A conserved γ-tubulin complex–binding domain in CDK5RAP2 stimulates the microtubule-nucleating activity of γ-TuRC.
Microtubule nucleation and organization by the centrosome require ␥-tubulin, a protein that exists in a macromolecular complex called the ␥-tubulin ring complex (␥TuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including ␥-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the ␥TuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for ␥TuRC attachment to the centrosome but not for ␥TuRC assembly. Perturbing CDK5RAP2 function delocalized ␥-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in ␥TuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly. INTRODUCTIONIn animal cells, the centrosome is the primary microtubule (MT) organizing center (MTOC), which plays a key role in the control of the temporal and spatial distribution of MT networks (Ou and Rattner, 2004;Doxsey et al., 2005;Luders and Stearns, 2007). Typically, centrosomes are positioned at the focus of a radial array of MTs during interphase and are incorporated into spindle poles during mitosis. Interphase centrosomes are composed of a pair of centrioles embedded in a cloud of electron-dense pericentriolar material (PCM). The centriole has a well-defined structure with MT triplets arranged into a cylinder, whereas the organization of the PCM is less apparent. Early studies have shown the existence of a salt (2 M KI)-insoluble scaffold/matrix underlying the PCM (Moritz et al., 1998;Schnackenberg et al., 1998). This matrix, formed with a high content of large coiled-coil proteins, provides binding sites for the tubulin family member ␥-tubulin and other proteins associated with centrosomal functions, such as MT nucleation. Through the cell cycle, the PCM varies in volume and the MT-nucleating activity, which are smallest in G1-phase and biggest during mitosis. In addition, proteins have a precise and cell cycle-specific placement within the PCM that has been observed as a tubular structure located along the surface of the centriole and around its proximal end but not around its distal end (Ou and Rattner, 2000;Ou et al., 2003). The outer surface of the PCM is dynamic and PCM proteins found in the cytoplasm transit to the PCM either by diffusion or via MTs.Distributed throughout the PCM, ␥-tubulin ...
The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes.
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