The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis, Although the mechanisms controlling centrosome cohesi on and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Na p1, a novel centrosomal protein, is part of a structure linking parental ce ntrioles in a cell cycle-regulated manner. To test this model, we have perf ormed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome spli tting, regardless of the cell cycle phase. Splitting occurs between parenta l centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overe xpression of truncated C-Nap1 mutants, but not full-length protein. Antibod ies raised against different domains of C-Nap1 prove that this protein diss ociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immuno-electron microscopy shows that C-Nap1 is confined to the proximal end domains of ce ntrioles, indicating that a putative linker structure must contain addition al proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.