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.
Cell polarization is achieved by an unequal distribution of cellular factors (mRNA, proteins) and/or organelles along a cellular axis. Signals inducing cell polarization (intrinsic, e.g. [1,2] or extrinsic, e.g. ) create distinct cellular domains which in turn determine the leading edge during migration, confer the directionality of transport and/or secretion of molecules within the tissue, or ensure the distribution of cell-fate determinants. Thus cell polarity is essential for the function of almost all cell types and, when perturbed, can lead to disease [4,5].
Cell polarization is also one of the prerequisites for asymmetric (unequal) cell division, a crucial biological process required to generate diversity during development. A cell divides asymmetrically when it gives rise to daughter cells with distinct developmental potentials. Examples of asymmetric cell division can be found within diverse species including bacteria (e.g. Caulobacter crescentus division ), unicellular eukaryotic organisms (e.g. Saccharomyces cerevisiae division ) and invertebrates (e.g. Caenorhabditis elegans and Drosophila melanogaster ), as well as in plants and algae (e.g. Arabidopsis thaliana and Fucus serratus ), and finally mammals .
The majority of unequal divisions create cells different in size. Most importantly, however, daughter cells inherit distinct cell-fate determinants that drive their diverse developmental programmes. The two main requirements for a successful asymmetric cell division are: (i) cell polarization along a predetermined axis and (ii) orientation of the mitotic spindle along the polarity axis (Figure 1).
The molecular mechanisms governing polarity and asymmetric cell division, although highly conserved and extensively investigated in different systems, are not yet completely understood. Studies on model organisms have contributed enormously to our understanding of the polarity network, which controls cell fate across species . In the present chapter we review the mechanisms regulating cell polarity establishment and maintenance, and asymmetric cell division in the C. elegans one-cell embryo.
Early development of the C. elegans embryo
The C. elegans early embryo is an excellent model to investigate the molecular mechanisms governing polarity and asymmetric cell division. The first embryonic division is asymmetric and completed approximately 30 min after fertilization. In addition, embryonic divisions are highly stereotypical and can be followed in real time using Nomarski optics [DIC (differential interference contrast); Figures 1A–1G].
The development of the embryo starts when the oocyte, arrested in prophase of meiosis I, is fertilized while passing through the spermatheca. Upon fertilization, both meiotic divisions are completed and two polar bodies are extruded from the zygote. The sperm, which provides the centrosomes to the embryo, fertilize the oocyte usually on the opposite side to the oocyte nucleus. Centrosomes and associated microtubules define the future posterior pole of the embryo and therefore the first axis of polarity, the anterior–posterior axis. Following sperm entry, the acto-myosin cytoskeleton undergoes rapid rearrangements, which result in strong ruffling of the cortex (Figures 1A, 1B, 1O and 1P). Meanwhile, the maternal and paternal pronuclei grow in size and become clearly noticeable by DIC (Figure 1A). While the ruffling of the cortex culminates in one pronounced invagination in the middle of the embryo (pseudocleavage), the female pronucleus starts migrating towards the posterior pole to meet the sperm pronucleus. The two pronuclei meet in the posterior half of the embryo [PNM (pronuclei meeting); Figure 1B] and migrate together towards the centre of the embryo while rotating 90° to align the centrosomes along the polarity axis (Figures 1C and 1J). Subsequently, nuclear envelopes break down (Figures 1D and 1K), the mitotic spindle sets up, elongates and is pulled to the posterior, while the posterior spindle pole exhibits oscillations along the short axis of the embryo (Figures 1E and 1L). Finally, the cytokinetic furrow ingresses in the posterior part of the embryo (Figure 1F), bisecting the spindle and creating two cells with unequal size, a bigger anterior (AB) and a smaller posterior (P1) cell (Figure 1G).
AB and P1 inherit distinct factors that specify different developmental fates (Figure 1N and see below). The anterior cell will divide symmetrically and give rise to somatic tissues. Conversely, the posterior cell will continue to divide asymmetrically to finally create two germ cell precursors and somatic cells. AB and P1 also differ in cell-cycle timing and spindle orientation. The AB cell divides approximately 2 min before P1 cells, and the mitotic spindle of AB cells is perpendicular, whereas the spindle of the P1 cell is parallel, to the polarity axis . All of these processes are under the control of the highly conserved PAR (PARtitioning defective) proteins .
PAR proteins: major polarity players
PAR proteins are major players orchestrating cell polarity and asymmetric division. They were identified in C. elegans in genetic screens for regulators of asymmetric division of the one-cell zygote. The par mutants are unable to establish and/or maintain polarity, they display developmental defects and eventually die . Until now seven master polarity proteins have been identified: PAR-1 to PAR-6 and PKC-3 (atypical protein kinase C, also known as aPKC). Subsequent studies in other organisms identified homologues of PAR proteins (with the exception of PAR-2), suggesting that they are part of a universal machinery governing cellular polarity (reviewed in ).
In C. elegans early embryos five PAR proteins are asymmetrically distributed forming physically and functionally distinct domains (Figures 1H–1N and 2). The anterior cortex is occupied by the dynamic complex of PKC-3 , and two PDZ-domain containing proteins, PAR-3 and PAR-6 (Figures 1H–1M and 2) [15⇓–17]. The posterior pole is occupied by PAR-1, a serine/threonine protein kinase , and PAR-2, a RING finger protein (Figures 1H–1M and 2) [19⇓–21]. The serine/threonine kinase PAR-4 and a member of the 14-3-3 family, PAR-5, are uniformly distributed through the cortex and in the cytoplasm [22,23]. The localization of PAR proteins in the one-cell embryo proceeds in two temporally and mechanistically distinct stages: the establishment and the maintenance phase (Figure 1) , as described below.
In newly fertilized embryos the PAR-3–PAR-6–PKC-3 complex is found all around the cortex, and PAR-1 and PAR-2 are in the cytoplasm [21,24]. A net of symmetrically distributed acto-myosin foci decorates the cortex of the entire embryo and drives cortical contractions and ruffling [11,25]. Cortical contractions depend on the activity of the small G-protein RHO-1, regulated by the GEF (guanine-nucleotide-exchange factor) ECT-2 (epithelial cell transforming sequence 2), and the GAPs (GTPase-activating proteins) RGA-3 and RGA-4 (RGA is Rho GAP), all of which are localized at the cortex [26⇓⇓–30].
Polarity establishment is initiated by the paternally provided centrosome, which brings the signal to break the cortical and cytoplasmic symmetries. The molecular nature of this signal is not clear [31,32]. Nevertheless, the sperm-donated centrosome and associated microtubules contact the posterior cortex and trigger the local inhibition of cortical contractions [21,27,33⇓⇓⇓–37], which results in a progressive relaxation of the posterior cortex and displacement of contractile centres towards the anterior pole, a process referred to as cytoplasmic flows (Figures 1A, 1B, 1O and 1P) [24,38,39]. The flows transport PAR-3–PAR-6–PKC-3 to the anterior and enable PAR-2, and subsequently PAR-1, to associate with the posterior cortex (Figures 1H and 1I) [24,38,40].
PAR proteins are important for polarity establishment. The anterior PAR proteins play a major role as polarity fails to be established in par-3, par-6 or pkc-3 mutants. Anterior PAR proteins also amplify cortical flows, further promoting their own transport towards the anterior pole [38,40]. In wild-type PAR-2 microtubule binding helps its own recruitment to the cortex nearest to the centrosomes, contributing to efficient polarity establishment . In the absence of ECT-2-dependent cortical flows, PAR-2 becomes essential to break embryonic symmetries [21,41]. PAR-4 positively regulates the acto-myosin contractility and thus contributes to polarity establishment (Figure 2) . Finally, PAR-5 also participates in the establishment phase as, in par-5-depleted embryos, the PAR-3 complex clears the posterior less efficiently [23,24].
An unanswered question is how PAR proteins interact with the cortex. C. elegans embryos lacking PAR-6 or PKC-3 display a residual cortical presence of PAR-3, whereas depletion of PAR-3 completely abolishes the peripheral localization of PAR-6 and PKC-3 [14,15]. PAR-3 could therefore serve as a scaffold protein. Studies in human cells and in Drosophila showed that PAR-3 can bind phospholipids in vitro [43,44] and has the ability to oligomerize [17,45,46]. Thus PAR-3 could be loaded on the cortex by interacting with phospholipids and subsequently recruit PAR-6 and PKC-3. At the posterior, PAR-2 loads on the cortex (possibly by phospholipid binding) and subsequently localizes PAR-1 .
Establishment of polarity and PAR cortical binding are also regulated by the Hsp90 (heat-shock protein 90) co-chaperone CDC-37 (Cdc is cell division cycle) . In embryos depleted of cdc-37 by RNAi (RNA interference) [cdc-37(RNAi)], PAR-3 initially clears up from the posterior, but PAR-6 and PKC-3 remain high around the cortex, indicating that CDC-37 is required for proper interaction between the anterior PAR proteins .
Thus cortical flows localize PAR-3, PAR-6 and PKC-3 to the anterior cortical half of the embryo. Concomitantly, PAR-2 and PAR-1 associate with the posterior cortex. At the end of the establishment phase (around the PNM stage), the anterior–posterior PAR domains are well defined, non-overlapping and each of them occupy approximately 50% of the embryonic cortex (Figure 1I) .
Although segregated to the anterior and posterior (Figure 1I), PAR proteins are not stably bound to the cortex, they can diffuse laterally and rapidly exchange with the cytoplasmic pool [40,48]. Polarity is maintained by a complex network of interactions, which counterbalances the dynamic behaviour of PAR proteins.
PAR-2 plays a major role in the maintenance phase. To prevent the invasion of the anterior complex towards the posterior, PAR-2 (i) inhibits the re-assembly of acto-myosin foci in the posterior pole, which, if it occurred, would trigger abnormal posterior-directed flows and the re-distribution of PAR-3–PAR-6–PKC-3 to the posterior cortex  and (ii) localizes to the posterior cortex PAR-1, which phosphorylates PAR-3 and thus contributes to exclude the anterior complex from the PAR-1–PAR-2 domain . In turn, PKC-3 phosphorylates PAR-2 and inhibits its anterior localization (Figure 2) .
PAR-5 is also involved in polarity maintenance, although its molecular targets are not known. It is plausible, however, that PAR-5 binds phosphorylated PAR-1 to exclude it from the anterior or PAR-3 to exclude it from the posterior, similar to Drosophila and mammalian cells [50⇓–52].
LGL-1, the homologue of Drosophila lgl (Lethal Giant Larvae), also participates in maintenance of polarity [53,54]. Although LGL-1 is not essential for embryonic development, its depletion increases the embryonic lethality and the polarity defects of a weak par-2 mutant, whereas its overexpression rescues PAR-2 depletion by restoring embryonic polarity [53,54]. LGL-1 co-immunoprecipitates with PAR-6 and PKC-3; however, it localizes to the opposite posterior cortex [53,54]. The model suggests that LGL-1 forms a complex with PKC-3 and PAR-6 at the boundary between PAR domains. This allows phosphorylation of LGL-1 by PKC-3 and makes the LGL-1–PKC-3–PAR-6 complex dissociate from the cortex, inhibiting spreading of the anterior complex to the posterior LGL-1–PAR-2 domain . In addition, similarly to PAR-2, LGL-1 contributes to inhibit the re-assembly of acto-myosin foci in the posterior of the embryo, and thus prevents posteriorly directed flows and re-distribution of the anterior complex to the posterior pole after the PNM stage [53,54].
CDC-37 is also involved in polarity maintenance as reducing its levels impairs the mutual exclusion between anterior and posterior complexes. As CDC-37-depleted embryos show lower PKC-3 levels, it is possible that, in cdc-37(RNAi) embryos, PAR-2 is less efficiently phosphorylated by PKC-3 and thus cannot be excluded from the anterior cortex ( and see below).
Polarity maintenance also depends on the small GTPase CDC-42 [26,55,56]. CDC-42 binds to PAR-6 and promotes its localization at the cortex, stabilizing the anterior complex during the maintenance phase [55–57].
Finally, spatio–temporal regulation of endocytosis is also crucial to maintain membrane asymmetries. DYN-1 (the orthologue of dynamin, a large GTPase ) regulates endocytosis and is enriched in the anterior cortex in one-cell embryos . In DYN-1-depleted embryos, the anteriorly enriched endocytic events, present in wild-type, are altered and polarity is lost. DYN-1 may regulate internalization and recycling of PAR-6, therefore restricting it to the anterior domain .
At the end of the maintenance phase, the cytokinetic furrow ingresses in the posterior half of the embryo (Figures 1F and 1M). To properly segregate cell-fate determinants into daughter cells, cell polarity must be coupled with the site of cytokinetic furrow ingression. To achieve this co-ordination, PAR proteins control the position of the mitotic spindle (see below). As an additional mechanism, the cytokinetic furrow senses the PAR-2 boundary and is able to correct its position (Figure 1M) . This mechanism depends on heterotrimeric G-proteins (see below) and ensures that the cytokinetic furrow matches the border between the posterior and the anterior PAR domains, asymmetrically separating them into the sister cells: the PAR-3–PAR-6–PKC-3 to the AB cell and PAR-2–PAR-1 to the P1 cell (Figure 1N) [24,60].
In summary, phosphorylation-dependent mutual inhibition of the anterior and posterior PAR proteins, regulation of endocytosis and myosin assembly/disassembly all contribute to maintain polarity in the one-cell embryo.
PAR protein downstream effectors
PAR protein asymmetry governs unequal segregation of several cytoplasmic proteins (including cell-fate determinants; Figure 1T) and thus guarantees their proper distribution into the daughter cells . Two CCCH zinc-finger proteins MEX-5 (MEX is Muscle Excess) and MEX-6 (highly similar in protein sequence and function hereafter referred as MEX-5/6)  and the mitotic kinase PLK-1 (Polo-Like Kinase 1) [62–65] form an anterior-to-posterior gradient in the one-cell embryo and are segregated to the AB cell. By contrast, other CCCH zinc-finger proteins form a posterior-to-anterior gradient and are subsequently segregated into the P1 cell [66,67]. A fundamental question is how cytoplasmic gradients are generated and maintained.
Previous studies show that a difference in diffusion rate can establish and maintain such gradients [68–71]. In the one-cell embryo, the diffusion coefficient of PIE-1 (PIE is Pharynx and Intestine Excess), a CCCH protein that represses transcription [72,73], is lower in the posterior than in the anterior, resulting in a posterior-to-anterior gradient. The authors postulate that the lower diffusion coefficient may be a consequence of PIE-1 binding to RNAs in the posterior cytoplasm .
The MEX-5 anterior-to-posterior gradient also depends on diffusivity [68,70,71], which is controlled by the PAR-1 kinase [68,70,71] and uniformly distributed PP2A (Protein Phosphatase 2A) . In the posterior, MEX-5 phosphorylated by PAR-1 is released from slow-diffusive RNA-containing complexes and thus exhibits faster mobility. In the anterior, however, PAR-1 is absent and MEX-5, dephosphorylated by PP2A, can associate with slow-diffusive complexes. Therefore, owing to the antagonistic action of PAR-1 and PP2A, MEX-5 diffuses faster from the posterior to the anterior pole than in the opposite direction, generating a concentration gradient .
The asymmetric localization of these proteins is crucial for embryonic patterning. For instance, PIE-1 is restricted to the germline lineage where it represses transcription [72,73]. MEX-5 binds to PLK-1 and promotes its localization to the anterior pole, and thus the AB cell . The enrichment of PLK-1 in AB is crucial for the asynchronous mitotic entry of AB and P1 [64,65], which in turn is important for the respective positioning and cell–cell contacts of descendant daughter cells . A feedback regulation between PAR proteins, MEX-5 and downstream effectors also exists. MEX-5 promotes the growth of the PAR-2 domain during polarity establishment , whereas PLK-1 activates MEX-5  and influences localization of PAR proteins [63,75]. Such a complex network of interactions is crucial for C. elegans development, and once perturbed leads to defective differentiation and death.
Positioning of the mitotic spindle and unequal cell division
The first division of the C. elegans zygote creates cells different in content and in size. To do so, the mitotic spindle of the one-cell embryo is firstly orientated along the polarity axis and subsequently pulled towards the posterior pole to dictate asymmetric cleavage and thus unequally segregate polarized factors (Figures 1E and 1L) . Interactions between the cortex and astral microtubules are important for both events. Forces generated at the cortex, which pull on astral microtubules, involve a set of conserved proteins: dynein (a microtubule minus-end motor), GOA-1 (GOA is G-Protein O, α subunit) and GPA-16 (GPA is G-Protein α subunit) (two redundant Gα proteins), GPR-1 and GPR-2 (GPR is G-Protein Regulator) (two almost identical Gα regulators, hereafter referred as GPR-1/2) and LIN-5 (LIN is abnormal cell LINeage) (a coiled-coil protein) (reviewed in [8,77]).
Although Gα subunits are symmetrically distributed, its activators display dynamic and cell-cycle-dependent localization (Figures 1Q–1S). Until metaphase GPR-1/2 and LIN-5 are enriched at the anterior cortex to align centrosomes with the anterior–posterior axis (Figure 1Q) [78–80], whereas, in metaphase and anaphase, GPR-1/2 and LIN-5 become enriched at the posterior and drive posterior spindle displacement (Figures 1R and 1S , reviewed in ). PKC-3 phosphorylates LIN-5 and thus inhibits cortical pulling forces in the anterior pole . In addition, pulling forces are diminished on the lateral–posterior cortex by LET-99 (LEThal 99) a DEP (Dishevelled, Egl-10 and Pleckstrin) domain protein, which negatively regulates the localization of GPR-1/2, therefore inhibiting lateral pulling on astral microtubules which is counterproductive for spindle positioning (Figures 1Q–1S, reviewed in ). In par mutants the asymmetric localization of LIN-5 and GPR-1/2 is lost and the spindle remains in the centre of the cell, resulting in a symmetric first cleavage . Therefore PAR proteins regulate asymmetric spindle positioning by controlling the localization and activity of LIN-5 and GPR-1/2.
PAR proteins regulate the process of asymmetric cell division. This does not only involve the establishment and the maintenance of cortical polarity, but also entails the proper distribution of downstream cell-fate determinants, alignment of the mitotic spindle with the axis of polarity, and co-ordination between cellular processes and cell-cycle progression. To do so, the cortical polarity of PAR proteins is transduced into the asymmetric distribution and/or activity of proteins controlling cell-cycle progression and cell fate.
PAR proteins act with partners and effectors to efficiently polarize cells. Identifying new partners and/or PAR effectors and addressing the co-ordination between PAR polarity and other processes during asymmetric cell division will lead us towards a better understanding of this process. Given that many proteins are highly conserved, it is plausible that similar mechanisms operate during mammalian development and asymmetric division of stem cells.
• Cell polarity and asymmetric cell division are crucial to generate diversity during development.
• Successful asymmetric cell division requires establishment of a polarity axis, subsequent distribution of cell-fate determinants and orientation of the mitotic spindle along the axis of polarity.
• PAR proteins are major regulators orchestrating cell polarization, spindle orientation and asymmetric cell division across species. In the one-cell embryo PAR proteins form two reciprocal and mutually exclusive domains [anterior (PAR-3–PAR-6–PKC-3) and posterior (PAR-2–PAR-1)].
• Our understanding of cell polarity and asymmetric division is still incomplete. Identifying new interactors of PAR proteins and studying how, for instance, spindle orientation, cytokinesis, polarity establishment and maintenance are co-ordinated in space and time will lead us towards a better understanding of differentiation and stem cell biology, as well as disease development.
We thank Lesilee Rose, Elsa Kress and Manoel Prouteau for critical reading of the chapter and many useful comments. We also thank the reviewers for their constructive criticisms. Research in the M.G. laboratory is supported by the Swiss National Science Foundation, by an EMBO YIP (Young Investigator Programme) grant and from the University of Geneva.
- © The Authors Journal compilation © 2012 Biochemical Society