Molecular characterization of the centrosome/nucleus-connector

中心体与细胞核的连接对适当的有丝分裂、核定位和细胞定向迁移至关重要。它是由一个蛋白质复合体介导的，该复合体包括无处不在的SUN家族的核膜蛋白和这些蛋白的几个未知的直接和间接结合伙伴，它们共同组成中心体-核连接器(CnC)。在这里，我们将使用Dictyostelialamobae来分离CnC蛋白复合体，并将鉴定和功能表征与中心体/核连接有关的新蛋白。这些细胞非常适合分析cnc突变，因为它们cnc的破坏不是致命的。此外，它们的中心体特征很好，SUN1是一个被批准的标记。我们的目标将通过对分离的NC组分进行质谱分析、提高用于功能分析的CNC特异性单抗、产生敲除或基因沉默突变体、对NC蛋白质动力学的活细胞成像以及对新的NC组分的亚细胞定位直到超微结构水平来实现。数据库搜索将揭示发现的Dictyostelialcnc组件的哺乳动物同源基因。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。除了对中枢Me/核附着的基本过程有更深入的了解外，我们的工作还将有助于解释发育神经元疾病的发病机制，如无脑或DYT1肌张力障碍。

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.