We showed that the two major actin filament nucleators during cytokinesis are the formin CYK-1 and the ARP2/3 complex and while CYK-1 is essential to nucleate the F-actin that forms the contractile ring, the ARP2/3 complex prevents excessive formin activity, which is detrimental for cytokinesis. Our results on the contribution of myosin to cytokinesis revealed that it is its motor activity, rather than the its ability to crosslink actin filaments or modulate actin levels, that drives contractile ring assembly and constriction. We also found that myosin functions together with Plastin, a non-motor F-actin crosslinker, to align the F-actin bundles circumferentially around the cell equator. We discovered a synergy between Plastin and Spectrins, as contractile ring formation failed when both proteins were co-inhibited, although the levels of actin and myosin were not affected. In a follow-up study we discovered that plastin and spectrins contribute distinctly to cytokinesis. When depleting one or the other in fragile rings, we found that spectrins stabilize the contractile ring by protecting it from rupturing and likely contribute directly to the repair process; while plastin contributes to cortical F-actin connectivity and to cortical tension that counteracts ring constriction. This was the first time that spectrins have been described to participate in the process of cell division. We also unraveled and characterized a robust repair mechanism that is in place during ring constriction to make the ring impervious to discontinuities in its structure. We discovered this phenomenon by devising an assay to cut constricting rings using laser microsurgery. Mechanical dissection of the ring response to laser cutting indicated that the constricting ring works against tension generated by the remaining cell cortex and dynamic components, such as the increase in surface area of the septum that grows behind the ring as constriction proceeds. In work nearing finalization we (in collaboration) built a global model for cytokinesis where both the contractile ring and the surrounding cortex are considered using, for the first time, experimental profiles of myosin. We found that contractile ring constriction velocity in the C. elegans zygote does not correlate with the total amount of myosin in the ring, and our 3D coarse-grained simulations corroborated by experimental data, showed that the ring constriction velocity can only be maintained over a range of myosin levels if myosin contributes to both active and viscous tensions in the cortical layer. In another story we discovered that the joint contribution of anillin in the contractile ring and the central spindle SPD-1 bundled microtubules is required to maintain active myosin in the ring during the second half of ring closure, ensuring the success of cytokinesis. We also completed a screen of loss of function phenotypes of actomyosin regulators in the C. elegans germline gonad. After pursuing a group of genes whose depletion gave rise to a phenotype of endomitosis, we identified anillin to be critical for the early development of the spermatheca. Dissection of the phenotype unraveled an unexpected requirement for two different myosins at different stages of spermatheca development, with each of them associating with specific actin networks organizations. These results were published in international peer-reviewed scientific journals and/or disseminated in presentations of our work to the scientific community by participating in national and international conferences and workshops, graduate, undergraduate and high school students, and the general public in fairs organized by the University of Porto.
