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

中心体是人类细胞的基本细胞器，其组织微管并具有基本功能，例如作为有丝分裂染色体分离中的纺锤体极的一部分。因此，中心体的功能障碍可导致包括小头畸形和癌症在内的疾病。中心体畸变，即额外中心体，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子和微管通路连接在一起，形成一个微管组织单位，该微管组织单位在G2/前期被激酶NEK 2分解。中心体接头由几种蛋白质组成，包括C-Nap 1、rootweight和CEP 68。最近，我们发现，保守的中心体蛋白ninein是一个进一步的中心体连接器组件的功能下游的C-Nap 1，但平行的rootaoke/CEP 68。然而，ninein的环状组织表明，它最有可能与另外的未知接头组分相互作用以实现接头功能。令人惊讶的是，缺乏中心体接头只会导致培养细胞中相对轻微的缺陷，这表明冗余机制补偿了接头损失。此外，初步的数据表明，中心体接头成为重要的染色体分离有丝分裂时，纺锤体的组装是更苛刻的，例如存在的额外的中心体。这种表型在与驱动蛋白马达蛋白HSET的抑制相结合时尤其明显，驱动蛋白马达蛋白HSET在有丝分裂中将额外的中心体拉到一起成为两个纺锤体极。这些观察结果使我们提出，在G2和有丝分裂的空间中心体组织的机制合作，并共同确保适当的纺锤体形成和染色体分离。在这个建议中，我们将首先检查ninein相互作用的蛋白质，我们已经确定了一个下拉/质谱方法的中心体连接器功能。与中心粒近端结合的augmin复合物是一个很有希望的候选者。其次，我们将分析合作的机制，确保空间组织的中心体，即，中心体连接器，微管通路，中心体核膜拴系和HSET电机的合作，在细胞中的染色体分离与正常和多余的中心体集。这将通过基因敲除突变体和化学抑制HSET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，我们将测试如何延长中心体凝聚力（NEK 2敲除）影响染色体分离时，特殊的中心体组织的其他原则失败。总之，这一提议将从一个新的角度阐明有丝分裂纺锤体的形成，此外还具有确定如何处理具有额外中心体的癌细胞的方法的潜力。

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.