Periodic Reporting for period 5 - ACTOMYO (Mechanisms of actomyosin-based contractility during cytokinesis)
Reporting period: 2022-01-01 to 2022-10-31
The main aim of this project is to deepen our understanding of the mechanisms of actomyosin-based contractility during cytokinesis. Cytokinesis completes cell division by physically partitioning the contents of the mother cell into the two daughter cells. Proper cell division plays an unquestionable role during cell proliferation, development and regeneration of tissues and failure in cytokinesis gives rise to tetraploid cells, which have been postulated to be a critical intermediate in the development of cancer. Hence, addressing fundamental questions about cytokinesis will provide mechanistic insight to understand cell homeostasis and disease. In metazoans, cytokinesis is primarily accomplished via the assembly and constriction of a contractile ring, which is a circular and continuous actomyosin structure that assembles around the cell equator beneath the plasma membrane, after the replicated chromosomes have segregated. Constriction of the ring closes the gap between the two daughter cells. Highlights of our work on the innerworkings of this structure include the finding that the contractile ring has the remarkable capacity for rapid local remodeling of its structure throughout constriction. Both ARP2/3-nucleated branched and formin-nucleated non-branched actin filament configurations positively contribute to cytokinesis and ARP2/3 prevents excessive formin activity, which is detrimental for cytokinesis. We definitively demonstrated that the motor activity of myosin is essential for cytokinesis in C. elegans embryos. We found that cytokinesis kinetics are dictated by a balance of myosin-dependent contractility and viscosity between the contractile ring and the surrounding cortex. Mechanisms involving crosstalk between anillin in the contractile ring and central spindle microtubules are in place to ensure that myosin remains active and contractile throughout ring constriction. Our investigation on the role of F-actin crosslinkers during cytokinesis unexpectedly led to the finding that spectrins, are required to protect contractile rings from rupturing during cell division. Importantly, we found that the joint action of the large and flexible spectrins with the short and rigid crosslinker plastin, are essential to organize and maintain the actomyosin network that forms the contractile ring.
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.
Our work has provided new insights into the cytokinesis process, namely into the stage of ring constriction, the least explored in the field. Importantly, our data show that cytokinesis is a highly robust process impervious to discontinuities in contractile ring structure. Contractile rings are able to keep constricting in the presence of a continuously interrupted structure and this notion is novel in the field and constitutes an important advance to envision and develop models for ring constriction. Force generation and propagation in cytokinesis or any other actomyosin-dependent process requires crosslinkers that keep the network interconnected. We discovered that the joint action of crosslinkers with distinct biophysical properties is essential to organize and stabilize the F-actin network that forms the contractile ring. We also showed for the first time that spectrins, well known for its roles in crosslinking F-actin in erythrocytes and axons, participate in the process of cell division. In collaboration we built a global cytokinesis model that recapitulates ring assembly and constriction in the context of the surrounding cortex, which will be key to continue investigating the process in the future.