Determination of protein associations within the human inner kinetochore in living cells using Acceptor Bleaching FRET and FLIM

哺乳动物细胞的DMA分离是由特定的亚染色体蛋白质复合体--着丝粒介导的一个重要的细胞过程。这个复合体的故障会导致非整倍体，并可能导致癌症。我们计划在活的人类细胞中，在时间和空间上阐明人类内部动粒蛋白的相互作用和可能的功能。体内荧光显微镜技术分析自然环境中的蛋白质，从而进行正确的蛋白质修饰。在我们申请的项目中，我们将通过FRET和FLIM来确定内部动粒蛋白的结合。关于分子结构和内部动粒复合体结构的信息很少，尽管结构现象似乎对动粒功能起着重要作用。该项目中进行的分析将确定内部动粒复合体的结构，并将有助于确定蛋白质功能(包括“核小体相关复合体”的蛋白质)。我们开发了一个内部动粒的工作模型，并在本项目中进行了测试和扩展。

DMA segregation of mammalian cells is an essential cellular process mediated by a specific sub-chromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. We plan to elucidate, in time and space in the living human cell, the interactions and possible functions of human inner kinetochore proteins. The in vivo fluorescence microscopy techniques analyse the proteins in their natural setting and thus granting correct protein modification. In the project we apply for, we will determine the association of the inner kinetochore proteins by FRET and FLIM. Little information is available on molecular structures and inner kinetochore complex architecture although structural phenomena seem to play an important role for kinetochore function. The analyses carried out in this project will determine the inner kinetochore complex architecture and will contribute to identify protein functions (including the proteins of the "nucleosome associated complex"). We developed a working model of the inner kinetochore which we test and extend in this project.