at the metaphase to anaphase transition when the APC is known to act, APCcdc20 activates separin in the one-cell embryo. Separin in turn promotes tight association between the paternal pronucleus/centrosome with the embryonic actin-rich cortex and also directs pronuclear/centrosome movement to the very posterior of the embryo. The closely positioned centrosome is able to interact with the posterior cortex and displace the PAR-3/PAR-6/PKC-3 complex, enabling PAR-2 to bind there and establishing anterior versus posterior cortical domains. Indeed, PAR-3 and PAR-6 become restricted anteriorly at roughly the same time as a tight association between the paternal pronucleus and the cortex is observed. Such a requirement for a tightly positioned interaction potentially explains why drugs that inhibit microtubules fail to cause polarity defects in C. elegans -- short-range interactions might not be affected by these drugs. Interestingly, the separin pathway in yeast is similarly required to maintain spindle pole bodies close the cortex (Rappleye, 2001).
Gastrulation in C. elegans embryos involves formation of a blastocoel and the ingression of surface cells into the blastocoel. Mutations in the par-3 gene cause abnormal separations between embryonic cells, suggesting that the
PAR-3 protein has a role in blastocoel formation. In normal development, PAR proteins localize to either the apical or
basal surfaces of cells prior to blastocoel formation; this localization is determined by cell contacts. Cells that ingress into the blastocoel undergo an apical flattening associated with an apical concentration of
non-muscle myosin. Evidence is provided that ingression times are determined by genes that control cell fate, though interactions with neighboring
cells can prevent ingression (Nance, 2002).
The events of gastrulation indicate that early embryonic cells have apical-basal polarity; for example, NMY-2, a C. elegans non-muscle myosin, accumulates at the apical surface of ingressing cells,
and the blastocoel forms at the basal surfaces. Apical-basal asymmetry is evident in embryonic cells of C. elegans as early as the 4-cell stage: PAR-3 and PAR-6
are localized to the apical surfaces, and PAR-2 and HMR-1(cadherin) are localized to the basal and lateral surfaces of cells. Drosophila and vertebrate homologs
of PAR-3 and PAR-6, together with the atypical protein kinase PKC-3, have been shown to function in establishing apical-basal polarity in epithelial cells and
neuroblasts. Large cell separations are observed between both basal and lateral surfaces of cells in par-3 mutants. This result suggests that par-3(+) may have a role in distinguishing the basal from lateral surfaces during blastocoel formation in wild-type embryos, where large separations only occur between basal surfaces (Nance, 2002).
Cell contacts restrict PAR-3 and PAR-6 to the contact-free, apical surfaces. This mechanism differs, at least in part, from the mechanism that
localizes these same proteins to the anterior surface of the 1-cell embryo. While PAR-2 has a role in determining PAR-3 localization at the 1-cell stage, PAR-2 is not required for the apical localization of PAR-3 at the 4-cell stage, nor is it required for blastocoel formation. HMR-1(cadherin) also is localized to cell contacts, but does not appear to have a role in PAR localization or blastocoel formation. Interestingly, genetic or immunological inhibition
of E-cadherin function in early mouse embryos does not prevent individual cells from becoming polarized, but rather causes a randomization in the axis of polarity. HMR-1 appears to be the only 'classical' cadherin with a ß-catenin binding site, similar to mouse E-cadherin, although the C. elegans genome sequence predicts several cadherin-related proteins whose functions and localization have not been determined (Nance, 2002).
Localization of PAR-3, or associated proteins, to the apical surface could in principle differentiate the basal surface from the lateral surface. For example, the localization of ion channels to the apical surface could create a gradient that affects the opposite (basal) surface differently from that of the lateral surfaces. Vectorial ion transport is essential for formation of the blastocoel in mouse embryos, and channel proteins appear to be localized with apical-basal polarity in trophectodermal cells lining the blastocoel. It will be interesting in future studies to determine how apical-basal polarity of the PAR proteins directs subsequent asymmetries. In the 1-cell embryo, the anterior-posterior polarity of the PAR proteins establishes a parallel gradient of MEX-5 in the cytoplasm. MEX-5, novel cytoplasmic protein that is localized through PAR activities to the anterior pole of the 1-cell stage embryo, in turn functions to prevent the anterior expression of posterior proteins. However, MEX-5 is uniformly distributed in somatic blastomeres that have an apical-basal polarity in PAR protein distribution, suggesting that MEX-5 does not mediate apical-basal polarity (Nance, 2002).
Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of
cellular polarity. Evidence suggests that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the
nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the
normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at
anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99
is a DEP (Disheveled, Egl-10 and Plekstrin) domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in
spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the
oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, these data
indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric
regulation of forces on the spindle (Tsou, 2002).
Trimeric G proteins and PAR-3 and PAR-3's binding partners also play a role in asymmetric division in Drosophila. In that system, the G
proteins function in localizing cell fate determinants in addition to orienting the spindle. In
Drosophila, the Inscuteable protein serves as the link between the polarity cues and the G proteins, as has been postulated for LET-99 in C. elegans.
LET-99 and Inscuteable have no sequence similarity or shared domains, but could be functioning similarly as adaptor proteins to organize protein
complexes. Drosophila does not appear to have an ortholog for LET-99, even in terms of domain organization, nor does C. elegans have a clear
Inscuteable ortholog. This lack of conservation could in part be due to differences in embryonic development. In C. elegans, as in many other
organisms, early divisions take place in large cells that require long astral microtubules to reach the cortex. In Drosophila, early divisions occur first in
cytoplasmic islands and then in small membrane domains within the syncitial blastoderm; similarly, the asymmetric divisions that require Inscuteable occur
in small cells. The strict maternal requirement for LET-99 suggests it is
specialized for functioning in large embryonic cells. Both the mouse and human genomes encode several proteins with a similar domain organization as
LET-99. It will be interesting to learn whether these DEP proteins function in any aspects of spindle positioning during the early development of these
organisms (Tsou, 2002).
Polarization of the one-cell C. elegans embryo establishes the animal's anterior-posterior (a-p) axis. Reduction-of-function anaphase-promoting complex (APC) mutations have been discovered that eliminate a-p polarity. The APC activator cdc20 (Fizzy in Drosophila) is required for polarity. The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there. The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome. Depletion of the protease separin, a downstream target of the APC, causes similar pronuclear and a-p polarity defects. It is proposed that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation (Rappleye, 2002).
Polarization of the C. elegans zygote along the anterior-posterior axis depends on cortically enriched (PAR) and cytoplasmic (MEX-5/6) proteins, which function together to localize determinants (e.g. PIE-1) in response to a polarizing cue associated with the sperm asters. Using time-lapse microscopy and GFP fusions, the localization dynamics of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 were studied in wild-type and mutant embryos. These studies reveal that polarization involves two genetically and temporally distinct phases. During the first phase (establishment), the sperm asters at one end of the embryo exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing the ring finger protein PAR-2 to accumulate in an expanding `posterior' domain. Onset of the establishment phase involves the non-muscle myosin NMY-2 and the 14-3-3 protein PAR-5. The kinase PAR-1 and the CCCH finger proteins MEX-5 and MEX-6 also function during the establishment phase in a feedback loop to regulate growth of the posterior domain. The second phase begins after pronuclear meeting, when the sperm asters begin to invade the anterior. During this phase (maintenance), PAR-2 maintains anterior-posterior polarity by excluding the PAR-3/PAR-6/PKC3 complex from the posterior. These findings provide a model for how PAR and MEX proteins convert a transient asymmetry into a stably polarized axis (Cuenca, 2003).
Previous studies have implicated the sperm-derived MTOC as the most likely source for the spatial cue that initially polarizes the zygote. Time-lapse analysis supports this view. Formation of the MTOC correlates temporally and spatially with the earliest evidences of polarity: (1) cessation of ruffling, (2) enrichment of GFP:PAR-2, and (3) loss of GFP:PAR-6 in the posterior cortex. The data also demonstrates that the primary effect of the polarizing cue is to clear the PAR-3/PAR-6/PKC-3 complex from the posterior cortex. This effect does not require PAR-2. In contrast, restriction of PAR-2 to the posterior requires PAR-6, PAR-3 and PKC-3, suggesting that PAR-2 does not sense the polarity cue directly but instead responds to local displacement of the anterior complex (Cuenca, 2003).
The establishment phase requires the class II non-muscle myosin, NMY-2: nmy-2(RNAi) prevents PAR-6 (and presumably associated PAR-3 and PKC-3) from sensing the polarity cue, causing it to remain uniformly distributed throughout the cortex. In NMY-2-depleted embryos, PAR-2 is prevented from accumulating at the cortex by PAR-6 (and/or its partners). This 'default' state of PAR-6 on/PAR-2 off is also observed in mutants lacking sperm asters and in mutants where the MTOC detaches from the cortex prematurely. These observations suggest that the initial symmetry-breaking event involves signaling between the MTOC and the actin cytoskeleton. Consistent with this view, one of the earliest signs of polarization is cessation of ruffling in the cortex nearest the MTOC. Cessation of ruffling correlates with MTOC formation, but does not appear to require PAR activity (cessation of ruffling was observed in all par mutants examined in this study). These observations suggest that modification of the actin cytoskeleton may be an obligatory step before the onset of PAR asymmetry. It is proposed that signaling from the MTOC modifies the actin cytoskeleton locally, which causes the PAR-3/PAR-6/PKC-3 complex to become destabilized, allowing PAR-2 to accumulate in its place (Cuenca, 2003).
The establishment phase also requires the 14-3-3 protein PAR-5. In its absence, PAR-6 responds only weakly, if at all, to the polarity cue and PAR-2 is no longer excluded from the cortex by the PAR-3/PAR-6/PKC-3 complex. Cessation of ruffling in the posterior, however, still occurs in par-5(RNAi) embryos, suggesting that PAR-5 is not required for the initial MTOC/actin cytoskeleton interaction. Although this interpretation is complicated by the fact that residual PAR-5 activity may persist in par-5(RNAi) embryos, it is proposed that PAR-5 functions primarily by regulating the ability of the PAR-3/PAR-6/PKC-3 complex to (1) exclude PAR-2 and (2) respond to changes in the cytoskeleton. The presence of a potential 14-3-3 binding motif in PAR-3 is consistent with the possibility that PAR-5 regulates the PAR-3/PAR-6/PKC-3 complex by binding to it directly (Cuenca, 2003).
Surprisingly, it was found that the predominantly cytoplasmic MEX-5 and MEX-6 also play a role during the establishment phase. In the absence of MEX-5 and MEX-6, the posterior domain occasionally does not form (15%-30% of embryos), and frequently (50% or more of embryos) is slow to reach its final configuration. These observations indicate that, although MEX-5 and MEX-6 are not absolutely required for PAR localization in the zygote, they do play a role in ensuring a robust response by the PAR-3/PAR-6/PKC-3 complex to the MTOC/actin cytoskeleton signal (Cuenca, 2003).
This aspect of MEX-5/6 function is negatively regulated by PAR-1. In par-1 mutants, MEX-5 and MEX-6 cause the posterior domain to extend further towards the anterior during the establishment phase. Since PAR-1 itself becomes enriched in the posterior domain, one attractive possibility is that PAR-1 and MEX-5/6 participate in a feedback loop that limits expansion of the posterior domain. It is proposed that at the beginning of the establishment phase, MEX-5 and MEX-6 levels are high throughout the zygote and help clear the PAR-3/PAR-6/PKC-3 complex from the region nearest the sperm asters. This clearing allows PAR-2 and PAR-1 to accumulate on the cortex, which in turn reduces MEX-5/6 activity and/or levels in the surrounding cytoplasm. Eventually, MEX-5/6 levels become too low to fuel further expansion of the posterior domain. It is not yet known whether the partial penetrance of the mex-5(-);mex-6(-) phenotype is due to redundancy with other factors, or is indicative of a minor role for the feedback loop in regulating PAR asymmetry (Cuenca, 2003).
The finding that the sperm-derived MTOCs play a role in initiating polarity raised the question of how polarity is maintained after pronuclear meeting, when the pronuclei/centrosome complex rotates and microtubules invade the anterior end of the embryo. In the absence of PAR-2, PAR-3 and PAR-6 and PKC-3 can become asymmetric before pronuclear meeting, but return into the posterior domain afterwards. This finding demonstrates two points: (1) the PAR-6/PAR-3/PKC-3 complex no longer responds to the MTOC-dependent cue after pronuclear meeting, and (2) PAR-2 is required after pronuclear meeting, but not earlier, to exclude the PAR-6/PAR-3/PKC-3 complex from the posterior. It is proposed that pronuclear meeting (and/or the end of prophase) triggers a change in the cytoskeleton, or in the PAR-6/PAR-3/PKC-3 complex, that turns off the MTOC-dependent polarity signal, or the ability to respond to it. From that point on, PAR-2 becomes essential to keep PAR-6/PAR-3/PKC-3 out of the posterior cortex. It is intriguing that PAR-6 briefly localizes to nuclei at pronuclear meeting, raising the possibility that it becomes modified at that time (Cuenca, 2003).
The existence of distinct establishment and maintenance phases is also supported by the observation that cdc-42 is required after prophase, but not earlier, for PAR-3, PAR-6 and PKC-3 asymmetry. Analysis of GFP:PAR-6 dynamics in par-1(RNAi) embryos suggests that PAR-1 also contributes to maintenance of PAR asymmetry after pronuclear meeting. How PAR-2, CDC-42 and PAR-1 function together to maintain the balance between anterior and posterior PAR domains remains to be determined (Cuenca, 2003).
PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study hybrid PAR proteins were generated that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but show defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appear between lateral surfaces of cells that are normally tightly adherent; cells that ingress during gastrulation fail to accumulate nonmuscle myosin at their apical surfaces, and ingression is slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis (Nance, 2003).
During the 4-cell stage of embryogenesis, the PAR proteins undergo a dramatic redistribution along the apicobasal axis. The results indicate that recruitment of PAR-3 to the apical cortex is a key step in this redistribution, analogous to previous observations on the role of PAR-3 at the 1-cell stage. PAR-3 localization to the apical cortex occurs independently of PAR-6 and PAR-2. Moreover, PAR-3 localization is crucial for recruiting PAR-6 and PKC-3 to the apical cortex, and restricting PAR-2 to basolateral surfaces. Localization of PAR-3 to the apical cortex is not sufficient for the colocalization of PAR-6 and PKC-3: PAR-6 does not colocalize with apical PAR-3 in pkc-3(RNAi) embryos, and PKC-3 does not colocalize with apical PAR-3 in par-6(ZF1) embryos, that is, in embryos in which PAR-6 is degraded after the first division. Thus both PAR-6 and PKC-3 must be present for either protein to associate with apical PAR-3. Biochemical studies of PAR-3, PAR-6 and PKC-3 homologs in mammalian cells have shown that these proteins can bind to one another directly, indicating that interactions between all three proteins might be necessary to stabilize a complex with apical PAR-3 (Nance, 2003).
C. elegans embryos have at least three distinct periods in which the PAR-3 complex must distinguish different cell surfaces. At the 1-cell stage, PAR-3 associates with the anterior surface, and at the 4-cell stage, PAR-3 associates with the apical surface. In late embryogenesis PAR-3 is localized asymmetrically in epithelial cells, and the apicobasal axis of the internal epithelia is inverted with respect to that of earlier embryonic cells. These localization patterns appear to be specified de novo during each period. Disruption of PAR asymmetry at the 1-cell stage by mutations in par-2 does not prevent apical localization of PAR-3 after the 4-cell stage. Similarly, the absence of the PAR-3 complex between the 4-400-cell stages in par-3(ZF1) embryos does not prevent the subsequent apical localization of PAR-3 during organogenesis (Nance, 2003).
The molecular cues used to localize the PAR-3 complex remain to be identified and, at some level, these are likely to vary. For example, sperm position and cell contacts specify polarity at the 1- and 4-cell stages, respectively. Although the mechanism of PAR localization has not been studied extensively in the epithelial cells of C. elegans, genetic studies in Drosophila have identified homologs of proteins in the C. elegans PAR-3 complex that regulate apicobasal polarity in epithelial cells. E-cadherin-mediated cell adhesion is required for apical PAR-3 complex localization in Drosophila epithelial cells, whereas HMR-1/E-cadherin is not essential for PAR-3 complex asymmetry at either the 1-cell or 4-cell stage in C. elegans. Apical localization of the PAR-3 complex in Drosophila epithelia is antagonized by a basolateral complex of proteins that includes Discs large and Scribble. The C. elegans homologs of the latter proteins, DLG-1/Discs large and LET-413/Scribble, are expressed in epithelial cells, and depletion of these proteins causes epithelial defects. However, these proteins do not appear to function in apicobasal polarity of early embryonic cells because they are either not expressed in the early embryo (DLG-1) or are not required for apical localization of PAR-3. Thus, identifying the molecular basis of cell-contact-dependent PAR localization remains an important goal for future studies on apicobasal PAR asymmetry (Nance, 2003).
Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage C. elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. The GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, are essential for generation of proper pulling forces. GPR-1/2 interact with guanosine diphosphate-bound GOA-1 and are enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2 (Colombo, 2003).
Signaling upstream of PAR-3 in C. elegans Epithelial tubes are a key component of organs and are generated from cells with distinct apico-basolateral polarity. A novel function during tubulogenesis is described for ZEN-4, the Caenorhabditis elegans ortholog of mitotic kinesin-like protein 1 (MKLP1; see Drosophila Pavarotti), and CYK-4, which contains a RhoGAP (GTPase-activating protein) domain. Previous studies have revealed that these proteins comprise centralspindlin (a complex that functions during mitosis to bundle microtubules), construct the spindle midzone, and complete cytokinesis. ZEN-4/MKLP1 functions postmitotically to establish the foregut epithelium. Mutants that lack ZEN-4/MKLP1 express polarity markers but fail to target these proteins appropriately to the cell cortex. Affected proteins include PAR-3/Bazooka and PKC-3/atypical protein kinase C at the apical membrane domain, and HMR-1/cadherin and AJM-1 within C. elegans apical junctions (CeAJ). Microtubules and actin are disorganized in zen-4 mutants compared to the wild-type. It is suggested that ZEN-4/MKLP1 and CYK-4/RhoGAP regulate an early step in epithelial polarization that is required to establish the apical domain and CeAJ (Portereiko, 2004).
The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood.
A method has been developed that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, it has been found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. This asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo. These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes (Labbé, 2003).
Microtubules are more stable at the anterior than the posterior cortex in wild-type C. elegans embryos. This difference in microtubule stability at the cortex is independent of the protein PAR-1 but depends on the proteins PAR-2, PAR-3, and Galpha. A role for PAR-3 in microtubule anchoring or stabilization had been hypothesized previously, based on observations of defects in spindle orientation and spindle positioning of par-3 mutant embryos. Based on these results, it is proposed that PAR-3 stabilizes microtubules and that PAR-2 affects microtubule stability by restricting PAR-3 activity to the anterior of the embryo. Because disruption of Galpha does not yield results that are consistently distinguishable from those obtained by disruption of par-2 or par-3, it is not possible to ascribe clearly a role for Galpha in stabilizing or destabilizing microtubules. However, the fact that disruption of Galpha results in equal microtubule residence time at the anterior and posterior cortex, clearly indicates that Galpha is required for asymmetric regulation of microtubule stability at the cortex. In C. elegans, Drosophila, and human cells, PAR-3 is found in a complex along with PAR-6, a protein with PDZ motifs, and an atypical protein kinase C, PKC-3. In C. elegans embryos, disruption of any individual member of this protein complex causes the mislocalization of the other members, and therefore it is currently not possible to directly test whether PAR-3 affects microtubule stability through the activity of PKC-3 or not. It will be of interest to determine whether microtubule dynamics at the cortex are regulated by atypical protein kinase C activity, as well as to identify whether this kinase can directly target microtubule-associated proteins (MAPs) to affect microtubule stability. PAR-6 also interacts with CDC-42 in C. elegans and in human cells. It is possible that the GTPase activity of CDC-42 could also directly or indirectly affect MAPs to modulate microtubule dynamics. Likewise, the DEP domain-containing protein LET-99 might affect the stability of microtubules at the posterior cortex of the embryo (Labbé, 2003).
These results demonstrate that PAR proteins and G protein signaling regulate microtubule dynamics at the cortex during spindle positioning in the early C. elegans embryo and are responsible for a 15% difference in microtubule stability between the anterior and posterior cortices. This 15% difference, although small, could perhaps contribute to posterior spindle displacement. Spindle-cutting experiments estimated that pulling on the posterior aster is 40% stronger than on the anterior aster during posterior spindle displacement. The regulation of microtubule dynamics along the anteroposterior axis of the embryo could modulate these pulling forces to influence posterior spindle displacement. One possibility that is consistent with current data is that a limiting number of motor proteins, such as dynein motors, are present at the cell cortex to pull a fraction of the microtubules, and decreasing the stability of microtubules in the posterior eliminates microtubules that do not engage dyneins. Such unengaged microtubules might obstruct pulling forces and impede movement of the spindle. In this regard, the large subunit of dynein (DHC-1) has been shown to localize to the entire cell cortex during metaphase and anaphase; however, whether active DHC-1 is present in limiting concentrations in the cortex has yet to be determined. Alternatively, asymmetrically localized minus end-directed motor activity at the cortex of the embryo might itself locally influence microtubule dynamics, and the difference observed in microtubule stability at the cortex might be a consequence of an asymmetry in motor activity. PAR proteins and G proteins also influence other microtubule-dependent processes in the embryo, such as spindle rocking and centrosome rotation at the 2-cell stage. The observed asymmetry in microtubule stability at the cortex might also contribute to the regulation of these processes. In the case of centrosome rotation, previous experiments have demonstrated that this process is sensitive to pharmacological agents that either stabilize or destabilize microtubules. This suggests that microtubule dynamics are important during this process (Labbé, 2003 and references therein).
An average microtubule residence time at the cortex of 15.5 ± 0.8 s in wild-type embryos has been measured. If microtubules were continuously growing during this time, this would generate pushing force on the cortex. A microtubule pushing-based model was recently proposed to explain nuclear positioning in S. pombe. In this case, plus-end microtubule growth generates pushing force toward the minus-end and causes microtubule buckling in the cytoplasm. Little, if any, buckling of microtubules is observed during posterior spindle displacement in C. elegans embryos. Furthermore, pulling forces on astral microtubules have been reported. These results suggest that microtubule plus-ends that interact with the cortex are stalled (not growing) and under tension. This raises the possibility that the microtubule plus-ends behave differently in the cytoplasm than they do when they make contact with the cell cortex (Labbé, 2003 and references therein).
Finally, a novel approach (Cortical Imaging of Microtubule Stability, CIMS) has been used to study the cortical stability of individual microtubules in a developmental system. Until now, most measurements of microtubule dynamics in vivo were done by using cells in culture or other relatively flat cells, which are better suited to image microtubules in a single plane of focus. CIMS has been used to study microtubule stability at the cortex of C. elegans embryos, which have a thickness of 20-30 microm. One of the main advantages of CIMS is that it reduces the imaging of a thick specimen to a thin region near the cell cortex. Therefore, it eliminates the problem of microtubules going in and out of the plane of focus. It also maximizes the optical resolution of fluorescence events by imaging near the objective lens, not deep into the specimen where light scattering occurs and thus optical resolution is decreased. Furthermore, it is possible to use CIMS to visualize certain microtubules along their lengths and thus quantify additional aspects of their dynamic behavior in living embryos. In preliminary experiments using this approach, an average growth rate of 35.7 microm/min and a shortening rate of 31.8 microm/min have been determined for microtubules at the cortex of C. elegans embryos undergoing mitosis. These rates are faster than those measured for microtubules in cultured cells but are similar to those observed in clarified Xenopus egg extracts. CIMS may prove useful for studying microtubule dynamics in other thick biological specimens, such as Drosophila, Xenopus, and echinoderm embryos (Labbé, 2003).
It is concluded that PAR proteins and G protein signaling regulate the stability of individual microtubules at the cortex of C. elegans embryos. This indicates that proteins that regulate asymmetric cell division also modulate microtubule dynamics at the cell cortex (Labbé, 2003).
PAR-3 is localized asymmetrically in epithelial cells in a variety of animals from Caenorhabditis elegans to mammals. Although C. elegans PAR-3 is known to act in early blastomeres to polarize the embryo, a role for PAR-3 in epithelial cells of C. elegans has not been established. Using RNA interference to deplete PAR-3 in developing larvae, a requirement for PAR-3 in spermathecal development was discovered. Spermathecal precursor cells are born during larval development and differentiate into an epithelium that forms a tube for the storage of sperm. Eggs must enter the spermatheca to complete ovulation. PAR-3-depleted worms exhibit defects in ovulation. Consistent with this phenotype, PAR-3 is transiently expressed and localized asymmetrically in the developing somatic gonad, including the spermathecal precursor cells of L4 larvae. The defect in ovulation can be partially suppressed by a mutation in IPP-5, an inositol polyphosphate 5-phosphatase, indicating that one effect of PAR-3 depletion is disruption of signaling between oocyte and spermatheca. Microscopy has revealed that the distribution of AJM-1, an apical junction marker, and apical microfilaments are severely affected in the distal spermatheca of PAR-3-depleted worms. It is proposed that PAR-3 activity is required for the proper polarization of spermathecal cells and that defective ovulation results from defective distal spermathecal development (Aono, 2004).
The PAR proteins are part of an ancient and widely conserved machinery for polarizing cells during animal development. A combination of genetics and live imaging methods were used in the model organism Caenorhabditis elegans to dissect the cellular mechanisms by which PAR proteins polarize cells.
Two distinct mechanisms by which PAR proteins polarize the C. elegans zygote have been demonstrated. (1) It is shown that several components of the PAR pathway function in intracellular motility, producing a polarized movement of the cell cortex. Evidence is presented that this cortical motility may drive the movement of cellular components that must become asymmetrically distributed, including both germline-specific ribonucleoprotein complexes and cortical domains containing the PAR proteins themselves. (2) PAR-1 functions to refine the asymmetric localization of germline ribonucleoprotein complexes by selectively stabilizing only those complexes that reach the PAR-1-enriched posterior cell cortex during the period of cortical motility. These results identify two cellular mechanisms by which the PAR proteins polarize the C. elegans zygote, and they suggest mechanisms by which PAR proteins may polarize cells in diverse animal systems (Cheeks, 2004).
To understand how PAR proteins function to generate cell polarity, advantage was taken of the potential to combine modern live-cell imaging techniques with an analysis of mutants in the C. elegans embryo. The results suggest a model in which PAR proteins establish polarity by two distinct mechanisms. (1) PAR-2, -3, -4, and -6 and MEX-5/6 establish polarity by generating an actomyosin-based movement of the cortex away from the point of sperm entry. This movement generates two distinct cortical domains -- a domain of new cortex with which PAR-2 dynamically associates in the posterior of the embryo and a domain of old cell cortex with which PAR-6 dynamically associates in the anterior of the embryo. This movement of the actin cortex to the anterior may drive the opposing flow of central cytoplasm and carry most of the P granules, which are enriched in the central cytoplasm after the beginning of flow, to the posterior. These movements do not result in the complete circulation of cortical and central cytoplasmic components, because the extent of cortical and central cytoplasmic flow is less than the full length of the embryo. (2) Around the time that these movements stop, PAR-1, localized to the posterior cell cortex, refines the pattern of P granule localization by stabilizing only those P granules that have reached the posterior cell cortex (Cheeks, 2004).
It has been proposed that cell polarization in the C. elegans zygote proceeds by distinct establishment and maintenance phases. The current results suggest a mechanism by which cell polarization is established -- by movement of the actin cortex and of cortical domains to which PAR proteins associate and by movement of central cytoplasm and P granules in the opposite direction. PAR-2 may be involved in both this establishment phase and in a second, maintenance phase of cell polarization because PAR-2 is required for the full extent of cortical and central cytoplasmic flow but is also required to later exclude anterior PAR proteins from the posterior cell cortex after pronuclear meeting (Cheeks, 2004).
The loss of cytoplasmic flow in many of the C. elegans par mutants may, in large part, explain their mutant phenotypes. For example, loss of the posterior cortical protein PAR-2 results in a partial failure of cortical flow. This would be expected to result in the generation of little new cortex in the posterior; consistent with this, anterior PAR proteins associate with most of the cell cortex in par-2 mutants. The small amount of cytoplasmic flow in par-2 mutants probably results in the incomplete localization of P granules previously observed in par-2 mutants. Mislocalized PAR-1 ectopically stabilizes these P granules (Cheeks, 2004).
Likewise, for anterior PAR proteins such as PAR-3 or PAR-6, loss of function results in a symmetric P granule distribution, most likely because the cytoplasmic flow that carries P granules posteriorly fails and because a resulting uniform distribution of PAR-1 stabilizes P granules in ectopic locations. The global distribution of posterior PAR proteins in these backgrounds suggests that PAR-2 is normally prevented from associating with old cortex by anterior PAR proteins. These findings show that the globally cortical protein PAR-4 functions in the same intracellular motility events as do some of the anteriorly or posteriorly localized PAR proteins. Embryos that lack the globally cortical protein PAR-5 have not been extensively analyzed because null alleles of par-5 do not yet exist, but preliminary recordings have demonstrated a partially penetrant phenotype in which cortical and central cytoplasmic flow fail to occur (Cheeks, 2004).
The par mutant phenotypes resemble those produced by loss of actomyosin contraction regulators such as the myosin II subunits NMY-2 and MLC-4 and loss of the actin binding protein POD-1, both of which result in the failure of cortical and central cytoplasmic flow. It has been proposed that actomyosin-based movement of the cell cortex in C. elegans and in other systems may be initiated and/or maintained by astral microtubules. Although PAR proteins regulate microtubule dynamics after the period of flow, no defects in astral microtubule distributions before the period of flow have been reported in C. elegans par mutants, suggesting that PAR proteins probably function in the cortical response to astral microtubules. PAR proteins might modify the actin cortex in a manner that allows the cortex to move, perhaps by allowing local depolymerization of the contractile actomyosin mesh at the posterior pole. Alternatively, because a small amount of flow could be seen in many of the par mutants, it is possible that PAR proteins modify the cortex in a way that allows further flow propagation to be initiated by astral microtubules, independently of the PAR proteins (Cheeks, 2004).
The mechanisms by which PAR proteins drive cortical and central cytoplasmic flow are not yet clear. CDC-42, which associates with the PAR-3/PAR-6/PKC-3 complex in C. elegans and in other organisms, has well-characterized roles in modulating the actin cytoskeleton. CDC-42 can induce actin polymerization via WASP and Arp2/3 in other systems. If actin polymerization drives cortical motility as it has been proposed to do in migrating cells, one would expect it to do so in the posterior of the embryo to drive cortical flow anteriorly. However, CDC-42 may function primarily in the anterior of the C. elegans embryo because cdc-42(RNAi) embryos have phenotypes that generally resemble loss of anterior PAR proteins instead of loss of posterior PAR proteins. How then might CDC-42 function in the anterior? Depolymerization of the actin meshwork near the astral microtubules in the posterior, along with a higher myosin contractility in the receding old cortex than in new cortex, may drive cortical flow, and there is precedence for CDC-42 regulating myosin II activity: in a variety of systems, CDC-42 activates p21-activated kinases, and p21-activated kinases can upregulate myosin II activity by phosphorylating myosin light-chain kinase (Cheeks, 2004).
Although these results suggest a general mechanism by which a cell can produce two distinct cortical domains, it is not clear how specific PAR proteins recognize new or old cortical domains. PAR-2 associates with the cell cortex before fertilization, and even in gonads before oocytes are cellularized, whereas PAR-3 and PAR-6 are not cortically enriched until the time of meiosis. Therefore, PAR-6 does not associate preferentially with old cortex simply as a result of associating with cortex earlier. Instead, it appears that PAR-2 is specifically excluded from the cortex during the period in which PAR-3 and PAR-6 first associate with the cortex; PAR-2 enrichment at the cell cortex has been reported to decrease as oocytes mature. The conclusion that PAR-2 is specifically excluded from the cortex by a PAR-3- and PAR-6-independent mechanism is supported by the dynamics of PAR-2 association with the cortex. As cortical flow begins, PAR-2 does not immediately associate with new cortex but instead does so with a 3-4 min delay. FRAP experiments on GFP::PAR-2 suggest that PAR-2 associates with the cell cortex far too dynamically to account for this 3-4 min delay on the basis of PAR-2 protein diffusion dynamics alone. Whether the cortex is modified or PAR-2 is modified at this time is not clear, but PAR-2 diffusion does not change significantly over time; PAR-2 is equally dynamic in the variable and transient anterior cap soon after fertilization, in the expanding posterior cap and at the two- and four-cell stages (Cheeks, 2004).
The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. The one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry (Munro, 2004).
It has been proposed how a local modulation of global cortical contractility could produce tension gradients in the cortex that would drive flows of cortical cytoplasm away from regions of low cortical tension and toward regions of high cortical tension. More specifically, it has been hypothesized that cortical flows in the one-cell C. elegans embryo might be caused by a local relaxation of cortical tension through an interaction between the sperm MTOC and the cortex. The results support this idea and reveal the cytoskeletal basis for contractile force generation and cortical flow: prior to the appearance of a distinct sperm pronucleus, F-actin and NMY-2 constitute a dynamic network of short-lived foci that form through transient local, myosin-dependent, contractions of the cortical meshwork. The association of surface invaginations with groups of foci and interfoci and the evident continuity of the actomyosin meshwork implies that neighboring foci are mechanically coupled to form a network of tensioned elements. Prior to appearance of the sperm MTOC, local contractions associated with focus formation drive small transient displacements of foci, but the network is globally stable. Appearance of the sperm MTOC is associated with an immediate, local cessation of focus formation, reflecting an apparent diminution of contractility or weakening of the otherwise symmetrical actomyosin network. The resulting global tension imbalance should cause an immediate and collective flow of remaining foci toward one another and away from the sperm MTOC as the entire meshwork contracts asymmetrically toward the opposite, anterior pole. Measurements of focus movements before and after appearance of the sperm MTOC confirm this prediction, and the loss of convergent flow in embryos lacking the regulatory myosin light chain MLC-4 confirms the contractile basis for these flows (Munro, 2004).
How do flows continue once initiated? The observations reveal a continuous local cycle of focus assembly/contraction followed by disassembly. The local contractions that produce each individual focus are short-lived, but at any time during flow, these contractions are distributed throughout the anterior cap and coupled to one another to form a continuously tensioned network. Thus the cortex appears to be a self-renewing contractile engine that continues to generate tension even as it contracts, rather than a pre-tensioned network that contracts once to release stored tension. Likewise, the persistent absence of foci in the posterior clear zone, and the constant flow speeds of filaments away from this zone, suggest a continued absence of contractility near the sperm MTOC. Thus, sustained, asymmetrical contractile force generation appears to sustain a continued cortical flow (Munro, 2004).
Previous studies suggest that factors closely associated with the sperm MTOC supply the cue that initiates cortical flow in the one-cell embryo, although the relative importance of the centrosome itself and the sperm astral microtubules remains controversial. The observations that focus dynamics remain unchanged near the newly formed sperm pronucleus, and that cortical flows never initiate in embryos depleted of essential centrosomal components (which also lack a sperm aster), support this hypothesis. Furthermore, they suggest that the sperm cue acts by modulating the actomyosin contractility cycle to produce a local reduction of cortical tension. Discovering the nature of this modulation is an important future goal (Munro, 2004).
NMY-2::GFP foci, PAR-6::GFP puncta, and yolk granules each move toward the anterior pole at the same velocity, indicating that all are components of the same cortical flow. The observation that the rear boundary of the anterior PAR-6 cap moves at the same speed as this flow suggests that cortical transport is the dominant mechanism for establishing an anterior PAR domain. Several lines of evidence suggest that the other members of the anterior PAR complex, PAR-3 and PKC-3, localize through the same cortical flow. PAR-3, PAR-6, and PKC-3 colocalize extensively in coimmunostaining experiments, and analysis of fixed embryos shows coincident distributions of NMY-2 and endogenous PAR-3. PAR-3 and PKC-3 are both essential for the cortical localization of PAR-6, and homologs of these proteins can complex in vitro. PAR-3 appears to provide the critical link that enables PAR-6 and PKC-3 to associate with the cortex, since it is the only member of the complex that can localize cortically in the absence of the others. PAR-3 is unlikely to bind NMY-2 directly since it remains associated with the cortex when NMY-2 is depleted from the embryo. However, PAR-3 cortical localization may involve actin or actin binding proteins since depleting cortical F-actin prevents PAR-3 from associating with the cortex (Munro, 2004).
The non-PAR proteins HMR-1/E-Cadherin, HMP-2/ß-catenin, and LAD-1/L1CAM each localize to the anterior pole of the embryo during cortical flow. None of these proteins have a known role in early embryonic polarity, but they or their homologs in other animals can associate indirectly with the cortical actin cytoskeleton. Thus cortical flow at the one-cell stage may be a feature common to all proteins associated with the actomyosin cytoskeleton (Munro, 2004).
Coordinate flows of NMY-2 and PAR-6 are associated with multiple examples of cortical PAR asymmetry in the early C. elegans embryo. This suggests that asymmetrical contractile flows underlie each asymmetry, although the cues that initiate these flows must at some level be different. As the sperm MTOC appears to be the cue for anterior/posterior polarity at the one-cell stage, centrosomes might function in subsequent anterior/posterior polarity for the lineage of cells that produce the germline; after the first cell division, there is a migration of the nucleus and centrosomes toward the posterior cortex of the germline precursor that precedes cortical flows of both yolk granules and NMY-2 and PAR-6. However, all other early embryonic cells exhibit apicobasal PAR asymmetry, and in these cells there is no obvious relationship between centrosomes/microtubules and the apicobasal axis. Instead, experiments on isolated cells suggest that contacts between cells determine the apicobasal axis. Thus there are likely to be multiple cues that cells can exploit to generate cortical flows, and it will be of interest to see how these converge on the actomyosin cytoskeleton (Munro, 2004).
The fact that depleting embryos of the anterior PAR proteins alters actomyosin dynamics and attenuates cortical flow implies that these proteins are not simply passive cargo transported by an independent flow. It suggests rather that they actively modulate cortical dynamics to promote cortical flow and thus their own transport. The net force acting on the NMY-2 foci must be the sum of active (e.g., contractile) forces that tend to move foci and passive (e.g., viscous and elastic) forces that resist these movements. The deep local furrowing observed in par-3 mutants argues against a simple decrease in contractile force generation in par-3 mutant embryos. An alternative possibility is that the anterior PAR complex could modulate cortical elements that passively resist focus movement, for example by modulating crosslinks within the actomyosin network itself or by modulating other cortical structures that interact mechanically with the actomyosin network to resist its deformation (Munro, 2004).
Studies in several labs suggest that the anterior PAR complex inhibits the association of PAR-2 with the cortex and that depletion of the anterior PAR complex from the posterior cortex removes this inhibition and allows PAR-2 to accumulate there. PAR-2 in turn is required to maintain the anterior localization of anterior PAR proteins after pseudocleavage in one-cell embryos, but the mechanism by which it acts is unknown. The gap observed between anterior and posterior PAR domains after pseudocleavage makes it unlikely that PAR-2 acts directly upon members of the anterior PAR complex. Instead, the observations suggest that the loss of anterior PAR asymmetry in par-2 (RNAi) embryos is caused by an aberrant flow of NMY-2::GFP and PAR-6::GFP toward the posterior pole that results from the ectopic return of NMY-2 to the posterior cortex after pseudocleavage. Thus it is proposed that PAR-2 contributes indirectly to maintaining the anterior localization of the anterior PAR complex, by inhibiting recruitment of NMY-2 to the posterior cortex, and thus by preventing inappropriate, NMY-2-driven, flows to the posterior. The same inhibition may also contribute to maintenance of anterior PAR asymmetries by promoting normal anterior-directed flows after pseudocleavage. However, PAR-2 does not appear to be highly conserved in animal evolution. Thus other mechanisms, including interactions among PAR-1, PAR-5, and the anterior PAR complex, or between PAR-1 and NMY-2, must also operate in animals to maintain PAR asymmetries once they are established (Munro, 2004).
The apicobasal polarity of epithelial cells is critical for organ morphogenesis and function, and loss of polarity can promote tumorigenesis. Most epithelial cells form when precursor cells receive a polarization cue, develop distinct apical and basolateral domains and assemble junctions near their apical surface. The scaffolding protein PAR-3 regulates epithelial cell polarity, but its cellular role in the transition from precursor cell to polarized epithelial cell has not been determined in vivo. This study used a targeted protein-degradation strategy to remove PAR-3 from C. elegans embryos and examine its cellular role as intestinal precursor cells become polarized epithelial cells. At initial stages of polarization, PAR-3 accumulates in cortical foci that contain E-cadherin, other adherens junction proteins, and the polarity proteins PAR-6 and PKC-3. Using live imaging, PAR-3 foci were shown to move apically and cluster, and it was shown that PAR-3 is required to assemble E-cadherin into foci and for foci to accumulate at the apical surface. It is proposed that PAR-3 facilitates polarization by promoting the initial clustering of junction and polarity proteins that then travel and accumulate apically. Unexpectedly, superficial epidermal cells form apical junctions in the absence of PAR-3, and PAR-6 was shown to have a PAR-3-independent role in these cells to promote apical junction maturation. These findings indicate that PAR-3 and PAR-6 function sequentially to position and mature apical junctions, and that the requirement for PAR-3 can vary in different types of epithelial cells (Achilleos, 2010).
Polarization of the C. elegans zygote is initiated by the guanine nucleotide-exchange factor (GEF) for the small GTPase Rho, ECT-2. ECT-2-dependent cortical flows mobilizes the anterior PAR proteins (PAR-3, PAR-6 and PKC-3) away from the future posterior end of the embryo marked by the sperm centrosome. This study demonstrates the existence of a second, parallel and redundant pathway that can polarize the zygote in the absence of ECT-2-dependent cortical flows. This second pathway depends on the polarity protein PAR-2. The RING-finger protein PAR-2 localizes to the cortex nearest the sperm centrosome even in the absence of cortical flows. Once on the cortex, PAR-2 antagonizes PAR-3-dependent recruitment of myosin, creating myosin flows that transport the anterior PAR complex away from PAR-2 in a positive-feedback loop. It is proposed that polarity in the C. elegans zygote is initiated by redundant ECT-2- and PAR-2-dependent mechanisms that lower PAR-3 levels locally, triggering a positive-feedback loop that polarizes the entire cortex (Zonies, 2010).
Vertebrate homologs of Drosophila Bazooka are required for asymmetric division Cell polarity is fundamental to the differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends on cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. The present study reports the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and shows that ASIP colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy reveals that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, these results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry (Izumi, 1998).
The asymmetric distribution of cellular components is
an important clue for understanding cell fate decision
during embryonic patterning and cell functioning after
differentiation. In C. elegans embryos, PAR-3 (Drosophila homolog: Bazooka) and aPKC
form a complex that colocalizes to the anterior periphery
of the one-cell embryo, and are indispensable for anterior-posterior
polarity, which is formed prior to asymmetric cell
division. In mammals, ASIP ('Atypical protein kinase C isotype
Specific Interacting Protein', aPAR-3 homolog) and
aPKCl form a complex and colocalize to the epithelial tight
junctions, which play critical roles in epithelial cell polarity.
Although the mechanism by which PAR-3/ASIP and
aPKC regulate cell polarization remains to be clarified,
evolutionary conservation of the PAR-3/ASIP-aPKC
complex suggests their general role in cell polarity
organization. The presence of the protein
complex in Xenopus laevis is shown in this study. In epithelial cells, XASIP and
XaPKC colocalize to the cell-cell contact region. They also colocalize to the animal hemisphere
of mature oocytes, whereas they localize uniformly in
immature oocytes. Moreover, hormonal stimulation of
immature oocytes results in a change in the distribution of
XaPKC 2-3 hours after the completion of germinal vesicle
breakdown, which requires the kinase activity of aPKC.
These results suggest that meiotic maturation induces the
animal-vegetal asymmetry of aPKC (Nakaya, 2000).
PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. An unexpected link between these two systems is described. A family of mammalian Par6 proteins have been identified that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homolog of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3 (Joberty, 2000).
Atypical protein kinase C (PKC) isotype-specific interacting protein (ASIP) specifically interacts with the atypical protein kinase C
isozymes PKClambda and PKCzeta. ASIP and atypical PKC, as well as their Caenorhabditis elegans counterparts (PAR-3 and
PKC-3, respectively), are thought to coordinately participate in intracellular signaling that contributes to the maintenance of cellular
polarity and to the formation of junctional complexes. The potential role of ASIP in other cellular functions of atypical PKC was
investigated by examining the effect of overexpression of ASIP on insulin-induced glucose uptake, previously shown to be mediated
through PKClambda, in 3T3-L1 adipocytes. When overexpressed in these cells, which contain PKClambda but not PKCzeta, ASIP is co-immunoprecipitated with endogenous PKClambda but not with PKCepsilon or with Akt. The subcellular localization of PKClambda is also altered in cells overexpressing ASIP. Overexpression of ASIP inhibits insulin stimulation of both glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but it does not inhibit glucose uptake induced by
either growth hormone or hyperosmolarity, both of which promote glucose uptake in a PKClambda-independent manner. Moreover,
glucose uptake stimulated by a constitutively active mutant of PKClambda, but not that induced by an active form of Akt, is inhibited
by ASIP. Insulin-induced activation of PKClambda, but not that of phosphoinositide 3-kinase or Akt, is also inhibited by
overexpression of ASIP. These data suggest that overexpression of ASIP inhibits insulin-induced glucose uptake by specifically interfering with signals transmitted through PKClambda (Kotani, 2000).
During early C. elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3. Together with the fact
that aPKC and a mammalian PAR-3 homolog, aPKC-specific interacting protein
(ASIP), colocalize at the tight junctions of polarized epithelial cells, this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. The overexpression of a dominant-negative mutant
of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3.
Immunocytochemical analyses, as well as measurements of paracellular diffusion
of ions or nonionic solutes, demonstrate that the biogenesis of the tight
junction structure itself is severely affected in aPKCkn-expressing cells.
Furthermore, these cells show increased interdomain diffusion of fluorescent
lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase,
suggesting that epithelial cell surface polarity is severely impaired in these
cells. aPKC associates not only with ASIP/PAR-3, but also with a mammalian homolog of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and that aPKC plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells (Suzuki, 2001).
At tight junctions (TJs), claudins with four transmembrane domains are
incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs
to the immunoglobulin superfamily, is also localized at TJs, but it remains
unclear how JAM is integrated into TJs. Immunoreplica electron microscopy
has revealed that JAM shows an intimate spatial relationship with TJ strands in
epithelial cells. In L fibroblasts expressing exogenous JAM, JAM is
concentrated at cell-cell adhesion sites, where there are no strand-like
structures, but rather characteristic membrane domains free of intramembranous
particles are detected. These domains are specifically labeled with anti-JAM
polyclonal antibody, suggesting that JAM forms planar aggregates through their
lateral self-association. Immunofluorescence microscopy and in vitro binding
assays have revealed that ZO-1 directly binds to the COOH termini of claudins and JAM
at its ZO-1's PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing
polarity-related protein, PAR-3, is directly bound to the COOH terminus of JAM,
but not to that of claudins. These findings led to a molecular architectural
model for TJs: small aggregates of JAM are tethered to claudin-based strands
through ZO-1, and these JAM aggregates recruit PAR-3 to TJs (Itoh, 2001).
The establishment and maintenance of cellular polarity are critical for the
development of multicellular organisms. PAR (partitioning-defective) proteins
were identified in Caenorhabditis elegans as determinants of asymmetric cell
division and polarized cell growth. Recently, vertebrate orthologs of two of
these proteins, ASIP/PAR-3 and PAR-6, were found to form a signaling complex
with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). ASIP/PAR-3 associates with the tight-junction-associated protein
junctional adhesion molecule (JAM) in vitro and in vivo. In fibroblasts and CHO cells overexpressing JAM,
endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over
expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3
localization at intercellular junctions and delays ASIP/PAR-3 recruitment to
newly formed cell junctions. During junction formation, JAM appears early in
primordial forms of junctions. These data suggest that the ASIP/PAR-3-aPKC complex
is tethered to tight junctions via its association with JAM, indicating a
potential role for JAM in the generation of cell polarity in epithelial cells (Ebnet, 2001).
Although ASIP is localized on tight junctions in cultured epithelial cells, it localizes on adherens junctions outlined by beta-catenin and afadin at the luminal surface, an apical end of the neuroepithelium in developing mouse central nervous systems. Mammalian homologs of other C. elegans polarity proteins, mPAR-6 and aPKC, also localize in the adherens junctions. In dorsal root ganglia of the peripheral nervous system, ASIP is found predominantly in the cytoplasm of ganglion cells. In dividing preneural cells at the ventricular (luminal) surface of the embryonic telencephalon, ASIP localizes in adherence junctions of luminal surface regardless of the axis of cell division. Therefore, only the daughter cell facing the lumen (apical daughter) may inherit ASIP when the division plate is oriented parallel to the surface. Given the roles of Bazooka, a Drosophila homolog of ASIP/PAR-3, in the asymmetric division of the Drosophila neuroblast, these observations suggest that ASIP, along with other polarity proteins and adherens junction proteins, plays an important role in neural cell
differentiation by means of asymmetric cell division (Manabe, 2002).
Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens. This study shows that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects.
It is concluded that the Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation (Hao, 2010).
The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. This study demonstrates a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. Phosphorylated serine 144 is identified as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3ζ results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding (Hurd, 2003).
In many cases, 14-3-3 binding motifs are substrates for phosphorylation by AGC family protein kinases such as PKA, PKB, and PKC. Unlike phosphorylation of Par3 serine 827, phosphorylation of serine 144 appears to be PKC independent. Interestingly, in a recent report, the activity of the PKB-activating kinase PI3K was shown to be necessary for the correct localization of Par3 to axons of hippocampal neurons. To date, few substrates for the Par1 and Par4 homologs have been identified, and one may speculate that these kinases may directly phosphorylate other members of the Par family of polarity proteins. Indeed, recently it has been demonstrated that Drosophila 14-3-3ζ and epsilon are able to interact with Par1 via a putative 14-3-3 domain distinct from the phosphoserine binding region of the protein. It has been proposed that 14-3-3 thus acts to target Par1 to its cellular substrates. This observation would suggest that 14-3-3 may act to functionally link the Par3/Par6/aPKC complex with mammalian Par1 homologs. As such, determination of the kinases responsible for phosphorylating Par3 may provide further insight into the precise regulation of cell polarity in multiple cell types (Hurd, 2003).
Phosphorylation of the polarity protein Par-3 by the serine/threonine kinases aPKCzeta/iota and Par-1 (EMK1/MARK2) regulates various aspects of epithelial cell polarity, but little is known about the mechanisms by which these posttranslational modifications are reversed. This study finds that the serine/threonine protein phosphatase PP1 (predominantly the alpha isoform) binds Par-3, which localizes to tight junctions in MDCKII cells. PP1alpha can associate with multiple sites on Par-3 while retaining its phosphatase activity. By using a quantitative mass spectrometry-based technique, multiple reaction monitoring, it was shown that PP1alpha specifically dephosphorylates Ser-144 and Ser-824 of mouse Par-3, as well as a peptide encompassing Ser-885. Consistent with these observations, PP1alpha regulates the binding of 14-3-3 proteins and the atypical protein kinase C (aPKC) zeta to Par-3. Furthermore, the induced expression of a catalytically inactive mutant of PP1alpha severely delays the formation of functional tight junctions in MDCKII cells. Collectively, these results show that Par-3 functions as a scaffold, coordinating both serine/threonine kinases and the PP1alpha phosphatase, thereby providing dynamic control of the phosphorylation events that regulate the Par-3/aPKC complex (Traweger, 2008).
Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. Nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin (Drosophila homolog: Canoe) and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. This paper deals with the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 is necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promotes the association of afadin with nectin, whereas afadin is not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone is not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulates it. This paper describes these novel roles of Par-3 in the formation of junctional complexes (Ooshio, 2007).
aPKC (atypical protein kinase C), PAR-3 and PAR-6 interact with each other to form a ternary complex and thus mutually regulate their functionality and localization. The biochemical nature of the aPKC-PAR-3 interaction has been investigated in detail to clarify its functional importance in cell polarity. The highly conserved 26 amino acid sequence 816-841, in PAR-3 was found to be necessary and sufficient for the tight association with aPKC. Among several conserved serine/threonine residues within the region, aPKC preferentially phosphorylates serine-827 in vitro, and this phosphorylation reduces the stability of the PAR-3-aPKC interaction. Several analyses using a phospho-serine 827 specific antibody have established that this phosphorylation by aPKC occurs in vivo. Over-expression of a point mutant of PAR-3 (S827A), which is predicted to form a stable complex with aPKC, causes defects in the cell-cell contact-induced cell polarization of epithelial MDCK cells, similarly to a dominant negative mutant of aPKC. These results imply that serine 827 in the aPKC binding site of PAR-3 is a target of aPKC and that the regulated interaction between a protein kinase, aPKC, and its substrate, PAR-3, plays an essential role in the establishment of cell polarity (Nagai-Tamai, 2002).
Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. Mammalian Lgl is shown to compete for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with a previous observation that the complex containing PAR-3 promotes it.These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity (Yamanaka, 2003).
Thus evidence is presented showing that the PAR-6β/aPKCλ complex interacts with either mLgl or PAR-3 in a mutually exclusive manner, forming two independent protein complexes. Notably, overexpression of mLgl-2 inhibits TJ formation; this finding is in direct contrast with the data found for PAR-3, whose overexpression, but not that of its mutant lacking the aPKC binding region, promotes TJ formation. This suggests that the two independent complexes have distinct functions in the establishment of epithelial cell polarity. This is consistent with the results of genetic studies of Drosophila in which Lgl is required for formation of the basolateral membrane domain through the inhibition of the formation of apical identity, whereas subapical Bazooka (PAR-3) is required for the formation of the apical membrane domain (Yamanaka, 2003).
In polarized epithelial cells, mLgl localizes to the lateral region, in contrast to the PAR-6β/aPKCλ/PAR-3 complex that localizes to the apical end of the lateral domain. Interestingly, mLgl-2 transiently codistributes with PAR-6β and aPKCλ during the initial phase of epithelial cell polarization, whereas PAR-3 stably codistributes with them at the apical end of the cell-cell contact region; this finding indicates that the balance between the two independent complexes changes during the initial phase of epithelial cell polarization. Further, overexpression of aPKCλ kn (kinase deficient aPKC) results in the abnormal codistribution of PAR-6β and mLgl-2 at the cell periphery; this finding suggests that aPKCλ activity is required for the segregation of PAR-6β and mLgl-2 localization during this process. Thus, the present results, as well as previous findings, led to the following working model. The cell-cell contact initially stimulates the localization of the protein complex containing PAR-6β, aPKCλ, and mLgl at the cell-cell contact region. The complex is 'inactive' for TJ formation. Once aPKCλ is activated, mLgl segregates from the PAR-6β/aPKCλ complex. This triggers the formation of the 'active' PAR-6β/aPKCλ/PAR-3 complex that promotes the formation of the epithelial junctional complex. Segregated mLgl remains in the lateral region and seems to contribute to the establishment of the basolateral membrane identity, because mLgl-1 has been reported to interact with syntaxin-4, a component of the basolateral exocytic machinery. Although the mechanism for activation of aPKCλ remains to be clarified, Cdc42 and/or Rac1 are strong candidates as activators of aPKC in MDCK cells, since the GTP-bound form of Cdc42 activates aPKCλ kinase activity through PAR-6 in vitro and cell-cell adhesion activates Cdc42 and Rac1 in epithelial cells (Yamanaka, 2003).
mLgl is phosphorylated by aPKCλ and this phosphorylation increases in response to cell-cell adhesion-mediated cell polarization. Further, a phosphomimicking mutant of mLgl-2 (3SE) fails to bind to aPKCλ. These results imply that aPKCλ-dependent phosphorylation of mLgl is involved in the regulation of its interaction with the PAR-6β/aPKCλ complex. In contrast, no difference could be detected between mLgl-2 wild-type and its 3SE mutant in their interactions with the PDZ domain of PAR-6β. In addition, overexpression of mLgl-2 mutants (3SA or 3SE) affects TJ formation similarly to that of wild-type. These results suggest the existence of another mechanism regulating the interaction of mLgl-2 with PAR-6β. Mammalian Crumbs/Stardust (Pals1) has been shown to interact with the PDZ domain of PAR-6β and this interaction is enhanced by activated Cdc42. Taken together with the present results, this suggests that the Crumbs/Pals1 complex might also be involved in the regulation of the interaction between mLgl and the PAR-6β/aPKCλ complex; the PAR-6β/aPKCλ complex, together with PAR-3, may involve the Crumbs/Pals1 complex to promote TJ formation. Thus, the dissociation of mLgl from the PAR-6β/aPKCλ complex likely triggers the interaction of the PAR-6β/aPKCλ complex with the Crumbs/Pals1 complex in addition to its interaction with PAR-3. The functional interactions proposed by this model are consistent with the results of recent genetic studies of Drosophila in which Lgl and Crumbs compete with each other to define respective membrane identity (Yamanaka, 2003).
The findings in this study suggest a notable analogy in the mechanism regulating epithelial polarity between Drosophila and mammals. This notion is supported by a recent observation in Drosophila that Lgl interacts with PAR-6 and aPKC and regulates the apicobasal polarity of Drosophila neuroblasts. In mammalian fibroblasts, mLgl-1 has been reported to form a protein complex with PAR-6α (also called PAR-6C) and aPKC and is involved in the polarized migration of wounded MEF cells. Further studies will further an understanding of the molecular mechanism underlying the establishment of cell polarity in a variety of biological contexts (Yamanaka, 2003).
Thus mammalian Lgl competes for PAR-3 in forming an independent protein complex with PAR-6 and aPKC in epithelial cells. During epithelial cell polarization, mLgl transiently colocalizes with PAR-6 and aPKC at the cell-cell contact region, and increased localization of mLgl and PAR-6 to the cell-cell contact region suppresses TJ formation. This finding contrasts with the data found for PAR-3, which promotes TJ formation and thus indicates that the balance between the two independent protein complexes regulates the establishment of epithelial cell polarity. It is also suggested that aPKC activity-mediated phosphorylation of mLgl is involved in the regulation of mLgl's interaction with PAR-6/aPKC. These findings provide new insight into the mechanism underlying the establishment of epithelial cell polarity (Yamanaka, 2003).
How a neuron becomes polarized remains an as yet unanswered question. Selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity (Shi, 2003).
Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, a network of protein interactions have been identified that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. It is proposed that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking (Wells, 2006).
Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. This study found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex (Bultje, 2009).
The results presented here demonstrate that the evolutionarily conserved cell polarity protein mPar3 and the Notch signaling pathway act together to regulate the asymmetric cell division of radial glial progenitor cells in the developing neocortex. Mammalian Par3 is not statically restricted to the apical membrane domain of radial glial cells; instead, its distribution is dynamic depending on the cell cycle progression. It is selectively localized to the ZO-1- expressing lateral membrane domain in the ventricular endfeet during interphase and then becomes dispersed during mitosis. This dynamic distribution of mPar3 can lead to asymmetric inheritance of mPar3 by the two daughter cells, which results in differential Notch signaling activation that depends on Numb/Numb-like and distinct daughter cell fate specification. While the daughter cell that inherits a greater amount of mPar3 develops high Notch signaling activity and remains a radial glial cell, the daughter cell that inherits less mPar3 harbors low Notch signaling activity and adopts either a neuronal or an intermediate progenitor cell (IPC) fate (Bultje, 2009).
The dynamic nature of mPar3 subcellular localization in radial glial progenitor cells has not been shown previously. In fact, the distribution of mPar3 in dividing radial glial progenitor cells has not been rigorously examined. A recent study suggests that the mPar protein promotes the proliferation of progenitor cells. However, it is unclear whether the mPar protein regulates asymmetric radial glial cell division. Precisely determining the subcellular distribution of mPar3 in dividing radial glial cells is of critical importance to understanding its function and the molecular control of asymmetric cell division. Given the enrichment of mPar3 in interphase radial glial cells at the luminal surface of the VZ, where the cell bodies of scarce dividing radial glial cells are located, it is rather challenging to distinguish mPar3 in the cell bodies of dividing radial glial cells from that in the ventricular endfeet of interphase radial glial cells. To overcome this difficulty, advantage was taken of the phospho-Vimentin antibody, which selectively labels radial glial cells in mitosis. Moreover, the cytoplasmic labeling seen with this antibody helps to define the cell contour and its cleavage furrow, thereby facilitating the determination of the precise distribution of mPar3 and the cleavage plane of individual dividing radial glial cells. It was found that at E14.5 in about half of radial glial cells with a defined cleavage plane (i.e., in anaphase/telophase), mPar3 shows asymmetric distribution and the axis of the mPar3 asymmetry is perpendicular to the cleavage plane; this would result in a preferential segregation of mPar3 into one of the two future daughter cells (Bultje, 2009).
Previous studies showed that about half of the divisions in the VZ of the developing mouse cortex at this developmental stage are asymmetric and neurogenic. Although the current analysis of mPar3 asymmetry in dividing radial glial cells is likely an underestimation, these data suggest that the subcellular distribution of mPar3 (i.e., symmetric versus asymmetric) may be critical for determining the mode of division of radial glial cells. Indeed, it was found that disrupting mPar3 asymmetry in radial glial cells either by depletion or by ectopic expression of mPar3 prevents asymmetric cell division and promotes symmetric cell division. While the precise mechanisms underlying the establishment of the mPar3 asymmetry remain to be uncovered, the findings strongly suggest that mPar3 and its subcellular distribution regulate the mode of radial glial cell division and daughter cell fate specification in the developing neocortex (Bultje, 2009).
Interestingly, while both suppression of mPar3 expression and ectopic mPar3 expression impair asymmetric radial glial cell division, their effects on daughter cell fate specification are rather different. Ectopic mPar3 expression promotes radial glial cell fate, whereas suppression of mPar3 expression facilitates neuronal production. These results indicate that the inheritance level of mPar3 influences daughter cell fate specification, although mPar3 itself being an unlikely cell fate determinant. Intriguingly, it was found that the expression level of mPar3 affects Notch signaling activity, a key cell fate regulator required for proper neocortical neurogenesis. While a high level of mPar3 expression leads to high Notch signaling activity, a low level of mPar3 expression results in low Notch signaling activity. Previous studies have shown that Notch signaling activity is high in radial glial progenitor cells, but low in differentiating cells such as neurons. However, it is unclear how differential regulation of Notch signaling activity is initialized in the daughter cells of dividing radial glial progenitors. This study found that asymmetric segregation of mPar3 can lead to differential Notch signaling activity in the two daughter cells (Bultje, 2009).
In Drosophila neuroblasts, the asymmetric localization of Numb, a negative regulator of Notch signaling, is fundamental for differential Notch signaling activity and cell fate diversity in the central nervous system. Furthermore, this asymmetry in Numb distribution depends on the asymmetric segregation of Bazooka, the mPar3 ortholog in Drosophila. In mammals there are two Numb homologs, Numb and Numb-like. Previous studies suggest that Numb is essential for the proper development of the mammalian brain. However, the correlation between Numb protein segregation and asymmetric daughter cell fate specification has not been definitively established. In addition, recent studies suggest that Numb is involved in trafficking and proper localization of the junctional protein cadherin in radial glial cells and thereby functions in maintaining the tissue architecture of the developing neocortex. This study found that mPar3 acts through Numb and Numb-like in regulating Notch signaling activity. Moreover, the data suggest that a direct interaction between mPar3 and Numb is critical. Despite that it is unclear whether Numb is asymmetrically distributed in dividing radial glial progenitor cells, these findings suggest that asymmetric inheritance of mPar3, which interacts with Numb/Numb-like, results in differential activation of Notch signaling in the two daughter cells of asymmetrically dividing radial glial progenitors in the developing neocortex. Moreover, a recent study showed that removal of Cdc42 in the developing neocortex leads to mislocalization of mPar3 and defects in neocortical neurogenesis. Given that mPar3 and activated Cdc42 interact with each other, the findings coupled with these observations suggest that the mPar protein complex and its interacting proteins, such as Cdc42 and Lgl, likely represent an essential molecular pathway that regulates Notch signaling activity and asymmetric cell division of radial glial progenitor cells in the mammalian neocortex (Bultje, 2009).
A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. This study found that, in mammalian cultured cells, Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. It is proposed that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation (Nakayama, 2008).
This study found here that PAR-3 is heavily phosphorylated at Thr833 in the central and rear regions of polarized migrating cells, and this phosphorylation is diminished by treatment with kinase inhibitor Y-27632, suggesting that RhoA/Rho-kinase phosphorylates PAR-3 there. Consistently, RhoA activity is higher in the central and rear portions than in the front area in the migrating cells. Knockdown PAR-3 (PAR-3 KD) induced the cells into having multiple small leading edges without front-rear polarity, and the effect of PAR-3 KD was rescued by RNAi-resistant PAR-3 but not with mimic phosphorylation of PAR-3 (PAR-3-833D). Treatment with Y-27632 also induced the cells into having multiple small leading edges. Thus, the phosphorylation of PAR-3 by Rho-kinase may prevent Rac1 activation and lamellipodia formation at the leading edge to control front-rear polarity and directional migration (Nakayama, 2008).
It was also found that Rho-kinase phosphorylated PAR-3 in the leading edge of polarized migrating cells. This is consistent with the previous observations that RhoA and Rho-kinase are activated in both leading edge and rear regions in the migrating cell. What is the physiological significance of PAR-3 phosphorylation in the leading edge? It is speculated that spatio-temporal on-off regulation of Rac1 activity is necessary for proper cell migration. During cell migration, the adhesion signal from integrin may activate Rac1 through PAR-3 in Cdc42-dependent and -independent manners. Integrin also activates RhoA and thereby Rho-kinase. Rho-kinase phosphorylates PAR-3 and in turn disrupts the PAR complex, which can prevent overactivation of Rac1. In support of this, the treatment of HeLa cells with Rho-kinase inhibitor enhanced lamellar length and cell migration. This pathway may serve as a local negative feedback signal to control the leading edge (Nakayama, 2008).
Maintenance of single-layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of β1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal-shaped endothelial cells with dysregulated levels and mislocalization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). β1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in β1-deficient vessels. Combined, these findings demonstrate that β1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation (Zovein, 2010).
Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical protein kinase C (PKC) play a key role in the signaling pathway, but the molecules that regulate upstream signaling are still not known. The guanine nucleotide exchange factor ECT2 (Drosophila homolog: Peeble) has been identified as an activator of the polarity complex. ECT2 interacts with Par6 as well as Par3 and PKCzeta. Coexpression of Par6 and ECT2 efficiently activates Cdc42 in vivo. Overexpression of ECT2 also stimulates the PKCzeta activity, whereas dominant-negative ECT2 inhibits the increase in PKCzeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 are regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKCzeta and possibly plays a role in epithelial cell polarity (Liu, 2004).
The polarity protein Par-3 plays critical roles in axon specification and the establishment of epithelial apico-basal polarity. Par-3 associates with Par-6 and atypical protein kinase C and is required for the proper assembly of tight junctions, but the molecular basis for its functions is poorly understood. Depletion of Par-3 elevates the phosphorylated pool of cofilin, a key regulator of actin dynamics. Expression of a nonphosphorylatable mutant of cofilin partially rescues tight junction assembly in cells lacking Par-3, as does the depletion of LIM kinase 2 (LIMK2; see Drosophila LIM-kinase1), an upstream kinase for cofilin. Par-3 binds to LIMK2 but not to the related kinase LIMK1. Par-3 inhibits LIMK2 activity in vitro, and overexpressed Par-3 suppresses cofilin phosphorylation that is induced by lysophosphatidic acid. These findings identify LIMK2 as a novel target of Par-3 and uncover a molecular mechanism by which Par-3 could regulate actin dynamics during cell polarization (Chen, 2006).
Cell polarity plays a key role in the development of the central nervous system (CNS). Interestingly, disruption of cell polarity is seen in many cancers. ASPP2 is a haplo-insufficient tumor suppressor and an activator of the p53 family.
The ASPP family of proteins, consisting of three members, ASPP1, ASPP2, and iASPP, is characterized by a common C-terminal sequence: ankyrin repeats,
SH3 domain, and proline-rich region containing protein. The best known function of ASPP2 is its binding to p53, p63, and p73, and the stimulation of their apoptotic functions by selectively enhancing their DNA binding and transactivation
activities on pro-apoptotic genes such as BAX and PIG3. In the past decade, ASPP2 has also been identified as a binding partner of a number of other proteins. ASPP2 may, therefore, integrate different signaling pathways to safe-guard normal development and suppress tumor growth. To investigate this possibility, a detailed analysis of CNS defects was carried out in ASPP2 Dexon3 mice and, in doing so, revealed a novel function of ASPP2 in controlling cell polarity and cell proliferation during CNS development. This study shows that ASPP2 controls the polarity and proliferation of neural progenitors in vivo, leading to the formation of neuroblastic rosettes that resemble primitive neuroepithelial tumors. Consistent with its role in cell polarity, ASPP2 influences interkinetic nuclear migration and lamination during CNS development. Mechanistically, ASPP2 maintains the integrity of tight/adherens junctions. ASPP2 binds Par-3 and controls its apical/junctional localization without affecting its expression or Par-3/aPKC lambda binding. The junctional localization of ASPP2 and Par-3 is interdependent, suggesting that they are prime targets for each other. These results identify ASPP2 as a regulator of Par-3, which plays a key role in controlling cell proliferation, polarity, and tissue organization during CNS development (Sottocornola, 2010).
Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. This study shows that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4 (see Drosophila Pp4-19C), promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. The cell polarity protein Par3, a negative regulator of neuronal differentiation, is identified as a Smek1 substrate, and it was demonstrated that Smek1 suppresses Smek1 activity. Smek1, which is predominantly nuclear in NPCs, was also shown to be excluded from the nucleus during mitosis, allowing it to interact with cortical/cytoplasmic Par3 and mediate its dephosphorylation by the catalytic subunit PP4c. These results identify the PP4/Smek1 complex as a key regulator of neurogenesis (Lyu, 2013).
In mammalian epithelial cells PAR-3 forms a ternary complex with aPKC and PAR-6, and is localized to the tight junction that
has been suggested as being important for creating cell polarity. To
gain insights into the mode of PAR-3 function in mammalian epithelial cells, the effect of PAR-3 over-expression was examined in MDCK cells. Although exogenous PAR-3-expression does not affect the epithelial polarity of confluent cells, it drastically transforms the morphology of cells at low density into a fibroblastic form with developed membrane protrusions. Time-lapse observations have revealed that PAR-3 over-expressing cells show intense motility, even after they have assembled into loose colonies, suggesting that the contact-mediated inhibition of cell migration (CIM) is suppressed. The expressions of E-cadherin and vimentin do not change with PAR-3 over-expression, suggesting that exogenous PAR-3 only disturbs the endogenous equilibrium of cellular states between a fundamental fibroblastic structure and an epithelial one. The co-expression of a dominant negative mutant of Rac1 and the addition of nocodazole strongly antagonize the effect of PAR-3 over-expression, suggesting the involvement of Rac1 activation and microtubule polymerizations. The data presented here suggest an intriguing link between the contact-mediated inhibition of cell migration and the regulation of cell polarity. The putative PAR-3 activities demonstrated here may function endogenously in the epithelial cell polarization process by being sequestered from the cytosol to the cell-cell junctional regions with aPKC and PAR-6 upon cell-cell adhesion (Mishima, 2002).
Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. The endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. In cultured mammalian cells, Numb binds to integrin-βs and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, a model is proposed in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge (Nishimura, 2007).
Numb localizes at a part of CCSs and functions in integrin endocytosis as a cargo-selective adaptor. Integrin is thought to be recycled from the tail to the front of migrating cells by endocytosis. However, many focal adhesions or focal complexes formed at the cell front disassemble behind the F-actin-rich lamellipodia. Numb mainly accumulated behind lamellipodia, although a certain population of Numb still remained and colocalized with integrin at the trailing edge. In addition, localization of Numb among CCSs correlated with the position of integrin adhesions, supporting the role of Numb in integrin endocytosis. Talin is a key molecule that tethers integrin to components of focal adhesions and actin stress fibers and is critical for focal-adhesion disassembly. Mutation of a conserved tyrosine residue within the integrin-β3 intracellular domain abolished the binding of both talin and Numb, suggesting that talin and Numb cannot bind to integrin simultaneously. Consistent with these observations, interaction of Numb with talin could not be detected. In addition, the binding of Numb to integrin does not activate the integrin extracellular domain, whereas the binding of talin does. The overexpression or knockdown of Numb does not directly affect cell adhesion. Thus, it appears that Numb does not actively promote focal-adhesion disassembly, but rather recruits free integrins without the components of focal adhesions to the AP-2 complex for internalization. Preferential localization of Numb around focal adhesions at the substratum-facing surface would facilitate recruitment of integrin during focal adhesion disassembly (Nishimura, 2007).
Recent genetic screening isolated Numb as a mutant defective for peripheral glia migration along axons in Drosophila (Edenfeld, 2007). Migration defects of postmitotic neurons have been described in Numb-knockout mice, indicating that Numb regulates particular cell migration in vivo. However, the defects of Numb knockdown on integrin endocytosis and cell migration are less marked than those of AP-2 and clathrin knockdown, suggesting that another adaptor molecule(s) may function in integrin endocytosis. A good candidate is disabled-2 (Dab2), which has a similar domain structure as Numb and binds to both components of clathrin-mediated endocytosis and to integrin-β. Dab2 is expressed in HeLa cells and positively controls cell adhesion and spreading. In contrast to Numb, Dab2 appears to preferentially localize to the apical surface. Thus, Numb and Dab2 may coordinately function in integrin endocytosis in different subcellular compartments for cell motility (Nishimura, 2007).
How does Numb localize at the substratum-facing surface and polarize toward the leading edge? Integrin adhesions could activate several intracellular signaling events and promote protein transport to adhesion sites by targeting microtubules and linking actin stress fibers. The actin cytoskeleton and/or adhesion itself are important for the preferential localization of Numb around adhesions. However, Numb still localized at the substratum-facing surface in the presence of cytochalasin-D, indicating that an additional mechanism may exist. Observations indicate that direct phosphorylation by aPKC may be a part of the regulatory mechanism underlying Numb localization at the substratum-facing surface. In addition, polarized localization of Numb toward the leading edge was lost upon aPKC knockdown. In support of these observations, asymmetric localization of Numb in Drosophila has been shown to be dependent on cortical actomyosin and the polarized localization/function of aPKC and PAR-3. Conclusive evidence will require isolation of the responsible motor(s) and anchor protein(s) for specific Numb localization (Nishimura, 2007).
Numb-full-3A, mutated at three phosphorylation sites, did not function as a constitutively active form that promotes integrin endocytosis and cell migration, but rather inhibited these processes. Similarly, both the phospho-mimic and nonphosphorylated form of μ2-adaptin, which is phosphorylated by AAK1, inhibit transferrin endocytosis, suggesting that clathrin-mediated endocytosis is tightly controlled by cycles of phosphorylation and dephosphorylation. Additional phosphorylation during endocytosis may be required for the dissociation of Numb from the binding proteins, integrin-β, and α-adaptin. Numb is indeed phosphorylated by several PKCs and CaMKs, and phosphatase inhibitor dramatically increases the phosphorylation level of Numb. Thus, local phosphorylation and dephosphorylation seems to allow Numb to localize defined CCSs around adhesion sites (Nishimura, 2007).
Trafficking of internalized integrin is regulated by growth factors and the extracellular matrix through several adaptors/kinases, including PI3-kinase, PKB/Akt, GSK3β, and PKCs. Several growth factors and adhesions indeed promote the recycling of integrins, leading to the upregulation of cell-surface expression, whereas treatment of cells with PDGF does not affect the internalization rate of integrin. The degree of colocalization and interaction of Numb with integrin-β1 was not significantly altered in HeLa cells before and after wounding. These data suggest that Numb functions constitutively in integrin endocytosis, although it is possibile that polarized migration promotes the internalization rate and amount of integrin endocytosis. It might be difficult to detect the changes in the interaction of Numb and integrin during migration due to the nature of rapid cycling of endocytosis and exocytosis and possibly due to the transient interaction. It has been reported that the inhibition of directional membrane trafficking causes membrane extension in all directions. Membrane trafficking controls the directionality of migrating cells. Taking into account the fact that Numb localization becomes polarized coincidently with directional migration, the subcellular region at which integrin is internalized and the subsequent coupling with the recycling processes could be important for efficient cell migration suitable for the particular environment (Nishimura, 2007).
Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. This study shows that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell-cell contact. When actomyosin is not downregulated at cell-cell contacts, migrating cells lose cohesion. A molecular mechanism is provided for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1-Par3/Par6 complex controls the localization of RhoE to cell-cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell-cell contacts, a loss of cell-cell cohesion and defective collective cell invasion (Hidalgo-Carcedo, 2011).
Collective movement requires the coordination of actomyosin organization between cells. Actomyosin contractility is high around the edge of the cell cluster and low between cells. At the margin of the group, actomyosin is organized in a supracellular structure analogous to the 'purse-string' observed in epithelial wound closure. Both the elevated actomyosin levels observed around the edges of groups of invading cancer cells and 'purse-string' wound closure are dependent on Cdc42. Force is transmitted between cells through cell-cell contacts near the edge of the group. However, if force is applied uniformly around the cell margin, the cell junctions become compromised and the coordination of movement between neighbouring cells fails. Consistent with this, contact inhibition of locomotion and cell-cell repulsion are associated with increased Rho-driven actomyosin contraction function after cell-cell contact. Therefore a mechanism is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. The localization of DDR1 to cell-cell contacts requires E-cadherin. Once localized at cell-cell contacts, DDR1 helps to recruit Par3 and Par6; these molecules are required for efficient collective invasion. Cell polarity regulators are required for optimal migration in two-dimensional scratch/wound assays, which have some aspects of a collective nature. Moreover, Par3 and Par6 are required for collective migration of border cells in the Drosophila embryo41. The DDR1-Par3/Par6 complex then controls the localization of RhoE. RhoE may be localized through the intermediary p190ARhoGAP, which can bind both Par6 and RhoE. Consistent with this, depletion of p190ARhoGAP gave a similar phenotype to that after DDR1 or Par3/Par6 depletion. Both RhoE and p190ARhoGAP can antagonize Rho-ROCK-mediated regulation of actomyosin. The Par3-dependent suppression of actomyosin that was observe reciprocates the suppression of Par3 function by the actomyosin regulator ROCK. It is likely that the reciprocal nature of these negative interactions serves to segregate Par3 and actomyosin robustly. The DDR1-dependent mechanism that is describe most probably acts together with other proteins to decrease Rho-ROCK function at cell-cell contacts, such as p120 catenin-dependent mechanisms. This analysis has not yet allowed determination of all the components of DDR1 complexes at cell-cell contacts (Hidalgo-Carcedo, 2011).
Various regulators of cell polarity become misregulated in cancer; this has been linked to increased metastasis. It is believed that disruption of DDR1-dependent Par3 localization to cell-cell contacts might be expected to favour blood-borne metastasis. The data do not exclude a positive role for DDR1 in metastasis as a collagen receptor. Indeed, it was found that interference with DDR1 function in metastatic MTLn3 cells decreased their ability to colonize lung tissue. DDR1 expression may therefore not correlate simply with metastatic ability, but it is important to consider whether it is acting in a cell-matrix or cell-cell adhesion context: in the former it may promote single-cell cancer invasion and processes such as lung colonization; in the latter it may only promote more local and lymphatic invasion and hinder haematogenous metastasis. It is likely that DDR1 engages in different molecular complexes depending on whether it is involved in cell-cell interactions or cell-matrix interactions. For example, the data suggest that DDR1 does not associate with myosin IIa at cell-cell contacts but it has been reported to associate with myosin IIa in other contexts (Hidalgo-Carcedo, 2011).
This study described a mechanism that is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. DDR1 helps to recruit Par3 and Par6; this complex then controls the localization of RhoE, which can antagonize Rho-ROCK-mediated regulation of actomyosin. Thus, DDR1 functions at cell-cell contacts to keep actomyosin activity at low levels. Without this decrease in actomyosin activity, cell cohesion cannot be maintained during collective cell migration (Hidalgo-Carcedo, 2011).
There is growing evidence that contact inhibition of locomotion (CIL) is essential for morphogenesis and its failure is thought to be responsible for cancer invasion; however, the molecular bases of this phenomenon are poorly understood. This study investigated the role of the polarity protein Par3 in CIL during migration of the neural crest, a highly migratory mesenchymal cell type. In epithelial cells, Par3 is localised to the cell-cell adhesion complex and is important in the definition of apicobasal polarity, but the localisation and function of Par3 in mesenchymal cells are not well characterised. In Xenopus and zebrafish it was shown that Par3 is localised to the cell-cell contact in neural crest cells and is essential for CIL. The dynamics of microtubules are different in different parts of the cell, with an increase in microtubule catastrophe at the collision site during CIL. Par3 loss-of-function affects neural crest migration by reducing microtubule catastrophe at the site of cell-cell contact and abrogating CIL. Furthermore, Par3 promotes microtubule catastrophe by inhibiting the Rac-GEF Trio, as double inhibition of Par3 and Trio restores microtubule catastrophe at the cell contact and rescues CIL and neural crest migration. These results demonstrate a novel role of Par3 during neural crest migration, which is likely to be conserved in other processes that involve CIL such as cancer invasion or cell dispersion (Moore, 2013).
ASIP/PAR-3 staining distributes to the subapical domain of
epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy has revealed that ASIP/PAR-3
concentrates at the apical edge of tight junctions, whereas ZO-1 distributes
alongside tight junctions. To clarify the meaning of this characteristic
localization of ASIP, the effects of overexpressed ASIP/PAR-3 on
tight junction formation were analyzed in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3 promotes cell-cell contact-induced tight junction
formation in MDCK cells when evaluated on the basis of transepithelial
electrical resistance and occludin insolubilization. The significance of the
aPKC-binding sequence in tight junction formation is also supported by the
finding that the conserved PKC-phosphorylation site within this sequence,
ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts
during the initial phase of tight junction formation in MDCK cells. Together,
these data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC (Hirose, 2002).
Cellular asymmetry is critical for the development of multicellular organisms. Homologs of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. It is concluded that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signaling proteins that are implicated in mammalian cell polarity (Lin, 2000).
The polarization of the anterior-posterior axis (A-P) of the C. elegans zygote
depends on the activity of the par genes and the presence of intact microfilaments. However, functional links between the PAR proteins and the cytoskeleton have not been fully explored. In mammalian cells, some PAR homologs form a complex with activated Cdc42, a Rho GTPase that is implicated in the control of actin organization and cellular polarity. A role for Cdc42 in the establishment of embryonic polarity in C. elegans has not been described. To investigate the function of Cdc42 in the control of cellular and embryonic polarity in C. elegans, RNA-mediated interference (RNAi) was used to inhibit cdc-42 activity in the early embryo. RNAi of cdc-42 disrupts manifestations of polarity in the early embryo, these phenotypes depend on par-2 and par-3 gene function, and cdc-42 is required for the localization of the PAR proteins. This genetic analysis of the regulatory relationships between cdc-42 and the par genes
demonstrates that Cdc42 organizes embryonic polarity by controlling the localization and activity of the PAR proteins. Combined with the recent biochemical analysis of their mammalian homologs, these results simultaneously identify both a regulator of the PAR proteins, activated Cdc42, and effectors for Cdc42, the PAR complex (Kay, 2001).
Recent analyses in mammalian cells show that activated Cdc42 forms a complex with homologs of the C. elegans PAR-3, PAR-6, and PKC-3 proteins. In C. elegans, mutation or RNAi of these three genes causes similar polarity defects, including the disruption of spindle orientation in two-cell embryos. To determine whether the activity of this complex is required for the cdc-42(RNAi) polarity phenotypes, spindle orientation was scored in par-3; cdc-42(RNAi) two-cell embryos in which par-3 activity was eliminated either by mutation or RNAi. The spindles in both cells in par-3; cdc-42(RNAi) embryos are usually longitudinal, like in par-3 mutants, showing that par-3(+) activity inhibits nuclear-centrosome rotation in cdc-42(RNAi) embryos (Kay, 2001).
par-3(+) inhibition of nuclear-centrosome complex rotation correlates with the localization of PAR-3 protein in wild-type and par mutant embryos; therefore, it was asked whether cdc-42(RNAi) affects PAR-3 localization. In wild-type one-cell embryos, PAR-3 is at the anterior cortex. In two-cell embryos, PAR-3 is found around the entire periphery of the anterior cell, which divides transversely, while PAR-3 is restricted to the anterior cortex of the posterior cell, which divides longitudinally. In cdc-42(RNAi) one-cell embryos, PAR-3 is often localized as in wild type, but is also observed to be uniform, scattered throughout the cortex. In two-cell cdc-42(RNAi) embryos, PAR-3 is variable and often detected in cortical patches. Importantly, the A-P distribution of PAR-3 between the blastomeres is variable; patches of PAR-3 cortical staining could favor either end of the embryo, be scattered throughout the entire circumference of both blastomeres, or be concentrated between cells. In wild-type embryos, PAR-3 colocalizes with PAR-6, and in mammalian cells, activated Cdc42 binds PAR-6 homologs. Therefore, it was asked whether cdc-42(RNAi) similarly disrupts the cortical patterning of PAR-6. Wild-type and cdc-42(RNAi) embryos were stained with an anti-PAR-6 antibody and patchy PAR-6 localization was observed around the cortex of blastomeres in the cdc-42(RNAi) zygotes and early embryos. cdc-42 is therefore required to pattern the localization of both components of this conserved complex (Kay, 2001).
In wild type, the cortical localization of the PAR-3/PAR-6 complex correlates with the inhibition of spindle rotation; blastomeres with PAR-3 all around the cortex have transverse spindles, while blastomeres either lacking or with asymmetrically localized cortical PAR-3 have longitudinal spindles. Interestingly, in 26 cdc-42(RNAi) two-cell embryos costained with an anti-PAR-3 antibody to detect the complex and with an anti-tubulin antibody to determine spindle orientations, no significant correlation between PAR-3 localization or levels and spindle orientation was noted. Among the 17 embryos in which PAR-3 was mislocalized, spindle orientation correlates in only seven embryos. This indicates that when cdc-42 activity is reduced, PAR-3 localization is not sufficient to inhibit nuclear-centrosome rotation (Kay, 2001).
PAR-3 localization to the anterior cortex depends on mutual antagonistic interactions with PAR-2, a RING finger domain protein that localizes to an adjacent nonoverlapping posterior cortical domain. To determine whether cdc-42(RNAi) disrupts the interactions between par-2 and par-3, their distributions were examined in cdc-42(RNAi) embryos and PAR-3 localization was assayed in par-2(it5); cdc-42(RNAi) double mutants. The localization of a PAR-2::GFP protein fusion was followed in wild-type and cdc-42(RNAi) early embryos. PAR-2::GFP localization in wild-type embryos mirrors that reported for immunolocalization of PAR-2. PAR-2::GFP is uniformly associated with the cortex after fertilization, but then becomes asymmetrically distributed from the posterior of the embryo to the cytokinetic furrow. PAR-2::GFP ultimately surrounds the newly formed posterior cell only to again recede to the posterior as the cell cycle proceeds. In cdc-42(RNAi) zygotes, PAR-2::GFP localization is initially uniform around the cortex of the cell; but, unlike in wild type, it does not become confined to the posterior cortex. Following cell division, PAR-2::GFP is initially detected on the posterior and lateral sides of the anterior cell and can be detected in variable patches at the cortex of this cell throughout the next division. In the posterior cell, the localization of PAR-2::GFP does not become confined to the posterior cortex as it does in wild type. These PAR-2::GFP localization patterns were confirmed using an antibody to detect PAR-2 in fixed cdc-42(RNAi) embryos. From these patterns, it appears that PAR-2 and PAR-3 must overlap in cdc-42(RNAi) embryos. To confirm this, cdc-42(RNAi); PAR-2::GFP embryos were stained with anti-PAR-3 antibodies and PAR-2::GFP localization was observed by GFP fluorescence. In cdc-42(RNAi) one-cell and two-cell embryos, PAR-2::GFP and PAR-3 are often observed in overlapping domains. The pattern and extent of overlap is variable between embryos, but in two-cell embryos, both proteins are detected where the cells contact one another. Because PAR-2 and PAR-3 overlap in cdc-42(RNAi) embryos, but not in wild type, cdc-42 is required for PAR-2 and PAR-3 to become localized to opposing domains (Kay, 2001).
To determine whether par-2(+) influences PAR-3 localization in cdc-42(RNAi) embryos, PAR-3 localization was examined in par-2(it5);cdc-42(RNAi) double mutant embryos. Surprisingly, cortical PAR-3 is often localized to the anterior, as in wild type. In addition, early cell cycle times and cleavage orientations in these double mutant embryos are often restored to normal, although they do arrest with morphological defects characteristic of cdc-42(RNAi) embryos fertilized between 15 and 20 hr after injection. These results must be interpreted cautiously, considering that neither cdc-42(RNAi) or the par-2(it5) allele is likely to eliminate gene function; however, they do suggest that cdc-42 control of embryonic polarity is mediated by its interaction with the pars. It can be concluded, however, that the activities of PAR-2 and CDC-42 antagonize each other in the control of PAR-3 cortical patterning (Kay, 2001).
The polarity phenotypes in cdc-42(RNAi) embryos are similar to the phenotypes that are caused by disrupting actin dynamics during the first cell cycle . Although gross alterations in actin cortical localization were not detected in cdc-42(RNAi) embryos, it is possible that cdc-42(RNAi) may disrupt more refined modifications of the cytoskeleton. Similarly, expression of dominant-negative or active Cdc42 in MDCK epithelial cells does not cause a gross change in actin morphology, and in Drosophila nurse cells does not inhibit actin filament assembly. Finally, given that the cdc-42(RNAi) polarity phenotypes can be fully suppressed by par-2 mutations, it suggests that cdc-42(RNAi) phenotypes are not simply a direct result of gross perturbations in the actin cytoskeleton (Kay, 2001).
The clearest morphological affect on embryonic polarity that results from cdc-42(RNAi) activity is variable spindle orientation in two-cell embryos. This variability is likely caused by incomplete inhibition of cdc-42 activity as indicated by a time course study. Consistent with this interpretation, cdc-42(RNAi) embryos from injected hermaphrodites cultured at a lower temperature display a less variable phenotype. Here, the variability allowed for the scoring of functional relationships between cdc-42 and the pars. par-3(+) is required to inhibit nuclear-centrosome rotation in cdc-42(RNAi) embryos, suggesting that mislocalized par-3 activity may contribute to the variable cdc-42(RNAi) phenotype. Consistent with this, in cdc-42(RNAi) embryos, PAR-3 distribution is variable. Unexpectedly, PAR-3 localization does not correlate with the inhibition of nuclear-centrosome rotation in these embryos, suggesting that PAR-3 levels and/or the subcellular location of PAR-3 may be important for this function. Alternatively, cdc-42(+) may be required for par-3 function, or may act independently of PAR-3 to control nuclear-centrosome rotation. It is also possible that the colocalization of PAR-2 with PAR-3 interferes with this PAR-3 function (Kay, 2001).
Previous observations suggest that mutual antagonistic interactions between PAR-2 and PAR-3 contribute to embryonic patterning. Superficially, cdc-42(+) appears to be required for this interaction, given that in cdc-42(RNAi) embryos, the PAR-2 and PAR-3 domains overlap. Nevertheless, since reducing par-2 function in cdc-42(RNAi) embryos largely restores PAR-3 localization to a wild-type pattern, par-2(+) must disrupt PAR-3 localization in cdc-42(RNAi) embryos. Likewise, reducing cdc-42 function in par-2 embryos restores normal PAR-3 patterning, indicating that cdc-42(+) stimulates cortical PAR-3 localization in par-2 mutants. Since reducing or eliminating these activities in parallel does not greatly disturb the PAR-3 localization pattern, it seems likely that the spatial coordination of their opposing activities functions to ensure a sharp boundary of PAR-3 along the A-P axis (Kay, 2001).
The mutual suppression between cdc-42(RNAi) and par-2 could be interpreted in several ways. cdc-42(RNAi) suppression of par-2 may be mechanistically similar to the suppression of par-2 by par-6 heterozygosity. Specifically, par-2(it5); par-6/+ hermaphrodites produce many viable progeny. It has been proposed that wild-type par-2 may only be required to inhibit posterior PAR-3 localization when cortical PAR-3 levels are high. Given that PAR-3 is still present at higher anterior than posterior levels in par-2 mutant zygotes, any perturbation that reduces cortical PAR-3 levels may be sufficient to reestablish a significant A-P asymmetry. Because par-6 encodes a PDZ domain protein that is required for cortical PAR-3 localization, heterozygosity for par-6 may have a direct effect on cortical PAR-3 levels. Recent studies indicate that mammalian PAR-6 homologs contain a Cdc42/Rac1 binding (CRIB) motif, which when bound to activated Cdc42 stimulates the interactions between PAR-6 and both PAR-3 and PKC-3. Thus, PAR-6 appears to be either a direct regulator of, or effector for, Cdc42 activity. In C. elegans, cdc-42(RNAi) disrupts the asymmetric cortical localization of PAR-6. Because the localization of both PAR-3 and PAR-6 appears patchy in cdc-42(RNAi) embryos, it is suggested that in C. elegans, CDC-42 acts as a membrane-associated protein that is important for the assembly of the cortical PAR complex. In addition, the assembly or maintenance of this complex may be important for the segregation of PAR-2 and PAR-3 to discrete A-P cortical domains. If viable and fertile, a cdc-42 null mutation may, like par-6, eliminate the cortical localization of PAR-3 and cause a par-3-like phenotype (Kay, 2001).
This genetic analysis of the regulatory relationships between cdc-42 and the par genes indicates that Cdc42 organizes embryonic polarity by controlling the localization and activity of the PAR proteins. Analyses of par-3; cdc-42(RNAi) double mutants suggest that cdc-42 controls par-3 activity. Consistent with this, cdc-42(RNAi) disrupts the asymmetric localization of both PAR-3 and PAR-6, further suggesting that in the C. elegans embryo, CDC-42 controls the asymmetric localization of the PAR-3/PAR-6 complex to establish or maintain embryonic polarity. Although previous genetic analysis indicates that PAR-3 localization in the posterior of the embryo is inhibited by par-2(+), cdc-42(+) appears to act independently of par-2 in the cortical patterning of PAR-3. In mammalian cells, activated Cdc42 interacts with PAR-6, suggesting that the genetic interactions in C. elegans may reflect a conserved interaction between the PAR-3/PAR-6 polarity-promoting complex and Cdc42. Such an association appears to simultaneously provide the missing links between these known mediators of cellular polarity; that is, identifying both a regulator of the PARs and effectors for Cdc42. In the C. elegans embryo, Cdc42 could provide either a primary polarity cue for the asymmetric localization of the presumed PAR-3/PAR-6/PKC-3 complex, or it may function secondarily to its distribution. In mammalian epithelial cells, overexpression of an activated form of Cdc42 causes the mislocalization of PAR-3, suggesting that Cdc42 can provide a primary localization cue for PAR-3. It would be interesting to determine whether activated CDC-42 has a similar ability in C. elegans embryos. If so, then the A-P localization of GTP-bound CDC-42 (activated) could provide the primary cue for the A-P localization of the PAR proteins (Kay, 2001).
Generation of asymmetry in the one-cell embryo of C. elegans establishes the
anterior-posterior axis (A-P), and is necessary for the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C. elegans
had not yet been investigated, however. C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. CDC-42 physically
interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. It is concluded that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit (Gotta, 2001).
Phenotypic and two-hybrid data suggest that CDC-42 might activate the PAR-3/PAR-6/PKC-3 complex through interaction with PAR-6. CDC-42 appears to be necessary for the activity of the complex as well as for its correct localization. It is also possible that the PAR-3/PAR-6/PKC-3 complex has a role in activating CDC-42, since its initial anterior localization seems CDC-42 independent. One way that CDC-42 and the PAR-3/PAR-6/PKC-3 complex might direct polarity is through the regulation of the actin cytoskeleton. In par-3 mutants and in cdc-42(RNAi) embryos, enrichment of actin at the anterior of early embryos is lost. Further, cosuppression of par-2 and cdc-42(RNAi) mutant phenotypes suggests that CDC-42 and PAR-2 have counterbalancing, antagonistic activities. Because PAR-2 has a RING finger, a motif that has been proposed to be involved in ubiquitin-mediated protein degradation, CDC-42 might normally activate a protein that is a target of PAR-2. Future biochemical and in vivo studies should help to reveal the nature of these interactions and identify downstream targets (Gotta, 2001 and references therein).
Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell
contacts -- claudins, occludin, and junctional adhesion molecule (JAM) -- plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity
establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca2+ depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays have confirmed that assembly of functional TJs is delayed
by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs , but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells (Gao, 2002).
A model is proposed in which Par6 exists in an equilibrium between an inactive and an active state. The active state binds to the PDZ1 domain of Par3 and may block the interaction of Par3 with junctional adhesion molecule (JAM), thereby preventing TJ assembly. The Par3-JAM interaction is proposed to be reversible, but recruitment of further components stabilizes the TJ, so that junctions will eventually form even in the presence of inhibitory Par6. Activation is induced by binding either Cdc42-GTP or the regulatory domain of aPKC. Activation can be artificially induced by removing the N-terminal domain of Par6 (which binds aPKC). This model accounts for the inhibitory effects of the overexpression of wild-type Par6 (which increases the concentration of the active form), of the DeltaN mutant (constitutively active), and of the PKCzeta (1-126) fragment (which will bind to and activate endogenous Par6) and for the reduced potency of Par6BdeltaPro136 (defective in Cdc42 binding). It also accounts for the inhibitory effects of Cdc42 (Q61L) (which will activate endogenous Par6) and of the N-terminal region of Par3 (which will compete with endogenous Par3 for JAM binding). Whether this model is indeed correct will require further investigation (Gao, 2002).
Mammalian polarity proteins have been studied predominantly in cell culture systems, and little is known about their functions in vivo. To address this issue, a shRNA lentiviral system was used to manipulate gene expression in mouse mammary stem/progenitor cells. Transplantation of Par3-depleted stem/progenitor cells into the mammary fat pad severely disrupted mammary development, and glands were characterized by ductal hyperplasia, luminal filling, and highly disorganized end bud structures that were unable to remodel into normal ductal structures. Unexpectedly, Par3-depleted mammary glands also had an expanded progenitor population. A novel function was identified for the atypical protein kinase C (aPKC)-binding domain of Par3 in restricting Par3 and aPKC to the apical region in mammary epithelia in vivo; it was found that mammary morphogenesis is dependent on the ability of Par3 to directly bind aPKC. These results reveal a new function for Par3 in the regulation of progenitor differentiation and epithelial morphogenesis in vivo and demonstrate for the first time an essential requirement for the Par3-aPKC interaction (McCaffrey, 2009).
The Par polarity proteins play key roles in asymmetric division of Drosophila melanogaster stem cells; however, whether the same mechanisms control stem cells in mammals is controversial. Although necessary for mammary gland morphogenesis, Par3 is not essential for mammary stem cell function. This study discovered that, instead, a previously uncharacterized protein, Par3-like (Par3L), is vital for mammary gland stem cell maintenance. Par3L function has been mysterious because, unlike Par3, it does not interact with atypical protein kinase C or the Par6 polarity protein. Par3L was found to be expressed by multipotent stem cells in the terminal end buds of murine mammary glands. Ablation of Par3L resulted in rapid and profound stem cell loss. Unexpectedly, Par3L, but not Par3, binds to the tumour suppressor protein Lkb1 and inhibits its kinase activity. This interaction is key for the function of Par3L in mammary stem cell maintenance. These data reveal insights into a link between cell polarity proteins and stem cell survival, and uncover a biological function for Par3L (Huo, 2014).
The loss of epithelial polarity is thought to play an important role during mammary tumor progression. Using a unique transgenic mouse model of ErbB2-induced mammary tumorigenesis, it has been demonstrated that amplification of ErbB2 is frequently accompanied by loss of the 14-3-3sigma gene. This study demonstrates that ectopic expression of 14-3-3sigma results in restoration of epithelial polarity in ErbB2-transformed mammary tumor cells. Targeted deletion of 14-3-3sigma within primary mammary epithelial cells increases their proliferative capacity and adversely affects their ability to form polarized structures. Finally, it was shown that 14-3-3sigma can specifically form complexes with Par3, a protein that is essential for the maintenance of a polarized epithelial state. Taken together, these observations suggest that 14-3-3sigma plays a critical role in retaining epithelial polarity (Ling, 2010).
The concept that 14-3-3sigma plays an instrumental role in the regulation of epithelial polarity in vivo is supported by the mammary epithelial disruption of this key tumor suppressor. Although mammary epithelial-specific disruption of 14-3-3sigma had little impact on the initial stages of ductal outgrowth, histological analysis of 14-3-3sigma-deficient epithelium revealed an increase in the number of luminal epithelial cells that correlated with an increase in the proliferative capacity of these cells. Furthermore, in contrast to well-organized single-layer epithelial organoids derived from wild-type mammary epithelium, the 14-3-3sigma-deficient organoids were filled structures that bore a remarkable similarity to mammary organoids that ectopically express ErbB2. Consistent with the importance of 14-3-3sigma in controlling polarity of luminal mammary epithelium, the mammary glands derived from the hypomorphic conditional 14-3-3sigma strain exhibited an identical luminal hyperproliferation phenotype, suggesting that a critical threshold of 14-3-3sigma is required to restrict luminal epithelial proliferation (Ling, 2010).
While the precise mechanisms that modulate epithelial polarity are unclear, proteomic analyses have revealed that 14-3-3sigma can interact with several proteins that play important roles in regulating cell contacts. Indeed, this study showed that 14-3-3sigma interacts with the Par3 component of the polarity complex, although the association with Par3 might not be direct. Loss of 14-3-3sigma leads to dislocation of Par3 from cell membranes, while it has little impact on Par3 protein levels in TM15, mouse primary mammary epithelium, or MDCK cells. Previous studies in Drosophila have indicated that 14-3-3 protein plays a crucial role in establishing epithelial polarity through its action on Par3 function (Benton, 2003). There is also recent evidence to suggest that Par3 plays a crucial function in regulating mammary epithelial biology. Disruption of Par3 in the mammary epithelium results in mammary epithelial hyperplasia with luminal filling (McCaffrey, 2009). Further evidence supporting the role of 14-3-3sigma in epithelial polarity derives from studies of a mouse mutant bearing a truncated 14-3-3sigma protein. Mice heterozygous for this truncated 14-3-3sigma gene have disrupted epithelial stratification in the skin, and homozygous fetuses die shortly after birth with severe skin abnormality. In addition, primary corneal epithelial cells expressing this dominant-negative protein failed to differentiate and form tight junctions (Xin, 2010). Taken together with the current observations, these data suggest that 14-3-3sigma plays a critical role in regulating epithelial polarity. In contrast to the positive effects of 14-3-3sigma on epithelial polarity, ectopic expression of 14-3-3zeta results in disruption of epithelial polarity of MDCK cells through binding Par3. Moreover, elevated expression of 14-3-3zeta can enhance the invasiveness of mammary tumor cell lines. It is conceivable that the opposing effects on epithelial polarity of these two closely related 14-3-3 proteins is due to their ability to localize Par3 to distinct apical and basal compartments within the cell (Ling, 2010).
The observation that ErbB2 tumors are associated with loss of 14-3-3sigma has important implications in understanding the genetic events involved in ErbB2-induced tumor progression. The fact that 14-3-3sigma is a major positive regulator of epithelial polarity suggests that loss of polarity may be an important step in tumorigenesis and metastasis. Consistent with this concept, it has been demonstrated recently that loss of the Scribble polarity regulator plays an important role in c-Myc-induced mammary tumors. Whether 14-3-3sigma plays a comparable role in ErbB2 mammary tumor progression remains to be addressed (Ling, 2010).
Asymmetric self-renewing division of neural precursors is essential for brain development. Partitioning-defective (Par) proteins promote self-renewal, and their asymmetric distribution provides a mechanism for asymmetric division. Near the end of neural development, most asymmetric division ends and precursors differentiate. This correlates with Par protein disappearance, but mechanisms that cause downregulation are unknown. MicroRNAs can promote precursor differentiation but have not been linked to Par protein regulation. This study tested a hypothesis that microRNA miR-219 promotes precursor differentiation by inhibiting Par proteins. Neural precursors in zebrafish larvae lacking miR-219 function retained apical proteins, remained in the cell cycle, and failed to differentiate. miR-219 inhibited expression via target sites within the 3' untranslated sequence of pard3 and prkci mRNAs, which encode Par proteins, and blocking miR-219 access to these sites phenocopied loss of miR-219 function. It is proposed that negative regulation of Par protein expression by miR-219 promotes cell-cycle exit and differentiation (Hudish, 2013).
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