Cytokinetic abscission in vivo

Cytokinetic abscission in vivo Cytokinesis, the final step of cell division, concludes with abscission, which cleaves the thin intercellular bridge between the two daughter cells. Accurate control of cell division and cytokinetic abscission is crucial for correct partitioning of the genetic material between the two daughter cells. The mechanisms of cytokinetic abscission have started being mapped, especially in human cultured cells. However, much less is known of how abscission is controlled in vivo in the context of multi-cellular living tissues. This project has aimed at addressing how cytokinetic abscission is controlled in vivo using the fruit fly as a key model organism. The fruit fly has emerged as a powerful model to study cell division and cytokinesis in a multi-cellular context. In the project we have applied a combination of advanced imaging, biochemical and molecular biological methods to study cytokinesis and to identify molecules involved in the process. We have complemented our work with studies of cytokinesis in human cultured cells to identify evolutionarily conserved mechanisms. In the project, we have elucidated molecular mechanisms of abscission as well as dynamics of the abscission process both in vivo and in human cells. Specifically, we have increased the understanding of how abscission factors are recruited to the midbody during cytokinetic abscission in vivo. We have uncovered that the so called centralspindlin complex plays an evolutionarily conserved role in recruiting the abscission machinery. Interestingly, the mechanism we discovered shows similarities to the mechanism used by viruses to recruit the machinery required for virus budding in human cells. We have also established live imaging to visualize dynamics of abscission factors and abscission timing in different types of stem cells in vivo. Our data highlight that the abscission process is highly dynamic and indicate that distinct cell types can undergo cytokinesis and abscission with different dynamics. In human cells, we have elucidated spatiotemporal dynamics of key abscission regulators using advanced imaging methods, i.e. super-resolution and live imaging. We have also applied electron microscopy to understand details about cytokinetic abscission at the ultrastructural level. Our results uncover previously uncharacterized dynamics of key abscission factors during cytokinetic abscission in human cells. Specifically, our data show that these key abscission factors can undergo motor protein-mediated transport to the intercellular bridge on vesicles, thereby contributing to their role in abscission. Moreover, we uncover that two key abscission factors undergo co-transport to the intercellular bridge and recruitment with similar dynamics to the midbody, suggesting that they not only undergo sequential recruitment, but also co-recruitment during abscission, which increases the understanding of the dynamics and progression of abscission in human cells. To decipher the molecular control of abscission in further detail, we are in the process of identifying abscission factors in vivo and in human cells. We have uncovered putative novel protein complexes and abscission factors and are currently investigating their complex formation, spatiotemporal dynamics and functional roles in cytokinetic abscission in different cell types in vivo as well as in human cells. We have elucidated a direct interaction between a key abscission factor and a novel interacting partner in Drosophila. We have generated reagents and are performing experiments to understand any putative coordinate spatiotemporal dynamics and functional roles of these proteins during cytokinetic abscission in different cell types in vivo, applying among others live imaging. We also have evidence of the involvement of a previously uncharacterized protein in cytokinesis and abscission in human cells. We are in the process of elucidating its recruitment dynamics and functions during cytokinesis and abscission. We hope that these studies will give novel insight into the molecular mechanisms of cytokinetic abscission in vivo and in human cells. Our established assays are enabling us understand in further detail the spatiotemporal dynamics and molecular mechanisms of abscission in different cell types in vivo as well as in human cultured cells. The project has increased the understanding of the spatiotemporal control of cytokinetic abscission, as well as given novel insight into the molecular mechanisms controlling the process, both in vivo and in human cultured cells. The project has also identified putative differential regulation of cytokinesis in different cell types, as well as evolutionarily conserved molecular mechanisms of cytokinetic abscission.

The overall effects of the project include increasing the knowledge about the mechanisms of cytokinetic abscission in vivo as well as in human cells. Specifically, concerning cytokinetic abscission in vivo, our findings contribute to the understanding of mechanisms of abscission factor recruitment to the midbody during cytokinesis. The findings in human cells contribute to the understanding of the spatiotemporal dynamics of proteins and their recruitment to the intercellular bridge during cytokinetic abscission. Overall, the findings contribute new knowledge in the cytokinesis research field. The project has also resulted in competence development for project participants, among others through method development and interdisciplinary collaboration, which will be valuable also in future projects. The project results have been, and will continue to be, disseminated in scientific journals, scientific seminars and presentations, both nationally and internationally.

Cytokinesis, the final step of cell division, concludes with abscission, which involves cleavage of the intercellular bridge between the two daughter cells. In recent years, key insights into the control of abscission have been gained in human cultured cell lines by a combination of advanced molecular biological and imaging technologies. However, the mechanisms controlling the final abscission step in vivo in the context of multi-cellular living tissues are much less understood. Major questions in the field concern how cytokinesis is controlled in a multi-cellular context and how cytokinesis might be differentially regulated in different cell types in vivo. This project aims at addressing these questions by elucidating the spatiotemporal and molecular control of cytokinetic abscission in powerful in vivo abscission models using Drosophila melanogaster as a model organism. We will complement our work with studies of cytokinesis in human cells to compare and contrast with our in vivo findings and to elucidate evolutionarily conserved mechanisms. To discover novel abscission regulators we aim to identify novel interactors of established abscission machinery components. The project will apply a multidisciplinary approach based on a combination of cutting-edge imaging technologies, genetic, cell biological, biochemical and molecular biological methods. We hope that our work will give novel insight into the mechanisms by which cytokinesis is controlled in different cell types dividing asymmetrically and symmetrically in vivo.