Final Report Summary - DIVIMAGE (Bridging spatial and temporal resolution gaps in the study of cell division)
Cell division is fundamental for the proliferation of all life forms. In animals, cell division proceeds by three distinct steps. First, mitosis mediates the segregation of one copy of the replicated genome to each of the two poles of the mitotic spindle. Then, a contractile ring composed of actin and myosin filaments mediates the ingression of a cleavage furrow between the two spindle poles. Nascent daughter cells remain connected by a membrane tube until they finally split apart by a process termed abscission. Mitosis and cytokinetic cleavage furrow ingression have been studied extensively in the past, but the mechanism of abscission is still poorly understood. Abscission involves the Endosomal Sorting Complex Required for Transport-III (ESCRT-III), which has been thought to assemble into spirals of persistent filaments that mediate membrane neck constriction by increasing their curvature over time. However, how different ESCRT-III components coordinately assemble and remodel polymer structures has remained unclear. We have studied ESCRT-III assembly and function using advanced light and electron microscopy techniques. By quantitative live-cell microscopy, we found that growing ESCRT-III assemblies continuously turn over their subunits with cytoplasmic pools. Further, our data indicate that dynamic subunit turnover is required to form functional ESCRT-III assemblies. By light and electron microscopy, we found that steady-state turnover of individual ESCRT-III subunits depended on Vps4, a factor that has been previously thought to function as recycling machinery only after completion of abscission. We further found that Vps4 promotes net growth of ESCRT-III polymers by alleviating from a growth inhibition mediated by the Vps2/Vps24 subunits. The interplay between different subunits and the dynamic organization of ESCRT-III polymers elucidated by our study represents a paradigm shift in our understanding of ESCRT-mediated membrane constriction. While highly relevant for cytokinesis, this also has broad implications for many other biological processes that involve the ESCRT-III machinery, as for example the budding of some viruses, the trafficking of vesicles inside cells, and nuclear envelope formation and repair.
Our study has been driven by technology advancement towards automated microscopy and computational data analysis. A major achievement of this project has been the development of novel computer vision and machine learning tools that are available to a broad research community. We have implemented the software platform CellCognition Explorer for novelty detection and deep learning of cellular phenotypes, and demonstrated broad applicability in several large-scale screens that revealed novel cell division regulators. Our work has therefore substantially advanced high-content screening technology and has provided groundbreaking insights into the molecular mechanisms underlying cell division.
Our study has been driven by technology advancement towards automated microscopy and computational data analysis. A major achievement of this project has been the development of novel computer vision and machine learning tools that are available to a broad research community. We have implemented the software platform CellCognition Explorer for novelty detection and deep learning of cellular phenotypes, and demonstrated broad applicability in several large-scale screens that revealed novel cell division regulators. Our work has therefore substantially advanced high-content screening technology and has provided groundbreaking insights into the molecular mechanisms underlying cell division.