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

脱落是细胞质分裂的最后一步，也是所有动物确保有丝分裂后细胞对成功物理分离的基本过程。此前，我们已经提出证据表明，HeLa细胞细胞间桥的次级收缩是由ESCRT-III依赖的细丝介导的。一些重要的方面，如最大程度的收缩和螺旋细丝的分子同一性，到目前为止还不能解决。因此，我们建议用连续切片电子断层扫描系统地分析HeLa细胞在三个连续时间点的脱落机制的超微结构。同时，我们还建议通过免疫电子显微镜定位ESCRT-III组分在HeLa细胞的细胞间桥中。我们还打算继续我们对线虫胞质分裂的时间分辨超微结构3D分析，因为我们最近在4细胞线虫胚胎中观察到了脱落过程中的细丝。对于线虫的细胞质分裂，我们建议在野生型和突变型(TSG-101 RNAi)4细胞胚胎中三维可视化细胞间桥，以阐明ESCRT机制在丝状体组装中的作用。对两个不同系统进行这种系统层析分析的最终目标是提供剥离机构的3D框架。我们希望获得显著的新的超微结构的见解，使我们能够进一步发展我们的细胞分裂的机制模型。

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.