Mixed synthetic oligonucleotides encoding a sequence conserved among tyrosine-specific protein kinases were used to probe the genome of the fission yeast Schizosaccharomyces pombe. A single gene (kin1+) was isolated that encodes a putative protein kinase closely related to the KIN1- and KIN2-encoded serine/threonine-specific protein kinases of Saccharomyces cerevisiae. kin1+ is transcribed into a 3.5-kilobase mRNA that contains an uninterrupted open reading frame encoding a polypeptide of 98 kDa. In contrast to results obtained with kin mutants of S. cerevisiae, disruption of the Sc. pombe kin1+ gene results in recessive morphological and growth defects. kin1-disrupted cells grow slowly on enriched medium and grow as spheres, in contrast to wild-type Sc. pombe cells, which grow as rods. Relative to kin1+ cells, kin1-disrupted cells are differentially sensitive to lysis by treatment with alpha- and beta-glucanases, suggesting an alteration in either the composition or the organization of their cell walls (Levin, 1990).
The kin1 protein kinase of the fission yeast Schizosaccharomyces pombe is a member of the PAR-1/MARK (partitioning-defective 1/microtubule-associated protein/microtubule affinity-regulating kinase) family important in eukaryotic cell polarity and cytoskeletal dynamics. kin1 plays a role in establishing the characteristic rod-shaped morphology of fission yeast. Cells in which kin1 was deleted are viable but are impaired in growth, and are rounded at one end or both ends. They are monopolar because after mitosis they fail to activate bipolar growth, and are delayed in cytokinesis, resulting in a high proportion of septated cells often with multiple septa. This phenotype can be partially rescued by heterologous expression of human MARK proteins, which restore bipolar growth in most cells, but do not correct the delay in cytokinesis. Using chromosomal epitope tagging, it has been shown that kin1p localizes to the cell ends, except during mitosis when it disappears from cell ends. After mitosis, kin1p first reappears at the new cell end. Overexpression of kin1 results in a loss of polarity, with partially or fully rounded cells. From these results it is suggested that kin1 is required to direct the growth machinery to the cell ends (Drewes, 2003).
Eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans have been isolated and analyzed. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to the absence of functional germ cells. It is proposed that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos (Kemphues, 1988).
The autonomous or cell-intrinsic developmental properties of early embryonic blastomeres in nematodes are thought to result from the action of maternally provided determinants. After the first cleavage of the C. elegans embryo, only the posterior blastomere, P1, has a cell-intrinsic ability to produce pharyngeal cells. The product of the maternal gene skn-1 is required for P1 to produce pharyngeal cells. The Skn-1 protein is nuclear localized and P1 appears to accumulate markedly higher levels of Skn-1 protein than its sister, the AB blastomere. The distribution of Skn-1 protein was examined in embryos from mothers with maternal-effect mutations in the genes mex-1, par-1, and pie-1. These results suggest that mex-1(+) and par-1(+) activities are required for the unequal distribution of the Skn-1 protein and that pie-1(+) activity may function to regulate the activity of Skn-1 protein in the descendants of the posterior blastomere P1 (Bowerman, 1993).
The first cleavage of C. elegans is asymmetric, generating daughter cells with different sizes, cytoplasmic components, and fates. Mutations in the par-1 gene disrupt this asymmetry. par-1 encodes a putative Ser/Thr kinase with similarity to kinases from yeasts and mammals. Two strong alleles have mutations in the kinase domain, suggesting that kinase activity is essential for par-1 function. PAR-1 protein is localized to the posterior periphery of the zygote and is distributed in a polar fashion preceding the asymmetric divisions of the germline lineage. Because PAR-1 distribution in the germline correlates with the distribution of germline-specific P granules, it is possible that PAR-1 functions in germline development as well as in establishing embryonic polarity (Guo, 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).
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 that 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. 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).
After fertilization in C. elegans, activities encoded by the maternally expressed par genes appear to establish cellular and embryonic polarity. Loss-of-function mutations in the par genes disrupt A/P asymmetries in early embryos and result in highly abnormal patterns of cell fate. Little is known about how the early asymmetry defects are related to the cell fate patterning defects in par mutant embryos, or about how the par gene products affect the localization and activities of developmental regulators known to specify the cell fate patterns made by individual blastomeres. Examples of such regulators of blastomere identity include the maternal proteins MEX-3 and GLP-1, expressed at high levels anteriorly, and SKN-1 and PAL-1, expressed at high levels posteriorly in early embryos. To better define par gene functions, the expression patterns of MEX-3, PAL-1 and SKN-1 were examined, and mex-3, pal-1, skn-1 and glp-1 activities were examined in par mutant embryos. Mutational inactivation of each par gene results in a unique phenotype, but in no case is a complete loss of A/P asymmetry observed. It is concluded that no one par gene is required for all A/P asymmetry and it is suggested that, in some cases, the par genes act independent of one another to control cell fate patterning and polarity. Discussed are the implications of these findings for the furtherance of an understanding of how the initial establishment of polarity in the zygote by the par gene products leads to the proper localization of more specifically acting regulators of blastomere identity (Bowerman, 1997).
The translation of maternal glp-1 mRNA is regulated both temporally and spatially in the early C. elegans embryo. To investigate the control of embryonic glp-1 expression, the distribution of GLP-1 protein was examined in selected maternal effect mutants that affect pattern or fate in the early embryo. Mutants that disrupt anterior-posterior asymmetry in the early embryo [par-1-par-6, emb-8, Par(q537)] disrupt the spatial but not temporal control of GLP-1 expression: GLP-1 is observed at the normal stage of embryogenesis in par-like mutants; however, it is uniformly distributed. In contrast, mutants that alter blastomere identity (skn-1, pie-1, mex-1, apx-1) do not affect the normal GLP-1 pattern. It is concluded that genes controlling the asymmetry of cellular components, including P granules, also control GLP-1 asymmetry in the early embryo. The finding that mutants that disrupt anterior-posterior asymmetry translate GLP-1 in all blastomeres suggests that loss of embryonic asymmetry causes translational activation of GLP-1 in the posterior (Crittenden, 1997).
Establishment of A/P polarity in the C. elegans embryo depends on filamentous (F-) actin. An F-actin-binding protein that is enriched in the anterior cortex of the one-cell embryo is hypothesized to link developmental polarity to the actin cytoskeleton. This protein, POD-1, as a new member of the coronin family of actin-binding proteins. A deletion has been genereated within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the A/P axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for A/P polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. It is concluded that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. Models have been put forth, attempting to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic A/P axis (Rappleye, 1999).
The KH domain protein MEX-3 is central to the temporal and spatial control of PAL-1 expression in the C. elegans early embryo. PAL-1 is a Caudal-like homeodomain protein that is required to specify the fate of posterior blastomeres. While pal-1 mRNA is present throughout the oocyte and early embryo, PAL-1 protein is expressed only in posterior blastomeres, starting at the four-cell stage. To better understand how PAL-1 expression is regulated temporally and spatially, MEX-3 interacting proteins (MIPs) have been identified and two that are required for the patterning of PAL-1 expression are described in detail. RNA interference of MEX-6, a CCCH zinc-finger protein, or SPN-4, an RNA recognition motif protein, causes PAL-1 to be expressed in all four blastomeres starting at the four-cell stage. Genetic analysis of the interactions between these mip genes and the par genes, which provide polarity information in the early embryo, defines convergent genetic pathways that regulate MEX-3 stability and activity to control the spatial pattern of PAL-1 expression. These experiments suggest that par-1 and par-4 affect distinct processes. par-1 is required for many aspects of embryonic polarity, including the restriction of MEX-3 and MEX-6 activity to the anterior blastomeres. PAL-1 is not expressed in par-1 mutants, because MEX-3 and MEX-6 remain active in the posterior blastomeres. The role of par-4 is less well understood. This analysis suggests that par-4 is required to inactivate MEX-3 at the four-cell stage. Thus, PAL-1 is not expressed in par-4 mutants because MEX-3 remains active in all blastomeres. It is proposed that MEX-6 and SPN-4 act with MEX-3 to translate the temporal and spatial information provided by the early acting par genes into the asymmetric expression of the cell fate determinant PAL-1 (Huang, 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).
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).
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 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).
The Caenorhabditis elegans vulva provides a simple model for the genetic analysis of pattern formation and organ morphogenesis during metazoan development. An essential role for the polarity protein PAR-1 in the development of the vulva has been discovered. Postembryonic RNA interference of PAR-1 causes a protruding vulva phenotype. Depleting PAR-1 during the development of the vulva has no detectable effect on fate specification or precursor proliferation, but instead seems to specifically alter morphogenesis. Using an apical junction-associated GFP marker, PAR-1 depletion was found to cause a failure of the two mirror-symmetric halves of the vulva to join into a single, coherent organ. The cells that normally form the ventral vulval rings fail to make contact or adhere and consequently form incomplete toroids, and dorsal rings adopt variably abnormal morphologies. PAR-1 undergoes a redistribution from apical junctions to basolateral domains during morphogenesis. Despite a known role for PAR-1 in cell polarity, no detectable differences has been observed in the distribution of various markers of epithelial cell polarity. It is proposed that PAR-1 activity at the cell cortex is critical for mediating cell shape changes, cell surface composition, or cell signaling during vulval morphogenesis (Hurd, 2003).
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).
Anteroposterior polarity in early C. elegans embryos is required for the specification of somatic and germline lineages, and is initiated by a sperm-induced reorganization of the cortical cytoskeleton and PAR polarity proteins. Through mechanisms that are not understood, the kinases PAR-1 and PAR-4, and other PAR proteins cause the cytoplasmic zinc finger protein MEX-5 to accumulate asymmetrically in the anterior half of the one-cell embryo. MEX-5 asymmetry requires neither vectorial transport to the anterior, nor protein degradation in the posterior. MEX-5 has a restricted mobility before fertilization and in the anterior of one-cell embryos. However, MEX-5 mobility in the posterior increases as asymmetry develops, presumably allowing accumulation in the anterior. The MEX-5 zinc fingers and a small, C-terminal domain are essential for asymmetry; the zinc fingers restrict MEX-5 mobility, and the C-terminal domain is required for the increase in posterior mobility. A crucial residue in the C-terminus, Ser 458, is phosphorylated in vivo. PAR-1 and PAR-4 kinase activities are required for the phosphorylation of S458, providing a link between PAR polarity proteins and the cytoplasmic asymmetry of MEX-5 (Tenlen, 2008).
The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work little is known how exocyst function is controlled. To identify regulators for exocyst function, a targeted RNAi screen was performed in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. Six kinase and seven phosphatase genes were found that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. In line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic SNARE proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the PP2A phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular GFP signal for the same proteins. Double RNAi experiments for par-1, pkc-3 and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in post-embryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that aPKC kinase and phosphatase PP2A regulate exocyst-dependent insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process (Jiu, 2013).
Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, this study found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 (Drosophila Lkb1) and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. These results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division (Benkemoun, 2014).
pEg3 is a Xenopus protein kinase related to members of the KIN1/PAR-1/MARK family. The founding members of this newly emerging kinase family are involved in the establishment of cell polarity and both microtubule dynamic and cytoskeleton organization. Sequence analyses suggest that pEg3 and related protein kinases in human, mouse, and Caenorhabditis elegans might constitute a distinct group in this family. pEg3 is encoded by a maternal mRNA, polyadenylated in unfertilized eggs and specifically deadenylated in embryos. In addition to an increase in expression, pEg3 is phosphorylated during oocyte maturation. Phosphorylation of pEg3 is cell cycle dependent during Xenopus early embryogenesis and in synchronized cultured XL2 cells. In embryos, the kinase activity of pEg3 is correlated to its phosphorylation state and is maximum during mitosis. Using Xenopus egg extracts it has been demonstrated that phosphorylation occurs at least in the noncatalytic domain of the kinase, suggesting that this domain might be important for pEg3 function (Blot, 2002).
Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro. A protein-serine kinase has been purified and characterized from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes the microtubules dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtuble-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease (Drewes, 1995).
The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. Recently, a novel protein kinase, termed p110 microtubule-affinity regulating kinase ('MARK'), has been shown to phosphorylate microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and to cause the detachment of tau from microtubules. p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells (Illenberger, 1996).
MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics. Describe here is the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes. They encode Ser/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+ and C. elegans par-1 genes that are involved in the establishment of cell polarity. Expression of both isoforms is ubiquitous, and homologous genes are present in humans. Catalytic activity depends on phosphorylation of two residues in subdomain VIII. Overexpression of MARK in cells leads to hyperphosphorylation of MAPs on KXGS motifs and to disruption of the microtubule array, resulting in morphological changes and cell death (Drewes, 1997).
The establishment of polarity in the embryo is fundamental for the correct development of an organism. The first cleavage of the C. elegans embryo is asymmetric with certain cytoplasmic components being distributed unequally between the daughter cells. Six par genes (partition-defective) have been identified that are involved in the process of asymmetric division. One of these genes encodes a highly conserved protein, PAR-1, which is a serine/threonine kinase that localizes asymmetrically to the posterior part of the zygote and to those blastocysts that give rise to the germ line. It was reasoned that the mammalian homolog of PAR-1 (mPAR-1) might be involved in the process of polarization of epithelial cells, which consist of apical and basolateral membrane domains. mPAR-1 is expressed in a wide variety of epithelial tissues and cell lines and is associated with the cellular cortex. In polarized epithelial cells, mPAR-1 is asymmetrically localized to the lateral domain. A fusion protein lacking the kinase domain has the same localization as the full-length protein but its prolonged expression acts in a dominant-negative fashion: lateral adhesion of the transfected cells to neighboring cells is diminished, resulting in the former cells being 'squeezed out' from the monolayer. Moreover, the polarity of these cells is disturbed, resulting in mislocalization of E-cadherin. Thus, in the C. elegans embryo and in epithelial cells, polarity appears to be governed by similar mechanisms (Bohm, 1997).
Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, a breakdown of the vimentin network is also observed, whereas actin fibers remain unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics (Ebneth, 1999).
One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. The relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs has been examined. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation (Schneider, 1999).
Tau is a microtubule-associated protein (MAP) that is functionally modulated by phosphorylation and that is hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of interest to identify the signaling pathways and enzymes capable of modulating tau phosphorylation in vivo. In SH-SY5Y human neuroblastoma cells and rat primary cortical cultures tau is phosphorylated at Ser262/356, within its microtubule-binding domain, by a staurosporine-sensitive protein kinase in response to the vicinal thiol-directed agent phenylarsine oxide. A 100-kDa protein kinase activity is present in SH-SY5Y cells that associates with microtubules, phosphorylates tau at Ser262/356, is activated by phenylarsine oxide, and is inhibited by the protein kinase inhibitor staurosporine. Isolation of individual protein bands from a polyacrylamide gel reveal two closely spaced proteins containing Ser262/356-directed protein kinase activity. These protein bands correspond to the 100-kDa microtubule/MAP-affinity regulating kinase (MARK), which phosphorylates tau within its microtubule-binding domain. Immunoblot analysis of the protein kinase bands confirm this finding, providing the first demonstration that activation of endogenous MARK results in increased tau phosphorylation within its microtubule-binding domain in situ (Jenkins, 2000).
Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. MARK2 affects the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites (Biernat, 2002).
MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubule-associated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. The activation of MARK was studied and the upstream kinase, MARKK, a member of the Ste20 kinase family, was identified. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation (Timm, 2003).
The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule binding domain of tau that is critical for microtubule binding. A novel human paralog, MARK4, shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%); MARK4 readily phosphorylates tau and the related microtubule-associated proteins MAP2 and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, a tandem-affinity purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. It is suggested that, while the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimers disease (Trinczek, 2003).
aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. In contrast, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. PAR-1b is shown to be essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. In contrast, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity (Suzuki, 2004).
In the C. elegans one-cell embryo as well as the Drosophila late oocyte, the complex segregate along the A-P axis: the aPKC/PAR-3/PAR-6 complex then localizes at the anterior cortex, whereas PAR-1 is at the posterior cortex. In Drosophila and mammalian epithelial cells, the complex segregates along the apicobasal axis: PAR-1 localizes at the basolateral membrane in contrast with the apical localization of the aPKC/PAR-3/PAR-6 complex. These observations raise questions whether the functional hierarchy of the aPKC/PAR-3/PAR-6 complex and PAR-1 is conserved evolutionarily. The functional relationship between aPKC, PAR-1b, and 14-3-3/PAR-5 suggested in this study is different from that suggested for Drosophila epithelial cells. In Drosophila follicle cells, PAR-1 inhibits the lateral invasion of aPKC, and the phospho-motif binding site of 14-3-3 binds to BAZ. These differences suggest the possibility that mammals and flies independently evolved similar but distinct mechanisms that regulate epithelial cell polarity using aPKC/PAR proteins. However, it is also possible that mutual regulations between PAR-1 and aPKC occur in both organisms, because most of the results in each study are not completely exclusive. For example, although the current study observed that PAR-1b depletion from MDCK cells did not induce the lateral leakage of aPKC and PAR-3, the possibility cannot be excluded that other mammalian PAR-1 proteins compensate for the PAR-1b function in these cells. To address this issue, perfectly corresponding experiments should be performed in each organism (Suzuki, 2004).
The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Human Par-1b (hPar-1b) has been shown to interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells. These findings suggest that one mechanism by which atypical PKC exerts its effect on the generation and/or maintenance of polarity may be to actively exclude Par-1 from particular subcellular compartments. Presumably the localization of Par-1 to different cellular domains would also allow it to interact with a different set of relevant effectors. Thus, phosphorylation of Par-1 by aPKC may enforce the mutual exclusion of Par-1 from the anterior cortex of C. elegans embryos and from epithelial cell tight junctions where Par-3/Par-6/aPKC complexes reside (Hurov, 2004).
The kinase PAR-1 plays conserved roles in cell polarity. PAR-1 has also been implicated in axis establishment in C. elegans and Drosophila and in Wnt signaling, but its role in vertebrate development is unclear. PAR-1 has two distinct and essential roles in axial development in Xenopus mediated by different PAR-1 isoforms. Depletion of PAR-1A or PAR-1BX causes dorsoanterior deficits, reduced Spemann organizer gene expression, and inhibition of canonical Wnt-β-catenin signaling. By contrast, PAR-1BY depletion inhibits cell movements and localization of Dishevelled protein to the cell cortex, processes associated with noncanonical Wnt signaling. PAR-1 phosphorylation sites in Dishevelled are required for this translocation, but not for canonical Wnt signaling. It is concluded that PAR-1BY is required in the PCP branch and mediates Dsh membrane localization while PAR-1A and PAR-1BX are essential for canonical signaling to β-catenin, possibly via targets other than Dishevelled (Ossipova, 2005).
Since the identification of PAR-1 as a Dishevelled-associated kinase, its precise role in Wnt signaling has been unclear. The question remains as to whether phosphorylation of Dsh by PAR-1 kinase is an essential process in Wnt-β-catenin signal transduction. The results show that the role of PAR-1 in Wnt signaling is complex and isoform dependent. Isoforms PAR-1A and PAR-1BX were identified that exert effects primarily on canonical Wnt signaling: they are physiologically essential in the Xenopus embryo for establishment of the organizer. This is an important finding because although PAR-1 has previously been implicated in canonical Wnt signaling, any understanding of its importance for endogenous Wnt signaling has remained ambiguous. A mutated Dishevelled that lacks PAR-1 sites is capable of driving a canonical β-catenin signal, which is still further potentiated by overexpressed PAR-1. This raises the possibility that PAR-1 acts on other components of the Wnt pathway that remain to be identified. Consistent with this notion, three other Dsh kinases in the canonical Wnt pathway, namely CKIα, CKIε, and CKII, have alternative substrates in addition to Dsh, namely β-catenin, APC, and TCF3, respectively (Ossipova, 2005).
A third PAR-1 isoform, PAR-1BY, differs from the other two in its role in vivo, being required for convergent extension but not for canonical Wnt signaling or organizer formation. The PAR-1B isoforms differ by only 10% at the amino acid level, but most of this difference is concentrated at the N terminus and in the spacer region around serine 646. The amino terminus of PAR-1BX is present in the Xenopus tropicalis homolog but is not conserved outside the Xenopus genus. The spacer differences are seen in sequenced vertebrates as alternative splicing of exons that are conserved, although whether this is the case in Xenopus laevis in particular will remain unknown until its genome is sequenced (Ossipova, 2005).
Finally, the data reveal a critical mechanism of action of PAR-1 in regulating Dsh, namely its translocation from cytoplasmic vesicles to the cell cortex. This translocation is typically concomitant with noncanonical PCP signaling, and indeed it has been shown that PAR-1 is not just sufficient for JNK regulation, but is also necessary endogenously. PAR-1 is shown to be (1) sufficient and necessary to drive this translocation; (2) that PAR-1 kinase activity is activated by Frizzleds, and (3) that the kinase activity and its targets in Dishevelled are essential, at least for exit from vesicular structures. These findings reveal a pathway leading from Frizzled receptors to elevation of PAR-1 kinase to phosphorylation of Dsh on specific serine and threonine residues to translocation of Dsh from vesicles to the cell cortex. PAR-1 is thus a vertebrate missing link between Frizzled and Dsh. The C-terminal DEP domain of Dsh is sufficient for translocation. It therefore seems likely that PAR-1-dependent phosphorylation, acting elsewhere, regulates some protein-protein interaction that retains Dsh in the cytoplasm. In Drosophila, Dishevelled isoforms lacking the PAR-1 target sites are able to rescue Wingless but not PCP mutants. A mechanism for this phenomenon has been identified, namely that PAR-1 is necessary for Dsh localization to the membrane and therefore fails to localize such Dsh mutants properly within the cell. Thus, the 'noncanonical isoform' of PAR-1 has a role consistent with its classification as a polarity protein, albeit planar cell polarity rather than the previously reported apicobasal polarity. Whether the 'canonical PAR-1' isoforms have polarity roles at all remains to be seen (Ossipova, 2005).
Aberrant phosphorylation of tau is associated with a number of neurodegenerative diseases, including Alzheimer's disease (AD). The molecular mechanisms by which tau phosphorylation is regulated under normal and disease conditions are not well understood. Microtubule affinity regulating kinase (MARK) and PAR-1 have been identified as physiological tau kinases, and aberrant phosphorylation of MARK/PAR-1 target sites in tau has been observed in AD patients and animal models. This study shows that phosphorylation of PAR-1 by the tumor suppressor protein LKB1 is required for PAR-1 activation, which in turn promotes tau phosphorylation in Drosophila. Diverse stress stimuli, such as high osmolarity and overexpression of the human beta-amyloid precursor protein, can promote PAR-1 activation and tau phosphorylation in an LKB1-dependent manner. These results reveal a new function for the tumor suppressor protein LKB1 in a signaling cascade through which the phosphorylation and function of tau is regulated by diverse signals under physiological and pathological conditions (Wang, 2007).
In both invertebrate and vertebrate embryonic central nervous systems, deep cells differentiate while superficial (ventricular) epithelial cells remain in a proliferative, stem cell state. The conserved polarity protein PAR-1, which is basolaterally localised in epithelia, promotes and is required for differentiating deep layer cell types, including ciliated cells and neurons. It has recently been shown that atypical protein kinase C (aPKC), which is apically enriched, inhibits neurogenesis and acts as a nuclear determinant, raising the question of how PAR-1 antagonises aPKC activity to promote neurogenesis. This study shows that PAR-1 stimulates the generation of deep cell progeny from the superficial epithelium of the neural plate and that these deep cells have a corresponding (i.e., deep cell) neuronal phenotype. Gain- and loss-of-function of PAR-1 increase and decrease, respectively, the proportion of epithelial mitotic spindles with a vertical orientation, thereby respectively increasing and decreasing the number of cleavages that generate deep daughter cells. PAR-1 is therefore a crucial regulator of the balance between symmetric (two superficial daughters) and asymmetric (one superficial and one deep daughter) cell divisions. Vertebrate PAR-1 thus antagonises the anti-neurogenic influence of apical aPKC by physically partitioning cells away from it in vivo (Tabler, 2010).
Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/β-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize β-catenin has remained unclear. Gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of β-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate (Ossipava, 2011).
Noncanonical Wnt ligands, such as Wnt5a and Wnt11, do not stabilizeα-catenin or activate TCF-dependent transcription, but regulate morphogenetic processes that involve changes n cell shape and motility, which are sometimes referred to as planar cell polarity (PCP). The signaling from Wnt5 or Wnt11 is thought to involve Ror and Ryk receptors, small Rho GTPases, Rho-associated kinase, c-Jun N-terminal kinases and intracellular calcium. Although noncanonical Wnt pathways have been shown to function in NC cell migration, their importance for NC specification has remained unclear (Ossipava, 2011).
Craniofacial defects in Wnt5a knockout mice, and in wnt11 (silberblick) and wnt5 (pipetail) zebrafish mutant embryos suggest possible roles for noncanonical Wnt signaling in NC development. The results of this study support the view that noncanonical signaling from Wnt11R is essential for NC specification in Xenopus embryos and that it might act by changing the localization and activity of the polarity kinase PAR-1 (Ossipava, 2011).
PAR proteins are conserved regulators of cell polarity that interact with several embryonic signaling pathways, including the Wnt pathway. PAR-1 associates with Dishevelled (Dvl, or Dsh) and participates in Frizzled-dependent Dvl recruitment. This study shows that PAR-1 is itself required for NC specification and can rescue NC defects in embryos with inhibited Wnt5 and Wnt11 signaling. These findings identify PAR-1 as a molecular target for noncanonical Wnt signaling and reveal an unexpected causal connection between cell polarization and the NC cell fate (Ossipava, 2011).
The axon initial segment (AIS) is a compartment that serves as a molecular barrier to achieve axon-dendrite differentiation. Distribution of specific proteins during early neuronal development has been proposed to be critical for AIS construction. However, it remains unknown how these proteins are specifically targeted to the proximal axon within this limited time period. This study revealed spatiotemporal regulation in mice driven by the microtubule (MT)-based motor KIF3A/B/KAP3 (see Drosophila Klp64D) that transports TRIM46 (see Drosophila Trim9), influenced by a specific MARK2 (Drosophila homolog: Par-1) phosphorylation cascade. In the proximal part of the future axon under low MARK2 activity, the KIF3/KAP3 motor recognizes TRIM46 as cargo and transports it to the future AIS. In contrast, in the somatodendritic area under high MARK2 activity, KAP3 phosphorylated at serine 60 by MARK2 cannot bind with TRIM46 and be transported. This spatiotemporal regulation between KIF3/KAP3 and TRIM46 under specific MARK2 activity underlies the specific transport needed for axonal differentiation (Ichinose, 2019).
A neuron is a differentiated and polarized cell that consists of a cell body, several dendrites, and a long axon. To achieve the functional polarized morphology, immature neurons undergo spatiotemporal differentiation processes crucial for axon specification: outgrowth of the future axon followed by the construction of the axon initial segment (AIS), with its specific components to facilitate and maintain axonal differentiation (Ichinose, 2019).
Microtubules (MTs) are extended polymers composed of ~13 protofilaments that consist of alphaand beta-tubulin dimers whose asymmetric structure produces the polarity of MTs with a fast-growing/shrinking plus end and a less dynamic minus end. The polarity of MTs contributes to polarized intracellular trafficking as rails of motor proteins, such as kinesin super family proteins (KIFs) and dynein. KIF3, a member of the kinesin-2 family, forms a heterotrimeric complex that consists of KIF3A, KIF3B, and its cargo-binding adaptor, kinesin-associated protein 3 (KAP3) encoded by kifap3. KIF3/KAP3 is also reported to contribute to MT organization as well as cargo transport. Thus, KIF3/KAP3 has a fundamental property to produce the polarized morphology of the cells as well as the specific distributions of organelles and proteins via elaborate coordination of related molecules, including MTs (Ichinose, 2019).
Previously, the molecular interplay of cytoskeletal proteins has been proposed to be a critical intrinsic factor to generate axonal polarity in developing neurons. In developing axons, the specific proteins are transported into the future AIS and organized to construct the unique compartment, in which MTs are oriented uniformly with the plus ends toward the distal axon and the minus ends toward the cell body (plus-end out MTs). In contrast, dendritic MTs, especially in the proximal region, display a mixed-polarity orientation (mixed-polarity MTs). To date, various models and molecules have been proposed to participate in the construction of the specific MT orientation in neurons. TRIM46 belongs to the class I members of the tripartite motif (TRIM) family. It comprises an N-terminal RING finger, which is a general feature of E3 ubiquitin ligases, a B-box, a coiled-coil, a C-terminal subgroup one signature (COS) box, and a C-terminal FN3 and B30.2-like domain. The TRIM family generally consists of large protein complexes that possess ubiquitin-protein isopeptide ligase activity. In the neuron, TRIM46 is reported to have unique MT crosslinking activity localized in the most proximal part of the axon, the AIS. TRIM46 contributes to axon specification and the establishment of neuronal polarity by building closely spaced, parallel, stabilized MT fascicles in a uniformly 'plus-end out' orientation. This specific MT orientation and stability is thought to be a directional cue for the polarized trafficking of cargoes by motor proteins into the axon. However, it has been unclear how the spatiotemporal distribution of TRIM46 is produced under the elaborate coordination of specific molecules during early neuronal developmental stages. This study investigated the spatiotemporal regulation of TRIM46 accumulation at the AIS driven by the MT-based motor KIF3A/B/KAP3 under the specific MARK2 phosphorylation cascade (Ichinose, 2019).
This study reports one of the spatiotemporal mechanisms underlying AIS construction, which is a critical process for axon establishment and neuronal development. At the proximal part of the future axon, in which a low amount of MARK2 is localized, nonphosphorylated KAP3 loads TRIM46 and transports it via KIF3/KAP3 along MTs to the limited region of the future AIS. TRIM46 facilitates plus-end-out MT orientation and axon specification. In contrast, in the dendrite, in which the amount of MARK2 is highly enriched, TRIM46 does not accumulate because KAP3 phosphorylated via MARK2 cannot bind with and transport TRIM46. This study has revealed that phosphorylation via MARK2 alters the protein-protein interaction between KIF3/KAP3 and TRIM46, which directly facilitates the formation of cell polarity (Ichinose, 2019).
Intracellular transport of cargoes generally requires specific regulation for 'cargo loading' and 'cargo unloading.' As shown in this study, KIF3/KAP3 can 'load' TRIM46 as its specific cargo bound for the future AIS via the transient nonphosphorylated form of KAP3 only at approximately day in vitro (DIV) 4 (DIV4). Interestingly, this strict regulation via phosphorylation might not affect the direction and localization of KIF3 itself because both the KAP3 S60A and S60E mutants localized in axons. The regulated phosphorylation would instead affect the cargo loading activity of KIF3/KAP3. For spatiotemporal regulation, MARK2 localized in the somatodendritic region at DIV4 and later. This specific localization is also explained by previous studies showing that KIF3 initially transports the PAR-3/PAR-6/aPKC complex to the future axon, which suppresses MARK2 activity at the future axon during early developmental stages. Downregulation of MARK2 activity would facilitate the loading of TRIM46 by KAP3 and induce transport to the proximal part of the future axon to build up the AIS. This regulation of the KIF3/KAP3/TRIM46 complex would form the positive feedback loop of loading and transport of specific cargoes to further accelerate axon differentiation. However, it remains unknown how KIF3/KAP3 can 'unload' TRIM46 at the AIS. From the global localization of the KIF3/KAP3/TRIM46 complex on MTs in COS-7 cells, it is speculated that KIF3/KAP3/TRIM46 itself does not distinguish the specific structural destination cues. Instead, other regulators would be required to 'unload' TRIM46 from KIF3/KAP3 outside of the AIS to accumulate TRIM46 at only the AIS. The most likely candidate is the phosphorylation of TRIM46 by CDK5. Results from an in vitro kinase assay against TRIM46 indicated that the N-terminal region of TRIM46 containing the RING domain is phosphorylated by CDK5. S106 was identified in the RING domain as a phosphorylation site by CDK5. S106 is specific to the TRIM46 subfamily and is not conserved in the other TRIM subfamilies, such as TRIM36. Interestingly, this RING domain is reported to be necessary for TRIM46 accumulation at the AIS. In addition, CDK5 is also reported to be a key regulator of the AIS. More evidence is required to elucidate the precise unloading mechanism of TRIM46 under the CDK5 cascade (Ichinose, 2019).
Interestingly, this study found that both KIF3A and KAP3 were reduced in the kif3b+/− neurons. There should be a balance between KIF3A/B and KAP3 maintained at steady state, of which the ratio was previously reported to be 1:1:0.7, and the excess amount might be proteolyzed in the kif3b+/− neurons. Indeed, this stuy also overexpressed recombinant tagged KAP3 in neuronal cells, but only low expression of KAP3 was observed. Therefore, the strict balanced expression would explain the low coefficiency between tagged KAP3 and TRIM46 compared to that between endogenous KAP3 and TRIM46. However, tagged KAP3 was observed much more clearly in the kif3b+/− neuron than in the kif3b+/+ neuron because the expression level of KIF3 was potentially decreased to half, and there might be extra room for overexpressed KIF3 in KIF3/KAP3 balance. It might be interesting to investigate how KIF3 heterotrimers maintain their molecular balance in the future (Ichinose, 2019).
Depletion of KAP3 and KIF5 affected the distribution of AIS components. KIF5 has been reported to transport AnkG, which anchors βIV spectrin and other membrane proteins, such as Voltage-gated sodium channel (NaV) and L1-CAM, at the AIS. Because accumulated AnkG maintains axon/dendrite differentiation, KIF5 may facilitate axon differentiation by transporting AnkG. In fact, the data showing that depletion of KIF5 reduced Nav accumulation at the AIS are consistent with these previous studies. However, the detailed mechanism by which KIF5 transports AnkG to the AIS is still unknown, although it has been previously shown that KIF5 prefers stabilized MTs enriched in the AIS. More detailed mechanisms between KIF5 and AnkG as well as the regulation between KIF5 and TRIM46-stabilized MTs at the AIS should be investigated to solve this problem. However, KAP3 depletion also causes the reduction of Nav accumulation at the AIS. Moreover, it also causes Nav accumulation at the distal axon and growth cone in severe cases, which phenocopies depletion of spectrin. Considering that KIF3 transports αII spectrin, our results are consistent with previous results. Because αII spectrin is able to dimerize with either βIV spectrin at the AIS or βII spectrin at the distal axon, KIF3 may facilitate not only AIS construction but also whole axon differentiation by transporting its cytoskeletal and membrane proteins, such as axonal voltage-gated potassium channels. However, another study suggests that TRIM46 affects AIS construction by coordinating with AnkG. Depletion of AnkG reduced TRIM46 accumulation at the AIS and vice versa. The detailed mechanism between TRIM46, AnkG, KIF3, and KIF5 should be clarified to reveal the crosstalk between these molecules for the construction of the AIS. For example, it would be interesting to test whether KAP3 transports TRIM46 to the AIS in AnkG-depleted neurons. Considering that KIF5 might prefer TRIM46-stabilized MTs, there would be positive feedback assembly of the AIS by cooperative transport between KIF5 and KIF3. This molecular coordination would set up a selective barrier for the polarized trafficking of vesicles and organelles into the axon; axon/dendrite differentiation is further accelerated via previously reported mechanisms. Another major MT motor, KIF1A, does not seem to regulate axonal differentiation because the localization of Nav at the AIS was not affected by KIF1A depletion. However, the length of the AIS visualized with Nav immunostaining was slightly increased. Increased AIS length was also observed in auditory-deprived chick neurons. Considering that KIF1A transports synaptic vesicle precursors, it is possible that reduced presynaptic activity by KIF1A depletion increased the AIS length (Ichinose, 2019).
As shown in this study, the spatiotemporal molecular interplay of KIF3/KAP3/TRIM46 under MARK2 phosphorylation facilitates axon specification. AIS construction is important not only for selective transport but also for initiation of action potentials. Thus, manipulating neuronal excitability in this region would be a potential target for the treatment of epilepsy and psychiatric disorders. To reveal the relation between the regulation of AIS construction and neuronal diseases, further precise analyses, including both electrophysiological approaches in vivo and structural in vitro reconstruction studies, will contribute to a deeper understanding (Ichinose, 2019).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.