Center on CLASPs: localisation and functions at the centrosome

Abstract

Microtubules are a crucial component of the cytoskeleton and play essential functions in intracellular transport, mitosis, cell motility and cell shape. Microtubule stability and dynamics are heavily regulated by microtubule-associated proteins, one of which are the cytoplasmic-associated linker proteins (CLASPs). CLASPs are plus-end tracking proteins and promote microtubule stability through preventing catastrophes, promoting rescues and mediating lattice repair. CLASPs are crucial for proper spindle formation and function in microtubule nucleation from the Golgi. CLASP was recently identified as a major regulator of microtubule density in interphase RPE1 cells, as severe CLASP depletion causes a strong reduction in microtubule density. In CLASP-depleted cells, microtubule nucleation from the Golgi was abolished and cytoplasmic microtubule nucleation was reduced. Interestingly, centrosomal microtubule nucleation was also significantly diminished, but the functions of CLASP at the centrosome are currently poorly understood. Here, we show using expansion microscopy that CLASP has distinct centrosomal subpopulations, localizing to the subdistal appendages, the centriole linker and procentrioles. In absence of CLASP, procentrioles are lost and the majority of the cells lose at least one mother centriole. Depletion of CLASP causes loss of key centrosomal proteins, such as γ-tubulin, Ninein and CPAP. Moreover, through pull down assays we detect a direct interaction between CLASP and CPAP. To study when in the cell cycle CLASP regulates centrosomal organization, we set out to synchronize cells throughout the depletion protocol in combination with EdU incorporation and detection. After releasing cells from a thymidine-induced arrest we observed a decrease in S-phase cells upon CLASP depletion, suggesting that a loss of CLASP induces a G1-phase arrest. These results indicate that CLASP is a key regulator of the centrosome. The distinct subpopulations imply an interesting array of functions of CLASP at the centrosome, for instance in centriole duplication, centriole maintenance, microtubule nucleation and microtubule anchorage.