The signal, however, reappeared quickly after bleaching, with 50% recovery occurring within 1 min and reaching 60% of the original fluorescence intensity with prolonged incubation (Fig. centrosomes require the presence of -tubulin, a highly conserved protein that exists in a macromolecular structure called the -tubulin ring complex (TuRC) [1]C[3]. It is therefore of significant interest to identify the molecules that link the TuRC to centrosomes. CDK5RAP2 is a centrosomal protein whose mutations lead to autosomal recessive primary microcephaly, a disorder caused by defective proliferation and cell-fate determination of neural progenitors during neurogenesis [4]C[6]. We previously showed that CDK5RAP2 associates with the TuRC and helps attach it to centrosomes [7], [8]. The TuRC-binding domain in CDK5RAP2 was delineated as a short sequence stretch that is highly conserved in -tubulin complex-targeting proteins of lower organisms, including centrosomin and fission CK-1827452 (Omecamtiv mecarbil) yeast Mto1p and Pcp1p [7]. Furthermore, CDK5RAP2’s TuRC-binding domain stimulates the microtubule-nucleating activity of the TuRC [8], and a disruption of CDK5RAP2 function results in the disorganization of interphase microtubules and the formation of anastral mitotic spindles [7]. Thus, CDK5RAP2 plays an essential role in the organization of microtubules by centrosomes. The centrosome’s pericentriolar material (PCM) dynamically exchanges its molecular contents with the cytoplasm, and several PCM components, such as the matrix proteins pericentrin and PCM-1, are recruited to centrosomes via microtubule-dependent mechanisms [9]C[11]. These mechanisms require the minus end-directed microtubule motor protein dynein in association with dynactin, a large molecular complex necessary for dynein functions [12],[13]. Cytoplasmic dynein consists of dynein heavy chains (DHCs), dynein intermediate chains (DICs), dynein light intermediate chains (DLICs) and dynein light chains (DLCs). Whereas the DICs interact with most of the other dynein components and with the dynactin complex, the DLICs and DLCs are thought to function in loading cargo onto dynein [14]C[17]. CDK5RAP2, like -tubulin and many other centrosomal proteins, is present in a large cytoplasmic pool, but how it is targeted to centrosomes has remained unclear. Here we report that CDK5RAP2 is CK-1827452 (Omecamtiv mecarbil) dynamically recruited to centrosomes in a microtubule-dependent manner and that CDK5RAP2 associates with dynein and depends on the dynein-dynactin complex for its localization at centrosomes. Results Dynamic attachment of CDK5RAP2 to centrosomes requires microtubules To examine the dynamics of centrosomal CDK5RAP2 by the fluorescence recovery after photobleaching (FRAP), we constructed a stable cell line expressing GFP-CDK5RAP2 at a level similar to that of the endogenous protein (Fig. 1A). GFP-CDK5RAP2 was enriched at centrosomes in these cells (Fig. 1B) and its signal at these sites was then eliminated by photobleaching. The signal, however, reappeared quickly after bleaching, with 50% recovery occurring within 1 min and reaching 60% of the original fluorescence intensity with prolonged incubation (Fig. 1C). This is in accord with previous observations of CDK5RAP2’s dynamic localization at centrosomes [18], [19]. Strikingly, when FRAP was carried out on these cells after treating them with nocodazole CK-1827452 (Omecamtiv mecarbil) to depolymerize microtubules, the GFP-CDK5RAP2 signal failed to recover at centrosomes during the recording period (Fig. 1B, noco-treated). These results demonstrate that intact microtubules are needed for the dynamic recruitment of CDK5RAP2 to centrosomes. Open in a separate window Figure 1 The dynamic recruitment of CDK5RAP2 to centrosomes MGC102953 depends on microtubules and dynein.(A) Immunoblotting of extracts prepared from MDA-MB-231 cells stably expressing GFP-CDK5RAP2. Cells were lysed and extracts were stained with an anti-CDK5RAP2 antibody to detect GFP-CDK5RAP2 and endogenous CDK5RAP2 in either the stable cell line or the parent cell line. Anti–tubulin was used as internal control. (B) GFP-CDK5RAP2 is localized on centrosomes. Cells transfected with GFP-CDK5RAP2 were stained with anti–tubulin to label centrosomes. (C) Centrosomal recruitment of CDK5RAP2. FRAP experiments were performed on cells expressing GFP-CDK5RAP2 that were treated with or without.