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

离断的三维重建：ESCRT依赖性丝的作用

• 批准号: 408902973
• 负责人: Professor Dr. Thomas Müller-Reichert
• 金额: --
• 依托单位: Experimentelles Zentrum
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/408902973?language=en
• 关键词: Three; dimensional; reconstruction; abscission; Role

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.

脱落是胞质分裂的最后一步，也是所有动物中确保有丝分裂后细胞对成功物理分离的重要过程。以前，我们已经提出的证据表明，在HeLa细胞的细胞间桥的二次收缩介导的ESCRT-III依赖的丝。一些重要的方面，如最大程度的收缩和螺旋丝的分子身份，迄今无法解决。因此，我们建议在这里系统地分析超微结构的HeLa细胞中的分裂机制在三个渐进的时间点，连续切片电子断层扫描。同时，我们还建议通过免疫电子显微镜将ESCRT-III组分定位在HeLa细胞的细胞间桥中。我们还打算继续我们的时间分辨超微结构的三维分析线虫胞质分裂，因为我们最近观察到丝在4细胞C。线虫胚胎对于线虫的胞质分裂，我们建议在野生型和突变体（tsg-101 RNAi）4-细胞胚胎中的3D中可视化细胞间桥，以澄清ESCRT机器在细丝组装中的作用，也在这个系统中。对两个不同系统进行这种系统的层析成像分析的最终目标是提供一个3D的发射机框架。我们期望获得显著新的超微结构的见解，使我们能够进一步发展我们的细胞动力学分裂的机制模型。