C. elegans Brat homologs regulate PAR protein-dependent polarity and asymmetric cell division

The evolutionary conserved PAR proteins control polarization and asymmetric division in many organisms. Recent work in Caenorhabditis elegans demonstrated that nos-3 and fbf-1/2 can suppress par-2(it5ts) lethality, suggesting that they participate in cell polarity by regulating the function of the anterior PAR-3/PAR-6/PKC-3 proteins. In Drosophila embryos, Nanos and Pumilio are homologous to NOS-3 and FBF-1/2 respectively and control cell polarity by forming a complex with the tumor suppressor Brat to inhibit Hunchback mRNA translation. In this study, we investigated the possibility that Brat could control cell polarity and asymmetric cell division in C. elegans. We found that disrupting four of the five C. elegans Brat homologs (Cebrats) individually results in suppression of par-2(it5ts) lethality, indicating that these genes are involved in embryonic polarity. Two of the Cebrats, ncl-1 and nhl-2, partially restore the localization of PAR proteins at the cortex. While mutations in the four Cebrat genes do not severely impair polarity, they display polarity-associated defects. Surprisingly, these defects are absent from nos-3 mutants. Similarly, while nos-3 controls PAR-6 protein levels, this is not the case for any of the Cebrats. Our results, together with results from Drosophila, indicate that Brat family members function in generating cellular asymmetries and suggest that, in contrast to Drosophila embryos, the C. elegans homologs of Brat and Nanos could participate in embryonic polarity via distinct mechanisms.     

Modeling the establishment of PAR protein polarity in the one-cell C. elegans embryo

At the one-cell stage, the C. elegans embryo becomes polarized along the anterior-posterior axis. The PAR proteins form complementary anterior and posterior domains in a dynamic process driven by cytoskeletal rearrangement. Initially, the PAR proteins are uniformly distributed throughout the embryo. After a cue from fertilization, cortical actomyosin contracts toward the anterior pole. PAR-3/PAR-6/PKC-3 (the anterior PAR proteins) become restricted to the anterior cortex. PAR-1 and PAR-2 (the posterior PAR proteins) become enriched in the posterior cortical region. We present a mathematical model of this polarity establishment process, in which we take a novel approach to combine reaction-diffusion dynamics of the PAR proteins coupled to a simple model of actomyosin contraction. We show that known interactions between the PAR proteins are sufficient to explain many aspects of the observed cortical PAR dynamics in both wild-type and mutant embryos. However, cytoplasmic PAR protein polarity, which is vital for generating daughter cells with distinct molecular components, cannot be properly explained within such a framework. We therefore consider additional mechanisms that can reproduce the proper cytoplasmic polarity. In particular we predict that cytoskeletal asymmetry in the cytoplasm, in addition to the cortical actomyosin asymmetry, is a critical determinant of PAR protein localization.     

Neuronal polarity in C. elegans

Neuronal polarity sets the foundation for information processing and signal transmission within neural networks. However, fundamental question of how a neuron develops and maintains structurally and functionally distinct processes, axons and dendrites, is still an unclear. The simplicity and availability of practical genetic tools makes C. elegans as an ideal model to study neuronal polarity in vivo. In recent years, new studies have identified critical polarity molecules that function at different stages of neuronal polarization in C. elegans. This review focuses on how neurons guided by extrinsic cues, break symmetry, and subsequently recruit intracellular molecules to establish and maintain axon-dendrite polarity in vivo.     

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.     

Cell polarity and gastrulation in C. elegans.

Gastrulation in C. elegans embryos involves formation of a blastocoel and the ingression of surface cells into the blastocoel. Mutations in the par-3 gene cause abnormal separations between embryonic cells, suggesting that the PAR-3 protein has a role in blastocoel formation. In normal development, PAR proteins localize to either the apical or basal surfaces of cells prior to blastocoel formation; we demonstrate that this localization is determined by cell contacts. Cells that ingress into the blastocoel undergo an apical flattening associated with an apical concentration of non-muscle myosin. We provide evidence that ingression times are determined by genes that control cell fate, though interactions with neighboring cells can prevent ingression.     

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.     

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.     

RAB-5 controls the cortical organization and dynamics of PAR proteins to maintain C. elegans early embryonic polarity

In all organisms, cell polarity is fundamental for most aspects of cell physiology. In many species and cell types, it is controlled by the evolutionarily conserved PAR-3, PAR-6 and aPKC proteins, which are asymmetrically localized at the cell cortex where they define specific domains. While PAR proteins define the antero-posterior axis of the early C. elegans embryo, the mechanism controlling their asymmetric localization is not fully understood. Here we studied the role of endocytic regulators in embryonic polarization and asymmetric division. We found that depleting the early endosome regulator RAB-5 results in polarity-related phenotypes in the early embryo. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we observed that PAR-6 is localized at the cell cortex in highly dynamic puncta and depleting RAB-5 decreased PAR-6 cortical dynamics during the polarity maintenance phase. Depletion of RAB-5 also increased PAR-6 association with clathrin heavy chain (CHC-1) and this increase depended on the presence of the GTPase dynamin, an upstream regulator of endocytosis. Interestingly, further analysis indicated that loss of RAB-5 leads to a disorganization of the actin cytoskeleton and that this occurs independently of dynamin activity. Our results indicate that RAB-5 promotes C. elegans embryonic polarity in both dynamin-dependent and -independent manners, by controlling PAR-6 localization and cortical dynamics through the regulation of its association with the cell cortex and the organization of the actin cytoskeleton.     

Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo

The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. We show that the one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry.     

Revisiting the role of microtubules in C. elegans polarity

Cells must break symmetry to acquire polarity. Microtubules have been implicated in the induction of asymmetry in several cell types, but their role in the Caenorhabditis elegans zygote, a classic polarity model, has remained uncertain. One study (see Tsai and Ahringer on p. 397 of this issue) brings new light to this problem by demonstrating that severe loss of microtubules impairs polarity onset in C. elegans.     

PAR proteins regulate microtubule dynamics at the cell cortex in C. elegans

BACKGROUND: The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood. RESULTS: We have developed a method that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, we found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. We further observed that this asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and that the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo. CONCLUSIONS: These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes.     

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.     

Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans

Secreted Wnt proteins influence neural connectivity by regulating axon guidance, dendritic morphogenesis and synapse formation. We report a new role for Wnt and Frizzled proteins in establishing the anteroposterior polarity of the mechanosensory neurons ALM and PLM in C. elegans. Disruption of Wnt signaling leads to a complete inversion of ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process, and vice versa. Different but overlapping sets of Wnt proteins regulate neuronal polarity in different body regions. Wnts act directly on PLM via the Frizzled LIN-17. In addition, we show that they are needed for axon branching and anteriorly directed axon growth. We also find that the retromer, a conserved protein complex that mediates transcytosis and endosome-to-Golgi protein trafficking, plays a key role in Wnt signaling. Deletion mutations of retromer subunits cause ALM and PLM polarity, and other Wnt-related defects. We show that retromer protein VPS-35 is required in Wnt-expressing cells and propose that retromer activity is needed to generate a fully active Wnt signal.     

PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos

Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.     

C. elegans PAR proteins function by mobilizing and stabilizing asymmetrically localized protein complexes

BACKGROUND: The PAR proteins are part of an ancient and widely conserved machinery for polarizing cells during animal development. Here we use a combination of genetics and live imaging methods in the model organism Caenorhabditis elegans to dissect the cellular mechanisms by which PAR proteins polarize cells. RESULTS: We demonstrate two distinct mechanisms by which PAR proteins polarize the C. elegans zygote. First, we show that several components of the PAR pathway function in intracellular motility, producing a polarized movement of the cell cortex. We present evidence 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. Second, 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. CONCLUSIONS: 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.     

Laminin is required to orient epithelial polarity in the C. elegans pharynx

The development of many animal organs involves a mesenchymal to epithelial transition, in which cells develop and coordinate polarity through largely unknown mechanisms. The C. elegans pharynx, which is an epithelial tube in which cells polarize around a central lumen, provides a simple system with which to understand the coordination of epithelial polarity. We show that cell fate regulators cause pharyngeal precursor cells to group into a bilaterally symmetric, rectangular array of cells called the double plate. The double plate cells polarize with apical localization of the PAR-3 protein complex, then undergo apical constriction to form a cylindrical cyst. We show that laminin, but not other basement membrane components, orients the polarity of the double plate cells. Our results provide in vivo evidence that laminin has an early role in cell polarity that can be distinguished from its later role in basement membrane integrity.     

Cell polarity and asymmetric cell division: the C. elegans early embryo

Cell polarity is crucial for many functions including cell migration, tissue organization and asymmetric cell division. In animal cells, cell polarity is controlled by the highly conserved PAR (PARtitioning defective) proteins. par genes have been identified in Caenorhabditis elegans in screens for maternal lethal mutations that disrupt cytoplasmic partitioning and asymmetric division. Although PAR proteins were identified more than 20 years ago, our understanding on how they regulate polarity and how they are regulated is still incomplete. In this chapter we review our knowledge of the processes of cell polarity establishment and maintenance, and asymmetric cell division in the early C. elegans embryo. We discuss recent findings that highlight new players in cell polarity and/or reveal the molecular details on how PAR proteins regulate polarity processes.     

Stabilization of cell polarity by the C. elegans RING protein PAR-2

Asymmetric localization of PAR proteins is a hallmark of polarized cells, but the mechanisms that create PAR asymmetry are not well understood. In the C. elegans zygote, PAR asymmetry is initiated by a transient actomyosin contraction, which sweeps the PAR-3/PAR-6/PKC-3 complex toward the anterior pole of the egg. The RING finger protein PAR-2 accumulates in a complementary pattern in the posterior cortex. Here we present evidence that PAR-2 participates in a feedback loop to stabilize polarity. PAR-2 is a target of the PKC-3 kinase and is excluded from the anterior cortex by PKC-3-dependent phosphorylation. The RING domain of PAR-2 is required to overcome inhibition by PKC-3 and stabilize PAR-2 on the posterior cortex. Cortical PAR-2 in turn prevents PAR-3/PAR-6/PKC-3 from returning to the posterior, in a PAR-1- and PAR-5-dependent manner. Our findings suggest that reciprocal inhibitory interactions among PAR proteins stabilize polarity by reinforcing an initial asymmetry in PKC-3.     

Elaborating polarity: PAR proteins and the cytoskeleton

Cell polarity is essential for cells to divide asymmetrically, form spatially restricted subcellular structures and participate in three-dimensional multicellular organization. PAR proteins are conserved polarity regulators that function by generating cortical landmarks that establish dynamic asymmetries in the distribution of effector proteins. Here, we review recent findings on the role of PAR proteins in cell polarity in C. elegans and Drosophila, and emphasize the links that exist between PAR networks and cytoskeletal proteins that both regulate PAR protein localization and act as downstream effectors to elaborate polarity within the cell.