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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - ACTOMYO (Mechanisms of actomyosin-based contractility during cytokinesis)

Teaser

Summary

The main aim of this project is to understand 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, ensuring that each daughter cell retains one copy of the replicated genome. 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 division as well as other actomyosin dependent processes, cell homeostasis and disease. In metazoans, cytokinesis is accomplished via the assembly and subsequent constriction of a contractile ring. The contractile ring assembles around the cell equator beneath the plasma membrane after the replicated chromosomes have segregated. Constriction of the ring progressively draws the plasma membrane inwards, closing the gap between the two daughter cells. It is known that the contractile ring is composed of actin and myosin filaments as well as other proteins that regulate actomyosin activity but the inner workings of the ring are still poorly understood. With this work we want to investigate the mechanisms and dynamics of the actomyosin contractile ring in the context of C. elegans embryos. The cytokinetic protein machinery is extremely well conserved in this simple yet powerful experimental system, and other advantages that make C. elegans embryos an unique system to study cytokinesis include: efficient protein depletion by RNA interference, quantitative live-imaging assays, stereotypic early divisions, robust gene editing methodologies, and established methods for assessing embryo development.

Work performed

Our work has been focused on determining the contribution of branched and non-branched actin filament populations as well as myosin motility and actin filament dynamics to cytokinesis. We showed that the two major actin filament nucleators during cytokinesis are the formin CYK-1 and the ARP2/3 complex and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and its activity is required throughout ring constriction. The ARP2/3 complex does not localize in the contractile ring but its depletion/inhibition delays contractile ring formation and constriction because of an excess in formin-nucleated cortical F-actin. We therefore proposed that the ARP2/3 complex negatively regulates CYK-1 activity and that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators. Our results on the contribution of non-muscle myosin II to cytokinesis show that it is myosin motor activity, rather than myosinâ€™s ability to cross-link actin filaments or modulate actin filament levels, that drives contractile ring assembly and constriction. Moreover, our data lead us to propose that the main determinant for formation of a compact and properly organized contractile ring is recruitment of sufficient motor-competent myosin to the cell equator. 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 when we aimed at cutting constricting cytokinetic rings by means of laser microsurgery. Laser cutting of the ring resulted in an immediate snapping event, demonstrating that the ring is under tension. Strikingly, the gap between severed ends was quickly repaired, with the ring recovering its original topology and resuming constriction. Surprisingly, many consecutive cuts, which prevented gap repair altogether, still allowed constriction although at a slow rate, which revealed that a continuous contractile ring structure is not a prerequisite for constriction. 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. These results have been published in international peer-reviewed scientific journals and we have been disseminating them by presenting our work to the scientific community by participating in national and international conferences and workshops, high school students that regularly visit our institution, and the general public in fairs organized by the University of Porto.

Final results

Our work so far 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 in vivo is a highly robust process impervious to discontinuities in contractile ring structure. Also, we showed that a continuous ring structure is not a prerequisite for constriction. These are novel ideas in the field that advance our understanding of cytokinesis and constitute important advances to envision and develop models for ring constriction. We also dedicated special attention to a central question in the field, which regards the force generation mechanisms that allow for shape changing during cytokinesis. We showed that myosin motor activity is essential for all stages of cytokinesis and that myosin ability to crosslink actin filaments is not sufficient for the process. Force generation and propagation in cytokinesis or any other actomyosin-dependent process require not only myosin motors but also crosslinkers that keep the network interconnected and we are currently investigating what is the role of non-motor crosslinkers during cytokinesis. In addition, we aim to build a global cytokinesis model that recapitulates ring constriction in the context of the whole surrounding cortex. Understanding the basic mechanisms of cytokinesis is going to prove central to improve diagnosing tools and therapies in disease where this essential cell process is compromised.