CLASPING AT INSIGHTS: Understanding the Role of CLASP at the Centrosome

Abstract

Microtubules are hollow cytoskeleton filaments critical for many cellular functions such as intracellular transport, cilia formation and cell division. Microtubules have two distinct polarised ends and often undergo rapid phases of shrinkage and growth at the fast-growing plus-ends. This dynamic instability is regulated by specialised microtubule associated proteins called plus-end tracking proteins (+TIPs). Cytoplasmic linker-associated proteins (CLASPs) are a class of +TIPs that suppress catastrophes and promote rescues by stabilising microtubule ends, as well as regulating repair in the microtubule lattice. CLASPs plays a key role in microtubule organisation and nucleation at the Golgi and are essential for spindle formation at the kinetochores in mitosis. Interestingly, it was recently found that CLASP depletion results in reduced centrosomal microtubule density and abnormal centriole formation. This suggests that CLASP plays an essential role at the centrosome, although its exact functions are still unknown. Here, using streptavidin-based pull-down assays, we show the CLASP C-terminal CLIP-ID domain is responsible for interactions with centriole elongation factor CPAP and centrosomal microtubule anchoring protein ninein. Reciprocally, the microtubule-binding domain of CPAP and coiled coil 2 domain of ninein are associated with CLASP. Furthermore, using immunofluorescence and ultrastructure expansion microscopy, we show that the CLIP-ID is sufficient and required for CLASP localisation to the subdistal appendages and centriolar linker of centrioles, but not to procentrioles. A ninein depletion results in decreased localisation of CLASP to the centrosome, although we also observed different levels of CLASP1 and CLASP2 presence at the centrioles in wildtype cells. This data suggests CLASP plays a variety of roles at the centrosome, including in microtubule nucleation and anchorage and centriole duplication and elongation.