Molecular characterization of the centrosome/nucleus-connector

中心体与细胞核的连接对于正确的有丝分裂、细胞核定位和定向细胞迁移至关重要。它是由一种蛋白质复合物介导的，该蛋白质复合物涉及普遍存在的SUN家族的核膜蛋白和这些蛋白质的几种未知的直接和间接结合伴侣，它们共同构成了中心体-核连接器（CNC）。在这里，我们将使用Dictyosteomaamoeastrum分离CNC蛋白复合物，并将确定和功能特性的新蛋白参与中心体/核附着。这些细胞非常适合分析CNC突变，因为它们的CNC的破坏不是致命的。此外，它们的中心体被很好地表征，并且SUN 1是其CNC的批准标记。我们的目标将通过分离的CNC组件的质谱分析，提高CNC特异性单克隆抗体的功能分析，产生敲除或基因沉默突变体，CNC蛋白质动力学的活细胞成像和新CNC组件的亚细胞定位到超微结构水平来实现。数据库搜索将揭示所发现的Dictyosteopathy CNC组件的哺乳动物直系同源物。它们在中心体核附着中的潜在作用将通过在哺乳动物细胞中的定位和RNAi研究来研究。除了对中心体/核附着的基本过程有更好的理解外，我们的工作将有助于解释发育性神经元疾病（如无脑畸形或DYT 1肌张力障碍）的发病机制。

The attachment of the centrosome to the nucleus is crucial for proper mitosis, nuclear positioning and directional cell migration. It is mediated by a protein complex involving nuclear envelope proteins of the ubiquitous SUNfamily and several unknown direct and indirect binding partners of these proteins, which together make up the centrosome-nucleus connector (CNC). Here we will use Dictyostelium amoebae to isolate CNC protein complexes and will identify and functionally characterize novel proteins involved in centrosome/nucleus attachment. These cells are well suited for analyses of CNC mutations, since disruption of their CNC is not lethal. Furthermore, their centrosome is well characterized and SUN1 is an approved marker for their CNC. Our goals will be achieved through mass spectrometrical analysis of isolated CNC components, raising of CNCspecific monoclonal antibodies for functional analyses, generation of knockout or gene silencing mutants, live cell imaging of CNC protein dynamics and subcellular localization of novel CNC components up to the ultrastructural level. Database searches will reveal mammalian orthologues of the discovered Dictyostelium CNC components. Their potential role in centrosome nucleus attachment will be investigated by localization and RNAi studies in mammalian cells. In addition to an improved understanding of the fundamental process of centroso me/nucleus attachment, our work will help to explain the pathogenesis of developmental neuronal diseases such as lissencephaly or DYT1 dystonia.