Table 1. Polarity establishment and maintenance proteins. Fertilization initiates a cascade of events that leads to breaking the symmetry of the oocyte, as evidenced by a local cessation of actomyosin contractility. What is the nature of the symmetry-breaking cue? It is not the sperm entry site nor paternal genetic material Sadler and Shakes, Goldstein and Hird, Instead, centrioles are key.
As in most metazoan organisms, the sperm contributes the sole pair of centrioles to the zygote. These centrioles recruit pericentriolar material PCM from maternal stores and thus assemble centrosomes that nucleate microtubules. After an initial phase during which centrosome maturation is suppressed McNally et al. Centrosomes can also break symmetry at a slight distance from the cortex, although subsequent AP polarity establishment is delayed in these instances, as is the case when microtubules are lacking and, as a consequence, centrosomes approach the cortex only after a lag time Bienkowska and Cowan, Microtubules from the meitoic spindle can also induce polarization in the absence of centrosomes in embryos arrested in metaphase of meiosis I Wallenfang and Seydoux, However, during embryogenesis centrosomes, or short microtubules that they nucleate, appear essential for symmetry breaking, as demonstrated by experiments in which centrosomes are ablated with a laser-microbeam Cowan and Hyman, b.
Compatible with interactions between centrosomes and the cell cortex being critical, symmetry breaking is compromised in embryos carrying mutations in the PAM-1 aminopeptidase, in which centrosomes remain far from the cortex Fortin et al. Interestingly, forcing centrosomes to remain at the cortex in pam-1 mutant embryos by depleting the dynein heavy chain DHC-1 rescues symmetry breaking, further indicating the importance of centrosome-cortex interactions in this process Fortin et al. Defective interaction between centrosomes and the cell cortex is also observed in embryos depleted of the deubiquitylating enzymes MATH and USP, and rescued also in these cases by the additional depletion of DHC-1 McCloskey and Kemphues, These observations raise the possibility that MATH and USP contribute to stabilizing one or several components critical for mediating centrosome-cortex interaction.
What are the mechanisms leading to the local cessation of cortical contractions following symmetry breaking? A critical event is the inactivation of RHO-1 in the vicinity of centrosomes. Interestingly, ECT-2 is distributed uniformly on the cortex before symmetry breaking, but removed in the vicinity of centrosomes concomitantly with the appearance of non-contractile cortex Motegi and Sugimoto, ; Schonegg and Hyman, How the presence of centrosomes results in local ECT-2 removal is not clear.
CYK-4 is present in sperm and persists in the newly fertilized zygote on membranous organelles in the vicinity of the centrosome, as well as on the cortex, where it could conceivably contribute to local RHO-1 inactivation. In line with this view, depletion of CYK-4 from sperm compromises symmetry breaking, with the cortical actomyosin network remaining uniformly contractile in a fraction of embryos Jenkins et al.
However, the mechanisms by which CYK-4 may contribute to polarity establishment remain to be further clarified, and it has been suggested that CYK-4 might be largely inactive during this stage Tse et al. In summary, centrosomes are critical for breaking symmetry of the oocyte and thus for setting into motion the sequence of events that results in establishing polarity along the AP axis. Although RHO-1 inactivation in the vicinity of the centrosome plays a major role in initiating AP polarity establishment, more recent work revealed the existence of a partially redundant mechanism relying on the PAR protein PAR-2 , which is discussed in Section 2.
This anterior movement entails a flow of cortical material away from centrosomes, which is compensated by posterior-directed streaming of cytoplasmic material Hird and White, Quantitative analysis and computer simulations confirm that the hydrodynamic properties of the cytoplasm can explain such compensatory streaming Niwayama et al.
What are the properties of the actomyosin network during the anteriorly-directed flow of cortical material? An answer comes from experiments in which the actomyosin network was severed locally with a laser microbeam and the resulting surface movements perpendicular to the cut monitored as a measure of the underlying cortical tension Mayer et al. Performing such severing along the longitudinal axis demonstrated that cortical tension orthogonal to the AP axis is twice as high in the anterior, contractile, region than in the posterior, non-contractile, region.
By contrast, performing such severing along the shorter axis established that cortical tension along the AP axis is uniform throughout the embryo. Thus, there is an anisotropy in cortical tension in the anterior, being higher orthogonal to the AP axis. Such cortical anisotropy is lacking in embryos that do not have anteriorly-directed cortical flows for instance, following defective centrosome assembly , compatible with cortical anisotropy being a consequence of such flows Mayer et al. A physical model of these phenomena that treats the cortex as a thin film of active viscous fluid demonstrates that local contractions of the actomyosin network can result in long-range movements of the cell cortex towards the embryo anterior Mayer et al.
Thus, anterior-restricted anisotropy in cortical contractility can be instrumental in polarity establishment. The partitioning of the cortex into a retracting anterior contractile domain and an expanding posterior non-contractile domain is accompanied by the asymmetric distribution of PAR proteins at the cell cortex.
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The six par genes for par titioning defective were identified amongst maternal-effect mutations as having defective AP polarity, resulting in the generation of daughter cells with altered fate, size, spindle positioning and cell cycle progression reviewed in Goldstein and Macara, ; Asymmetric cell division and axis formation in the embryo ; Rose and Kemphues, a ; Kemphues and Strome, The anterior PAR complex marked by GFP::PAR-6 is present throughout the cortex initially, but recedes from the posterior following symmetry breaking, concomitant with the anteriorly-directed movement of the contractile cortex Munro et al.
Movie 2. Time is indicated in seconds from the beginning of the recording. Although anteriorly-directed movement of the contractile actomyosin network marked by NMY-2 ::GFP foci occurs concomitantly with expansion of the posterior domain marked by GFP::PAR-2, the dynamics of these two cortical components differs Petrasek et al.
This suggests that PAR-2 distribution is somehow uncoupled from actomyosin dynamics, which is also in line with the finding that PAR-2 can bind phospholipids Motegi et al. These experiments indicate that exchange between the cytoplasmic and the cortical pool, while occuring Cheeks et al. Together, these studies illustrate that PAR proteins constitute a dynamic ensemble, whose kinetics must be taken into consideration when reflecting upon the mechanisms of polarity establishment. Anteriorly directed movement of the actomyosin network is critical for polarity establishment, as evidenced, for instance, by the fact that movements of cortical GFP::PAR-6 are attenuated in embryos partially depleted of NMY-2 Munro et al.
Polarity establishment is also altered in other cases in which cortical contractility is compromised, such as in embryos with impaired RHO-1 activity, including those depleted for NOP-1 as mentioned earlier, or of the TAO kinase KIN Spiga et al. Reciprocally, par genes are required for efficient anteriorly-directed cortical flows.
In par-2 RNAi embryos, by contrast, cortical flows are normal initially Cheeks et al. In summary, polarity establishment entails contraction of the active actomyosin network towards the embryo anterior, accompanied by the segregation of anterior and posterior PAR proteins into two largely mutually exclusive domains. Recent work has unveiled a role for PAR-2 during the early steps of polarization that is masked by the principal mechanism discussed above relying on local inactivation of RHO-1 Motegi et al.
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Embryos carrying a partial loss-of-function allele of ect-2 , ect-2 ax , exhibit inefficient anteriorly-directed cortical flows and delayed PAR-3 clearance from the posterior cortex, but eventually achieve a polarized distribution of PAR-3 Zonies et al. Because par-2 is essential for such delayed polarization in ect-2 ax embryos, it was proposed that there is a partially redundant pathway dependent on PAR-2 for polarity initiation that is revealed when the actomyosin network is compromised Zonies et al.
What are the mechanisms that enable PAR-2 to exert this function? Live imaging revealed that PAR-2 associates with centrosomes, whereas biochemical experiments established that PAR-2 is a microtubule binding protein Motegi et al. Thus, in vivo , association of PAR-2 with microtubules emanating from the centrosomes at the posterior would prevent the action of PKC-3 , which would otherwise inhibit PAR-2 cortical localization Motegi et al.
In search of a mechanism underlying this protective effect, it was discovered that PAR-2 binds phospholipids in vitro. This binding is curbed upon phosphorylation of PAR-2 by PKC-3 , but this negative regulation does not operate when microtubules are present in addition. Furthermore, membrane-bound PAR-2 recruits more PAR-2 , creating a positive feedback loop that promotes expansion of the posterior domain.
Whilst important when the principal pathway of polarity establishment is lacking, this PARdependent pathway is not essential under normal circumstances. Thus, although embryos expressing only a mutant version of PAR-2 that cannot bind microtubules initiate polarity with slower kinetics, they manage to establish it correctly thereafter Motegi et al. In summary, initiation of polarity establishment in the vicinity of centrosomes is aided by a PARdependent pathway that relies on the ability of this protein to bind microtubules and thus be shielded from PKCmediated phosphorylation.
The phase of polarity establishment is followed by a maintenance phase during which centrosome ablation no longer interferes with AP polarity Cowan and Hyman, b , although microtubules have been suggested to contribute to refining polarity during this phase Ai et al. During polarity maintenance, mutual inhibition between the anterior and posterior cortical domains is critical and par-2 is essential to maintain the anterior PAR complex restricted to the embryo anterior.
Thus, in par-2 mutants, GFP::PAR-6, which had been restricted to the anterior during the establishment phase, returns to the posterior to occupy the entire cortical circumference Cuenca et al.
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What, then, are the mechanisms preventing the spread of anterior PAR proteins to the posterior and reciprocally? In turn, the presence of PAR-2 at the cell cortex promotes the recruitment of PAR-1 through a direct interaction between the two proteins Motegi et al.
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Apart from the actomyosin network and the PAR proteins, other components participate in generating AP polarity in the one-cell embryo. LGL-1 is enriched on the posterior cortex, and its depletion, while not resulting in a phenotype on its own, affects the distribution of cortical NMY-2 and enhances the lethality incurred from partial PAR-2 inactivation. Therefore, LGL-1 can be thought of as defining a second pathway that contributes to polarity establishment in the absence of PAR What restricts LGL-1 cortical distribution to the posterior?
Phosphorylation by PKC-3 is key here as well.
Mutating three PKC-3 predicted phosphorylation sites to non-phosphorylatable residues leads to uniform cortical distribution of GFP::LGL-1, whereas the converse phosphomimetic variant does not localize Hoege et al. Furthermore, embryos expressing a mutant version that cannot interact with CDC as sole source of PAR-6 exhibit polarity defects mirroring those of partial CDC depletion Aceto et al.
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This reporter revealed that cortical CDC GTP is enriched initially on the posterior, then on the anterior, before becoming undetectable. Although cdc RNAi embryos have been reported to have defects primarily in the maintenance phase of polarization Gotta et al. Additional components, including ones that are important for basic cellular processes, are harnessed to ensure polarity maintenance. For instance, the dynamin GTPase, a universal modulator of endocytosis, membrane trafficking, and actin dynamics, participates in polarity maintenance in one-cell C.
Just like dynamin, the early endosomal protein RAB-5 is enriched on the anterior side during the maintenance phase, in a manner that depends on AP polarity cues Andrews and Ahringer, However, pleiotropic phenotypes often complicate analyzing the consequences of depleting components required for basic cellular processes. For instance, RAB-5 depletion also alters the actin cytoskeleton Hyenne et al.
Overall, whether trafficking events are instructive for directing AP polarity or merely elements that contribute in a more passive manner through their general requirement for cell physiology remains to be clarified. In summary, the maintenance of AP polarity relies principally on the mutual inhibition of anterior and posterior cortical domains, which, together with associated components, ensures the robust maintenance of polarity along the AP embryonic axis.
The wealth of information regarding the relationships between anterior and posterior polarity components, as well as knowledge about their dynamics and that of the underlying actomyosin network, have fueled mathematical modeling of how AP polarity is achieved in one-cell C. An initial model captured the essence of the behavior of the actomyosin network, as well as of the PAR proteins and of their relationships Tostevin and Howard, This model consists of reaction-diffusion equations that rest on the assumption that PAR protein dynamics at the cell cortex result from their diffusion within the cortical plane and from exchange with the cytoplasmic pool.
A further assumption is that association of anterior PAR proteins with the contractile actomyosin cortex increases over time. In the output from this model, anterior PAR proteins exclude posterior PAR proteins and reciprocally , generating effective positive feedback loops that lead to mutually exclusive cortical distributions. The molecular tenets of such mutual exclusion remain open in the model, but could correspond for instance to phosphorylation of PAR-2 by PKC-3 in the case of inhibition of the posterior complex by the anterior one.
This initial model left open several questions, including how a postulated asymmetry in the distribution of microtubules that is essential in the model to maintain polarity relates to the situation in vivo. Further modeling indicates that mutual inhibition can maintain distinct domains even without a contribution of the actomyosin network, provided oligomerization of the anterior PAR proteins is included Dawes and Munro, Interestingly, PAR-3 proteins are known to dimerize in other systems and have thus been postulated to correspond to the molecular basis for such oligomerization.
This revised model features a non-linear relationship in mutual cortical exclusion, which predicts that polarity should be lost abruptly below a threshold level of anterior PAR proteins, which is indeed observed experimentally Dawes and Munro, In this model, the cell cortex is considered as a thin film that transports embedded molecules towards the anterior by advection, much like a river would carry leaves floating on the water surface. Compatible with this model, simulations indicate that advection by such fluid flow can indeed displace anterior PAR proteins transiently and thus be critical for polarization of one-cell C.
Movie 3. Simulation of a mathematical model of polarity establishment. Model developed by Goehring et al.