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

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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.