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Abstract

The centrosome is the main microtubule organizing center (MTOC) of animal cells. It consists of mother and daughter centrioles that are surrounded by the so called pericentriolar matrix (PCM), a cloud of proteins. During interphase, cells contain only one main MTOC consisting of two centrosomes kept together via centrosome cohesion. The current understanding of centrosome cohesion involves two distinct pathways: the centrosome linker and microtubule (MT)-based cohesion. The best described and most studied centrosome linker proteins are C-Nap1 and Rootletin and its interactor CEP68. C-Nap1 forms a ring-like structure at the proximal end of both centrioles and organizes the Rootletin/CEP68 filaments. In late G2/prophase the Never in mitosis A-related kinase 2 (NEK2) efficiently phosphorylates C-Nap1 and Rootletin to dissolve the centrosome linker, which then leads to centrosome separation in G2/prophase. Afterwards the two centrosomes are move apart to form the opposite poles of the mitotic spindle. Over the time, many different proteins have been proposed as potential centrosome linker components, but the exact function and the structure of the centrosome linker is still unclear. During this study, I identified Ninein as a centrosome linker component downstream of C-Nap1 that links the two centrosomes together in RPE1 cells. In contrast, in HCT116, U2OS and Caco-2 cells Rootletin and Ninein establish centrosome cohesion together. Overamplified centrosomes, which occur either naturally in Caco-2 cancer cells or after PLK4-induced centrosome amplification use the linker for centrosome clustering in interphase. Under these conditions, Rootletin showed centrosome linker function in RPE1 cells. Surprisingly, despite linker disassembly in G2/prophase, the absence of C-Nap1 extends the duration of metaphase in cells with centrosome amplification. This is evidenced through the continued activation of the spindle assembly checkpoint (SAC), as suggested by the signal accumulation of MAD1 and BUB1 at kinetochores. The reduced MT nucleation activity of centrosomes lacking C-Nap1 and the prolonged nuclear envelope breakdown in linker-deficient prophase cells are likely to be a reason for the observed mitotic defects. Consequently, C-Nap1 deficient cells with centrosome amplification show an increase in multipolar spindle formation and chromosome mis-segregation. These defects become more pronounced with partial inhibition of the kinesin HSET, a protein that typically clusters supernumerary centrosomes during mitosis. In conclusion, this study sheds light on the cell type-dependent differences of the centrosome linker and the function in clustering overamplified centrosomes during interphase. Moreover, the results in C-Nap1 deficient cells with multiple centrosomes indicate an unknown impact of the centrosome linker on pseudo-bipolar spindle formation and chromosome segregation in mitosis.