Polarity establishment, asymmetric division and segregation of fate determinants in early C. elegans embryos

Polarity establishment, asymmetric division, and acquisition of cell fates are critical steps during earlydevelopment. In this review, we discuss processes that set up the embryonic axes, with an emphasis onpolarity establishment and asymmetric division. We begin with the first asymmetric division in theC. elegansembryo, where symmetry is broken by the local inactivation of actomyosin cortical contractility. Thiscontributes to establishing a polarized distribution of PAR proteins and associated components on the cellcortex along the longitudinal embryonic axis, which becomes the anterior-posterior (AP) axis. Thereafter, APpolarity is maintained through reciprocal negative interactions between the anterior and posterior corticaldomains. We then review the mechanisms that ensure proper positioning of the centrosomes and the mitoticspindle in the one-cell embryo by exerting pulling forces on astral microtubules. We explain how a ternarycomplex comprised of Gα(GOA-1/GPA-16), GPR-1/GPR-2, andLIN-5is essential for anchoring the motorprotein dynein to the cell cortex, where it is thought to exert pulling forces on depolymerizing astralmicrotubules. We proceed by providing an overview of cell cycle asynchrony in two-cell embryos, as well asthe cell signaling and spindle positioning events that underly the subsequent asymmetric divisions, whichestablish the dorsal-ventral and left-right axes. We then discuss how AP polarity ensures the unequalsegregation of cell fate regulators via the cytoplasmic proteinsMEX-5/MEX-6and other polarity mediators,before ending with an overview of how the fates of the early blastomeres are specified by these processes.

Asymmetric Spindle Positioning and Intracellular Trafficking in C. Elegans Embryos

Kalyani Thyagarajan

Asymmetric cell division is critical for generating daughter cells that possess distinct fates during development and in stem cell lineages. Correct orientation of the mitotic spindle with respect to the polarity axis is crucial to this process. In the one-cell stage embryo of the nematode C. elegans, polarity cues define an anterior-posterior axis, and the mitotic spindle is positioned asymmetrically towards the posterior of the embryo in response to these cues, resulting in daughter cells that differ not only in fates, but also in sizes. Although the way by which force is generated has becoming increasingly well understood, the mechanisms by which polarity cues regulate the asymmetric distribution of active cortical force generators that pull on astral microtubules to position the spindle remained unclear at the onset of my thesis. During the course of our work, we found that intracellular trafficking plays a crucial role in the distribution and levels of proteins involved in asymmetric spindle positioning. Firstly, we found that the distribution of the Gβ protein GPB-1, a negative regulator of pulling forces, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. GPB-1 transits through the endosomal network in a dynamin-, clathrin- and RAB-5-dependent manner. We also found that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis, and thus symmetric pulling forces during mitosis. Secondly, we found that the clathrin heavy chain modulates spindle-positioning forces as well as the cortical distribution of positive (GPR-1/2 and DHC-1) and negative (GPB-1 and LET-99) regulators of pulling forces. Although the mechanisms by which it does so remain unclear, our data suggests that clathrin plays multiple roles in the one-cell stage C. elegans embryos, including in recycling GPB-1 to the plasma membrane. Overall, our results lead us to propose that the clathrin heavy chain and the modulation of Gβγ trafficking play a critical role during asymmetric division of one-cell stage C. elegans embryos.

EPFL2011

Mechanisms of centrosome separation in C. elegans

Alessandro De Simone

Centrosomes are the major microtubule organizing centers of animal cells. The two centrosomes present at the onset of mitosis must separate in a timely fashion along the nuclear envelope to ensure proper bipolar spindle assembly and thus genome stability. Microtubuleassociated motors of the kinesin-5 family are required for centrosome separation in several systems, but are partially redundant or entirely dispensable in others, where the minus-end directed motor dynein plays an important role. The mechanisms by which dynein powers centrosome separation are incompletely understood. Furthermore, the nature of the symmetry-breaking mechanisms that imbalance the forces acting on centrosomes to favor theirmovement away from each other are not known. We addressed these questions using a combination of 3D time-lapse microscopy, image processing and computational modeling to dissect centrosome separation in the polarized one-cell C. elegans embryo that entirely relies on dynein for this process. First, we have characterized the quantitative features of centrosome separation in the wild-type. Next, we compared centrosome separation between wild-type and mutant/RNAi conditions. Our analysis revealed that centrosome separation is powered by the combined action of dynein at the nuclear envelope and at the cell cortex. Moreover, we demonstrated that cortical dynein requires actomyosin contractility to separate centrosomes. These observations suggest that cortical dynein acts by harnessing anterior-directed actomyosin cortical flows initiated earlier in the cell cycle by the centrosomes themselves. To confirmthismodel, we successfully tested experimentally two of its key predictions, namely that dynein complexes flow toward the anterior together with the cortex and that the velocity of centrosome separation correlates with that of the flow of the nearby cortex. Taken together, these results demonstrate that centrosome separation is driven by nuclear and cortical dynein, where the latter acts by transmitting forces produced by the cortical actomyosin flow. To test whether this model is sufficient to explain centrosome separation, we developed a 3D computationalmodel of cytoskeleton dynamics. Indeed, predicted centrosome separation agrees quantitatively with the experimental observations in wild-type and mutant/RNAi conditions. Moreover, the qualitative predictions of the model are robust for parameter changes. Furthermore, computational simulations demonstrate that forces are intrinsically organized to move centrosomes away from each other without the need of any extrinsic symmetry-breaking mechanism. Indeed, in the case of nuclear dynein-driven separation, the position of centrosomes between the nuclear envelope and the cortex results in an asymmetric microtubule aster that leads to centrosome outward movement. In the case of cortical dynein, cortical flows are triggered by centrosomes and always move away from them, such that their forces are always directed to separate centrosomes. Therefore, this separation mechanism functions irrespective of the initial position of centrosomes along the cortex. In conclusion, in this thesis we uncover a novel organizing principle in which dynein, coupled with cell geometry and flow pattern, serves to robustly separate centrosomes and thus ensure genome stability.

EPFL2016

lis-1 is required for dynein-dependent cell division processes in C. elegans embryos

Pierre Gönczy

We investigated the role of the evolutionarily conserved protein Lis1 in cell division processes of Caenorhabditis elegans embryos. We identified apparent null alleles of lis-1, which result in defects identical to those observed after inactivation of the dynein heavy chain dhc-1, including defects in centrosome separation and spindle assembly. We raised antibodies against LIS-1 and generated transgenic animals expressing functional GFP-LIS-1. Using indirect immunofluorescence and spinning-disk confocal microscopy, we found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations, including the cell cortex, the vicinity of microtubule asters, the nuclear periphery and kinetochores. We established that lis-1 contributes to, but is not essential for, DHC-1 enrichment at specific subcellular locations. Conversely, we found that dhc-1, as well as the dynactin components dnc-1 (p150Glued) and dnc-2 (p50/dynamitin), are essential for LIS-1 targeting to the nuclear periphery, but not to the cell cortex nor to kinetochores. These results suggest that dynein and Lis1, albeit functioning in identical processes, are targeted partially independently of one another.