Spatial organisation of centrosomes by centrosome cohesion and centrosome nuclear envelope interactions.

通过中心体内聚力和中心体核膜相互作用进行中心体的空间组织。

• 批准号: 298572189
• 负责人: Professor Dr. Elmar Schiebel
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/298572189?language=en
• 关键词: Spatial; organisation; centrosomes; centrosome; cohesion

Centrosomes are essential organelles of human cells that organize microtubules and have essential functions for example as part of the spindle poles in mitotic chromosome segregation. Accordingly, malfunction of centrosomes can cause diseases including microcephaly and cancer. Centrosome aberrations, namely supernumerary centrosomes, are particularly important in cancer cells. Centrosomes duplicate once per cell cycle. During interphase of the cell cycle the two centrosomes of a cell are linked together by the centrosome linker and a microtubule pathway into one microtubule organizing unit that becomes resolved in G2/prophase by the kinase NEK2. The centrosome linker consists of several proteins including C-Nap1, rootletin and CEP68. Recently, we discovered that the conserved centrosomal protein ninein is a further centrosome linker component that functions downstream of C-Nap1 but parallel of rootletin/CEP68. However, the ring-like organisation of ninein indicates that it most likely interacts with addition, yet unknown linker components to achieve linker function. Surprisingly, lack of the centrosome linker only causes relative mild defects in cultured cells suggesting that redundant mechanisms compensate for the linker loss. In addition, preliminary data indicate that the centrosome linker becomes important for chromosome segregation in mitosis when spindle assembly is more demanding for example by the presence of supernumerary centrosomes. This phenotype is especially pronounced in combination with inhibition of the kinesin motor protein HSET that in mitosis pulls supernumerary centrosomes together into two spindle poles. These observations lead us to propose that mechanisms of spatial centrosome organisation in G2 and mitosis cooperate and jointly ensure proper spindle formation and chromosome segregation. In this proposal, we will first check ninein interacting proteins that we have identified by a pull down/mass spectrometry approach for centrosome linker function. The augmin complex that also associates with the proximal end of centrioles is one promising candidate. Second, we will analyse cooperation of mechanisms that ensure spatial organisation of centrosomes, namely, the centrosome linker, the microtubule pathway, centrosome-nuclear envelope tethering and the HSET motor for cooperation in chromosome segregation in cells with a normal and supernumerary centrosome set. This will be done with gene knockout mutants and chemical inhibition of HSET in combination with live cell imaging of mitotic spindle assembly and chromosome segregation. Third, we will test how prolonged centrosome cohesion (NEK2 knockout) affects chromosome segregation when other principals of special centrosome organisation fail. Taken together, this proposal will illuminate mitotic spindle formation from a new angle and in addition has the potential of identifying ways how to tackle cancer cells with supernumerary centrosomes.

中心体是人类细胞中组织微管的重要细胞器，具有重要的功能，例如在有丝分裂染色体分离中作为纺锤杆的一部分。因此，中心体的功能障碍可导致小头畸形和癌症等疾病。中心体畸变，即多余的中心体，在癌细胞中特别重要。中心体每个细胞周期复制一次。在细胞周期的间期，细胞的两个中心体通过中心体连接体和微管途径连接在一起，成为一个微管组织单元，在G2/前期由激酶NEK2分解。中心体连接体由几种蛋白质组成，包括C-Nap1、rootletin和CEP68。最近，我们发现保守的中心体蛋白九蛋白是另一个中心体连接元件，其功能位于C-Nap1的下游，但与根蛋白/CEP68平行。然而，ninin的环状组织表明，它最有可能与未知的附加连接元件相互作用，以实现连接功能。令人惊讶的是，在培养细胞中，中心体连接体的缺失只会导致相对轻微的缺陷，这表明冗余机制弥补了连接体的缺失。此外，初步数据表明，当纺锤体组装要求更高时，例如由于存在多余的中心体，中心体连接体对有丝分裂中的染色体分离变得重要。在有丝分裂中将多余的中心体拉入两个纺锤极的运动蛋白HSET抑制时，这种表型尤其明显。这些观察结果使我们提出G2和有丝分裂的空间中心体组织机制相互合作，共同确保正确的纺锤体形成和染色体分离。在本提案中，我们将首先检查我们通过拉下/质谱方法鉴定的中心体连接体功能的九种相互作用蛋白。与中心粒近端相关的augmin复合体是一个有希望的候选者。其次，我们将分析确保中心体空间组织的机制合作，即中心体连接体，微管途径，中心体-核膜系结和HSET马达在具有正常和多余中心体集的细胞中进行染色体分离的合作。这将通过基因敲除突变体和HSET的化学抑制，结合有丝分裂纺锤体组装和染色体分离的活细胞成像来完成。第三，我们将测试中心体内聚时间延长（NEK2敲除）如何影响染色体分离，当其他特殊中心体组织原理失效时。综上所述，这一建议将从一个新的角度阐明有丝分裂纺锤体的形成，此外还具有确定如何处理具有多余中心体的癌细胞的方法的潜力。