Three-dimensional reconstruction of abscission:Role of ESCRT-dependent filaments

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Abscission is the last step of cytokinesis and an essential process in all animals to ensure successful physical separation of post-mitotic cell pairs. Previously, we have presented evidence that a secondary constriction at the intercellular bridge in HeLa cells is mediated by ESCRT-III-dependent filaments. A number of important aspects, such as the maximum extent of constriction and the molecular identity of the helical filaments, could not be addressed so far. Therefore, we propose here to systematically analyze the ultrastructure of the abscission machinery in HeLa cells at three progressive time points by serial-section electron tomography. In parallel, we also propose to localize ESCRT-III components in intercellular bridges of HeLa cells by immuno-electron microscopy. We also aim to continue with our time-resolved ultrastructural 3D analysis of cytokinesis in nematodes, as we recently observed filaments during abscission in 4-cell C. elegans embryos. For cytokinesis in nematodes, we propose to visualize intercellular bridges in 3D in both wild-type and mutant (tsg-101 RNAi) 4-cell embryos to clarify the role of the ESCRT machinery in filament assembly also in this system. Ultimate goal of such a systematic tomographic analysis of two different systems is to provide a 3D framework of the abscission machinery. We expect to obtain significantly new ultrastructural insights, enabling us to further develop our mechanistic model of cytokinetic abscission.