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

• Applicant: Professor Dr. Elmar Schiebel
• Subject Area: Cell Biology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 298572189

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.

• DFG Programme: Research Grants