CDC-42 and RHO-1 coordinate acto-myosin contractility and PAR protein localization during polarity establishment in C. elegans embryos

In C. elegans one-cell embryos, polarity is conventionally defined along the anteroposterior axis by the segregation of partitioning-defective (PAR) proteins into anterior (PAR-3, PAR-6) and posterior (PAR-1, PAR-2) cortical domains. The establishment of PAR asymmetry is coupled with acto-myosin cytoskeleton rearrangements. The small GTPases RHO-1 and CDC-42 are key players in cytoskeletal remodeling and cell polarity in a number of different systems. We investigated the roles of these two GTPases and the RhoGEF ECT-2 in polarity establishment in C. elegans embryos. We show that CDC-42 is required to remove PAR-2 from the cortex at the end of meiosis and to localize PAR-6 to the cortex. By contrast, RHO-1 activity is required to facilitate the segregation of CDC-42 and PAR-6 to the anterior. Loss of RHO-1 activity causes defects in the early organization of the myosin cytoskeleton but does not inhibit segregation of myosin to the anterior. We therefore propose that RHO-1 couples the polarization of the acto-myosin cytoskeleton with the proper segregation of CDC-42, which, in turn, localizes PAR-6 to the anterior cortex.     

CDC-42 controls early cell polarity and spindle orientation in C. elegans.

BACKGROUND: 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. RESULTS: Here, we show that 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. We further show that C. elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. CONCLUSIONS: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit.     

Cyclin E-Cdk2 temporally regulates centrosome assembly and establishment of polarity in Caenorhabditis elegans embryos

Establishment of polarity in C. elegans embryos is dependent on the centrosome. The sperm contributes a pair of centrioles to the egg and these centrioles remain incapable of polarizing the cortex while the egg completes meiosis. Coincident with the establishment of polarity, the centrioles recruit centrosomal proteins, several of which are required for polarity, suggesting that the temporal regulation of centrosome assembly may control the initiation of polarization. We found that cyclin E-Cdk2 is required for the establishment of polarity. Cyclin E-Cdk2 controls the recruitment of centrosomal proteins specifically at the time of polarity establishment. Cyclin E is required for several examples of asymmetric cell division and fate determination in C. elegans and Drosophila. Here, we suggest a possible mechanism for cyclin E-Cdk2-dependent differentiation: the establishment of cortical polarity by the centrosome.     

线虫(Caenorhabditis elegans)受精卵中细胞极性的建立

早期线虫胚胎提供了一个研究发育过程的极佳模型。线虫胚胎的第一次分裂是不对称的,产生的两个子细胞在尺度的大小和发育命运上均有不同,而这些不同是由第一次有丝分裂周期中胞质决定子的不均匀分布造成的。通常相信,在受精过程中,精子所携带的中心体介导了对极性建成至关重要的胞质流动的产生。同时,细胞骨架成分被认为参与了胞质成分的定位事件。关于par基因的研究目前进展迅速,大多数par基因的突变都导致了线虫早期胚胎分裂不对称性的丧失。     

Reversal of cellular polarity and early cell-cell interaction in the embryos of Caenorhabditis elegans

During early embryogenesis of Caenorhabditis elegans the serial stem cell-like cleavages of the germ line cells P0-P3 generate a number of somatic founder cells with different developmental potentials. Observations on partial embryos show that in the first two of these unequal divisions in the germ line the somatic daughter cell comes to lie anterior to the new germ line cell. In the following two, however, the somatic daughter cell comes to lie posterior to the new germ line cell, suggesting a reversal of polarity in the germ line. By the use of a laser microbeam, egg fragments can be extruded from young embryos; the fragments often cleave like partial twins. Depending on whether the fragment is derived from the posterior region of the uncleaved zygote P0 or its daughter P1, the mirror image duplications that are generated are joined at their larger soma-like cells or at their smaller germ line-like cells, respectively. This result is best explained as a reversal of polarity taking place in the germ line cell P2. This notion is strengthened by the finding that partial embryos derived from the posterior region of the P2 cell in late interphase do not undergo stem cell-like (i.e., unequal) cleavages in contrast to those derived from P0 or P1. Finally, an apparent early cell-cell interaction is described which is inconsistent with the classical notion of "mosaic" nematode development: removal of the germline cell P2 results in an altered developmental pattern of its somatic sister cell EMS. A working model is presented linking reversal of polarity and cell-cell interaction and offers an explanation for the unique behavior of the EMS cell in normal development.     

Long astral microtubules and RACK-1 stabilize polarity domains during maintenance phase in Caenorhabditis elegans embryos

Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity.     

A Highlights from MBoC Selection: CGEF-1 and CHIN-1 Regulate CDC-42 Activity during Asymmetric Division in the Caenorhabditis elegans Embryo

The anterior–posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.     

A posterior centre establishes and maintains polarity of the Caenorhabditis elegans embryo by a Wnt-dependent relay mechanism

Cellular polarity is a general feature of animal development. However, the mechanisms that establish and maintain polarity in a field of cells or even in the whole embryo remain elusive. Here we provide evidence that in the Caenorhabditis elegans embryo, the descendants of P₁, the posterior blastomere of the 2-cell stage, constitute a polarising centre that orients the cell divisions of most of the embryo. This polarisation depends on a MOM-2/Wnt signal originating from the P₁ descendants. Furthermore, we show that the MOM-2/Wnt signal is transduced from cell to cell by a relay mechanism. Our findings suggest how polarity is first established and then maintained in a field of cells. According to this model, the relay mechanism constantly orients the polarity of all cells towards the polarising centre, thus organising the whole embryo. This model may also apply to other systems such as Drosophila and vertebrates.     

Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans

The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans.During development and in tissue homeostasis, multicellular organisms frequently use apoptosis to eliminate cells that are useless or potentially dangerous. Apoptotic cells are readily recognized, internalized and degraded by neighboring or specialized engulfing cells. Rapid clearance of unwanted cells avoids the release of harmful intracellular contents into the surroundings that can lead to inflammation and autoimmune disease.¹The nematode C. elegans serves as a simple yet powerful genetic model organism to study cell corpse clearance in vivo. Many genes involved in recognition, internalization or degradation of apoptotic corpses have been identified through forward and reverse genetic screens in the past two decades.² Loss of engulfment activity not only results in the persistence of cell corpses, but also leads to the survival of some cells destined to die,³ and – in some cases – leads to impaired cell migration.⁴Phenotypic, genetic and biochemical analyses of the early ‘classical'' ced (cell death abnormal) genes led to the identification of three partially redundant signaling cascades that cooperate to regulate cytoskeletal rearrangements and the migration of the engulfing cell around the apoptotic corpse.^{5, 6, 7, 8, 9} In the first pathway, the transmembrane protein CED-1/MEGF10 has been proposed to act as a cell corpse receptor¹⁰ that binds to exposed phosphatidylserine (PS), either directly or indirectly through the action of the bridging molecule TTR-52/TTR.^{11, 12} The lipid transporter homolog CED-7 also plays a role at this stage, at least in part by promoting the exposure of PS in the outer leaflet of the doomed cell.¹³ The adaptor protein CED-6/GULP transduces the signal(s) from CED-1 downstream to CED-10/Rac1 and DYN-1/Dynamin to drive cytoskeletal rearrangements and phagosome maturation.^{8, 14, 15, 16} In the second pathway, activation of CED-10 is promoted by the bipartite GEF (guanine exchange factor) complex composed of CED-12/Elmo–CED-5/Dock180.^{17, 18, 19, 20} This GEF complex in turn is regulated by CED-2/CrkII and the small GTPase MIG-2/RhoG. In the third pathway, the cytoskeletal regulator ABL-1/Abl suppresses corpse clearance by inhibiting ABI-1/Abl-interacting protein.²¹ Active GTP-loaded CED-10 promotes the extensive cytoskeletal rearrangements that are essential for proper cell corpse internalization.⁸ This process is negatively regulated by the GTPase-activating protein (GAP) SRGP-1/srGAP1 that facilitates GTP hydrolysis in CED-10.²²Here we present the identification and characterization of cdc-42 (cell division control protein-42) as an additional mediator of engulfment signaling regulated by SRGP-1 (Slit/Robo GTPase activating protein 1). Our epistatic analyses, performed with cdc-42(lf) mutants and cdc-42 overexpression experiments, suggest that cdc-42 acts downstream or in parallel to the ced-1/6/7 and in parallel to the ced-2/5/12 signaling cascades. Using a functional and rescuing GFP::CDC-42 protein, we show that CDC-42 is recruited to the cell membrane surrounding apoptotic corpses, and that this localization requires the integrin-α PAT-2 but not the canonical ced-1/6/7 or ced-2/5/12 cascades.Taken together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment upstream of the canonical Rac GTPase CED-10, possibly by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling. Our data confirm and significantly expand on recent results published by Hsieh et al.,²³ who independently identified CDC-42 as an engulfment regulator downstream of integrin-α PAT-2.     

CDC-42 regulates PAR protein localization and function to control cellular and embryonic polarity in C. elegans.

BACKGROUND: The polarization of the anterior-posterior axis (A-P) of the Caenorhabditis elegans zygote depends on the activity of the par genes and the presence of intact microfilaments. Functional links between the PAR proteins and the cytoskeleton, however, have not been fully explored. It has recently been shown that 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. RESULTS: To investigate the function of Cdc42 in the control of cellular and embryonic polarity in C. elegans, we used RNA-mediated interference (RNAi) to inhibit cdc-42 activity in the early embryo. Here, we demonstrate that RNAi of cdc-42 disrupts manifestations of polarity in the early embryo, that these phenotypes depend on par-2 and par-3 gene function, and that cdc-42 is required for the localization of the PAR proteins. CONCLUSIONS: Our 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.     

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior–posterior body axis via the inhibition of dorsal–ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain‐of‐function mutant animals for C. elegans RhoG MIG‐2. We identified serine 139 of MIG‐2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG‐2^S139. Live imaging analysis of genome‐edited animals indicates that MIG‐2^S139 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild‐type cells, while MIG‐2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG‐mediated polarization. Therefore, we propose that the Hippo–RhoG feedback regulation maintains cell polarity during directional cell motility.     

A conserved role of Caenorhabditis elegans CDC-48 in ER-associated protein degradation

Protein degradation mediated by the ubiquitin/proteasome system is essential for the elimination of misfolded proteins from the endoplasmic reticulum (ER) to adapt to ER stress. It has been reported that the AAA ATPase p97/VCP/CDC48 is required in this pathway for protein dislocation across the ER membrane and subsequent ubiquitin dependent degradation by the 26S proteasome in the cytosol. Throughout ER-associated protein degradation, p97 cooperates with a binary Ufd1/Npl4-complex. In Caenorhabditis elegans two homologs of p97, designated CDC-48.1 and CDC-48.2, exist. Our results indicate that both p97 homologs interact with UFD-1/NPL-4 in a similar CDC-48(UFD-1/NPL-4) complex. RNAi mediated depletion of the corresponding genes induces ER stress resulting in hypersensitivity to conditions which induce increased levels of unfolded proteins in the ER lumen. Together, these data suggest an evolutionarily conserved retro-translocation machinery at the endoplasmic reticulum.     

Cell polarity in the early Caenorhabditis elegans embryo.

Beginning with the first mitotic division in a Caenorhabditis elegans embryo, asymmetric cleavages establish much of the body plan. Although they share a common axis of polarity, at least three kinds of asymmetric cell division occur: two are under intrinsic control, while a third requires an inductive signal and may operate repeatedly throughout development.     

The Caenorhabditis elegans ect-2 RhoGEF gene regulates cytokinesis and migration of epidermal P cells

A reduction-of-function mutation in ect-2 was isolated as a suppressor of the Multivulva phenotype of a lin-31 mutation. Analysis using markers indicates that this mutation causes defects in both the cytokinesis and migration of epidermal P cells, phenotypes similar to those caused by expressing a rho-1 dominant-negative construct. ect-2 encodes the Caenorhabditis elegans orthologue of the mouse Ect2 and Drosophila Pebble that function as guanine nucleotide exchange factors (GEFs) for Rho GTPases. The ect-2Colon, two colonsGFP reporter is expressed in embryonic cells and P cells. RNA interference of ect-2 causes sterility and embryonic lethality, in addition to the P-cell defects. We have determined the lesions of two ect-2 alleles, and described their differences in phenotypes in specific tissues. We propose a model in which ECT-2GEF not only activates RHO-1 for P-cell cytokinesis, but also collaborates with UNC-73GEF and at least two Rac proteins to regulate P-cell migration.     

CDC-25.1 controls the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively regulating CDK-1 in Caenorhabditis elegans

In Caenorhabditis elegans, cdc-25.1 loss-of-function mutants display a lack of germline proliferation. We found that the proliferation defect of cdc-25.1 mutants was suppressed by wee-1.3 RNAi. Further, among the seven cdk and seven cyclin homologs examined, cdk-1 and cyb-3 RNAi treatment caused the most severe germline proliferation defects in an rrf-1 mutant background, which were similar to those of the cdc-25.1 mutants. In addition, while RNAi of cyd-1 and cye-1 caused significant germline proliferation defects, RNAi of cdk-2 and cdk-4 did not. Compared with the number of germ nuclei in wee-1.3(RNAi) worms, the number in wee-1.3(RNAi);cdk-1(RNAi) and wee-1.3(RNAi);cyb-3(RNAi) worms further decreased to the level of cdk-1(RNAi) and cyb-3(RNAi) worms, respectively, indicating that cdk-1 and cyb-3 are epistatic and function downstream of cdc-25.1 and wee-1.3 in the control of the cell cycle. BrdU labeling of adult worms showed that, while 100% of the wild-type germ nuclei in the mitotic region incorporated BrdU when labeled for more than 12 h at 20°C, a small fraction of the cdc-25.1 mutant germ nuclei failed to incorporate BrdU even when labeled for 68 h. These results indicate that CDC-25.1 is required for maintaining proper rate of germline mitotic cell cycle. We propose that CDC-25.1 regulates the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively controlling CDK-1, which forms a complex primarily with CYB-3, but also possibly with CYD-1 and CYE-1.     

The DH-PH region of the giant protein UNC-89 activates RHO-1 GTPase in Caenorhabditis elegans body wall muscle

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing a DH domain followed by a PH domain at its N terminus, which is characteristic of guanine nucleotide exchange factor proteins for Rho GTPases. To obtain evidence that the DH-PH region has activity toward specific Rho family small GTPases, we conducted an experiment using the yeast three-hybrid system. The DH-PH region of UNC-89 has exchange activity for RHO-1 (C. elegans RhoA), but not for CED-10 (C. elegans Rac), MIG-2 (C. elegans RhoG), or CDC-42 (C. elegans Cdc42). The DH domain alone has similar activity for RHO-1. An in vitro binding assay demonstrates interaction between the DH-PH region of UNC-89 and each of the C. elegans Rho GTPases. Partial knockdown of rho-1 in C. elegans adults showed a pattern of disorganization of myosin thick filaments similar to the phenotype caused by unc-89 (su75), a mutant allele in which all of the isoforms containing the DH-PH region are missing. Taken together, we propose a model in which the DH-PH region of UNC-89 activates RHO-1 GTPase for organization of myosin filaments in C. elegans muscle cells.     

Heads or tails: cell polarity and axis formation in the early Caenorhabditis elegans embryo

In C. elegans, the first embryonic axis is established shortly after fertilization and requires both the microtubule and microfilament cytoskeleton. Cues from sperm-donated centrosomes result in a cascade of events that polarize the distribution of widely conserved PAR proteins at the cell cortex. The PAR proteins in turn polarize the cytoplasm and position mitotic spindles. Lessons learned from C. elegans should improve our understanding of how cells become polarized and divide asymmetrically during development.