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

中心体是人类细胞的重要细胞器，可组织微管并具有重要功能，例如作为有丝分裂染色体分离中纺锤体极的一部分。因此，中心体的功能障碍会导致包括小头畸形和癌症在内的疾病。中心体畸变，即多余中心体，在癌细胞中特别重要。每个细胞周期中心体复制一次。在细胞周期的间期，细胞的两个中心体通过中心体接头和微管途径连接在一起，形成一个微管组织单元，该微管组织单元在 G2/前期被激酶 NEK2 分解。中心体连接子由多种蛋白质组成，包括 C-Nap1、rootletin 和 CEP68。最近，我们发现保守的中心体蛋白 ninein 是另一个中心体连接元件，其功能在 C-Nap1 下游，但与 rootletin/CEP68 平行。然而，ninein 的环状组织表明它最有可能与附加的、未知的连接器组件相互作用以实现连接器功能。令人惊讶的是，中心体连接体的缺乏仅导致培养细胞中相对轻微的缺陷，这表明冗余机制补偿了连接体的损失。此外，初步数据表明，当纺锤体组装要求更高（例如由于存在多余中心体）时，中心体接头对于有丝分裂中的染色体分离变得重要。这种表型在与驱动蛋白运动蛋白 HSET 的抑制相结合时尤其明显，该蛋白在有丝分裂中将多余的中心体拉在一起形成两个纺锤体极。这些观察结果使我们提出，G2 和有丝分裂中的空间中心体组织机制相互配合，共同确保适当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下拉/质谱方法鉴定的九蛋白相互作用蛋白，以了解中心体连接子功能。与中心粒近端相关的augmin复合体是一种有希望的候选者。其次，我们将分析确保中心体空间组织的机制的合作，即中心体连接体、微管途径、中心体-核膜束缚和 HSET 马达，以在具有正常和多余中心体组的细胞中进行染色体分离。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，我们将测试当特殊中心体组织的其他原理失败时，延长的中心体凝聚力（NEK2 敲除）如何影响染色体分离。总而言之，该提议将从一个新的角度阐明有丝分裂纺锤体的形成，此外还有可能确定如何用多余中心体处理癌细胞的方法。

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.