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  • Immunofluorescence Cells were fixed with paraformaldehyde PF

    2019-07-11

    Immunofluorescence. Cells were fixed with 4% paraformaldehyde (PFA) in PBS and extracted by 0.5% Triton X-100-PBS, or fixed in cold methanol. The cells were immunostained with mouse monoclonal Adox weight against α-tubulin (Sigma), γ-tubulin (Sigma), Ran (Upstate), rabbit polyclonal antibodies against pericentrin (Abcam) and Crm1 (raised by injecting rabbits). DNA was stained with DAPI, and cells were mounted between slide and coverslip with mowiol (Sigma). Western blotting. Cell lysates or Xenopus egg extracts were immunoblotted with rabbit polyclonal antibody against Crm1 (1:2000), mouse monoclonal antibody against γ-tubulin (1:5000), rabbit polyclonal antibody against GFP (PTGLAB) (1:1000) and mouse monoclonal antibody against α-tubulin (1:5000). Centrosome isolation. Centrosomes were isolated from XTC cells according to a protocol by Mitchison and Kirschner [15]. Centrosome fractions were analyzed by Western blot with antibody against γ-tubulin. Microtubule re-growth assay. XTC cells were treated with 1μM nocodazole for 2h at 23°C to depolymerize microtubules. The cells were washed with PBS and allowed to recover in fresh media for 5min at 23°C and then processed for immunofluorescence.
    Results
    Discussion The main function of Crm1 is to export NES-containing proteins from the nucleus to the cytoplasm [4]. The newly discovered roles of CRM1 in centrosomal duplication indicate that Crm1 does not only regulate nucleocytoplasmic transporting of proteins [7]. In this study, we found that Crm1 stably located at centrosomes during the whole cell cycle. The truncated mutants overexpression results showed that the N-terminal region of Crm1 is responsible for its location at centrosomes. It has been noticed that the N-terminal region of CRM1 (i.e. CRIME domain) shares significant homology with the N-terminus of many other importin-β family proteins and is required for the binding to RanGTP in the formation of export complex [16], [19], and that the central region (i.e. CCR domain) of Crm1 is highly conserved and required for its binding to NES cargoes [16]. It also has been reported that centrosomes contain a fraction of Ran GTPase stably associated with the anchoring protein AKAP450 [14]. In this study, we observed that Crm1 knockdown by RNAi had no effect on Ran localization at centrosomes. As the centrosome-binding region of Crm1 tested in this study is exactly the CRIME domain, we propose that RanGTP might play a role in targeting Crm1 to centrosomes through interacting with its CRIME domain and leaving its rest region Adox weight to timely attract NES substrates to centrosomes. Overexpression of Crm1 (1–112) reduces the amount of pericentrin and γ-tubulin at centrosomes, suggesting that Crm1 might regulate the localization of pericentrin and γ-tubulin at centrosomes. Pericentrin is a conserved centrosomal component, which provides the structural scaffold to anchor numerous centrosomal proteins, including γ-tubulin [11], [12], [13]. Moreover, it was reported that pericentrin is sensitive to LMB, the specific inhibitor of Crm1 [14], indicating that pericentrin might be an NES-containing protein. We also found that pericentrin could accumulate into nucleus when treated with LMB, while γ-tubulin did not. We propose that Crm1 (1–112), which contains the RanGTP binding domain but lack of NES binding domain, might compete with endogenous Crm1 to enrich at centrosomes, but cannot target NES proteins, including pericentrin, to the centrosomes. In fact, we found that other truncated mutants that have CRIME but no CCR domain also inhibited the localization of pericentrin and γ-tubulin at centrosomes (data not shown). As pericentrin and γ-tubulin form a complex at centrosomes in vertebrate cells [18], we suggest that the reduction of the amount of γ-tubulin at centrosomes is due to the mislocalization of pericentrin. It has been reported that pericentrin/γ-tubulin complexes are necessary for microtubule organization and nucleation [18]. In microtubule re-growth experiments, high level overexpression of the mutant Crm1 (1–112) deduced the level of pericentrin/γ-tubulin at centrosomes and influenced the ability of the cells to nucleate microtubules from their centrosomes, suggesting that Crm1 might regulate the function of centrosomes by interacting with pericentrin.