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Biochem Biophys Res Commun
2009 Jul 03;3843:383-8. doi: 10.1016/j.bbrc.2009.04.154.
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A fraction of Crm1 locates at centrosomes by its CRIME domain and regulates the centrosomal localization of pericentrin.
Liu Q
,
Jiang Q
,
Zhang C
.
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Crm1 plays a role in exporting proteins containing nuclear export signals (NESs) from the nucleus to the cytoplasm. Some proteins that are capable of interacting with Ran/Crm1 were reported to be localized at centrosomes and to function as centrosome checkpoints. But it remains unclear how Crm1 locates at centrosomes. In this study, we found that a fraction of Crm1 is located at centrosomes through its N-terminal CRM1, importin beta etc. (CRIME) domain, which is responsible for interacting with RanGTP, suggesting that Crm1 might target to centrosomes through binding centrosomal RanGTP. Moreover, overexpression of the CRIME domain, which is free of NES binding domain, resulted in the dissociation of pericentrin and gamma-tubulin complex from centrosomes and the disruption of microtubule nucleation. Deficiency of Crm1 provoked by RNAi also decreased the spindle poles localization of pericentrin and gamma-tubulin complex, coupled with mitotic defects. Since pericentrin was sensitive to Crm1 specific inhibitor leptomycin B, we propose that the centrosomal Crm1 might interact with pericentrin and regulate the localization and function of pericentrin at centrosomes.
Fig. 1. A fraction of Crm1 locates at centrosomes. (A) Western blot analysis of the endogenous Crm1 in Xenopus egg extracts, XTC and HeLa cells using rabbit anti-Crm1 polyclonal antibody. (B) Crm1 is one of components in isolated centrosomes from XTC cells by discontinuous sucrose gradient fractionation. Fractions were collected from bottom to top of the centrifugation tube and analyzed by western blotting with anti-Crm1 and anti-γ-tubulin antibodies. (C) and (D) Subcellular localization of Crm1 during the cell cycle in XTC and HeLa cells. XTC or HeLa cells were fixed by methanol and double immunostained by anti-Crm1 (green) and anti-γ-tubulin (red) antibodies. DNA was stained with DAPI (blue). Bar, 10 μm. (E) Subcellular localization of Crm1 in nocodazole-treated HeLa cells. Hela cells were treated with 1 μM nocadazole for 2 h, and fixed for immunostaining with Crm1 (green) and α-tubulin (red). Bar, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The N-terminal region 1–112aa of Crm1 is essential for its localization to centrosomes. (A) Schematic diagrams of Crm1 truncated mutants. −, +/− and + represent 0%, less than 50%, and more than 80% of cells showing centrosomal localization, respectively. At least 200 cells were evaluated for each kind of transfected cells in every experiment. Data are averages of three independent experiments. (B) Subcellular localization of GFP-tagged truncated Crm1 fragments in XTC cells. XTC cells were transiently transfected with GFP-Crm1 (1–112), GFP-Crm1 (1–417), GFP-Crm1 (417–601), GFP-Crm1 (601–1071), GFP-Crm1 (1–112), GFP-Crm1 (112–417), GFP-Crm1 (1–310), GFP-Crm1 (310–417) and GFP-Crm1 (112–310), respectively. Cells were immunostained with anti-γ-tubulin antibody (red). The square areas in the merge images are zoomed on the right column. Bar, 10 μm. (C) Western blot analysis of truncated GFP-Crm1 mutants expressed in XTC cells by anti-GFP antibody. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Overexpression of GFP-Crm1 (1–112) reduces centrosomal pericentrin and γ-tubulin and inhibits microtubule re-growth from centrosomes. (A and B) NIH3T3 and XTC cells were transfected with GFP-Crm1 (1–112) (green), fixed and immunostained with anti-pericentrin antibody (red) (A) or anti-γ-tubulin antibody (red) (B). The arrows indicate the centrosomes in transfected cells. The white square areas indicate the centrosomal pericentrin in transfected cells and are zoomed in the top left corner. The yellow square areas indicate the centrosomal pericentrin in non-transfected cells and are zoomed in the top right corner. Bar, 20 μm. (C) High levels of GFP-Crm1 (1–112) overexpression inhibit microtubule re-growth from centrosomes. XTC cells were transfected with GFP-Crm1 (1–112) and then treated with 1 μM nocodazole for further 2 h to depolymerize microtubules. Put back in fresh medium for 5 min, cells were fixed and immunostained with anti-α-tubulin antibody to monitor the microtubules re-growth. Top, cells were treated with nocodazole for 2 h and cultured in fresh medium for 0 min. Middle, treated cells with low levels of GFP-Crm1 (1–112) were cultured in fresh medium for 5 min. Bottom, cells with high levels of GFP-Crm1 (1–112) were cultured in fresh medium for 5 min. Arrows show the centrosomes in transfected cells and arrowheads show the centrosomes in non-transfected cells. Bar, 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Crm1 deficiency caused by RNAi induces pericentrin and γ-tubulin displacement from spindle poles and mitotic defects. (A) HeLa cells transiently transfected with control or pSUPER-Crm1 (Crm1 RNAi). After 72 h, cells were analyzed by Western blot to determine the level of Crm1. The same blot was probed with anti-α-tubulin antibody as a loading control. (B) mRFP-H2B and pS-Crm1 co-transfected (1:20) cells were immunostained with anti-Crm1 antibody (green). mRFP-H2B is a positive marker for Crm1-RNAi cells. Bar, 20 μm. (C–G) Control cells were co-transfected with mRFP-H2B and control pSUPER vector, while Crm1 RNAi-cells were co-transfected with mRFP-H2B and pS-Crm1. After 72 h cells were fixed by methanol and stained with anti-Crm1 (C), anti-pericentrin (D), anti-γ-tubulin (E) and anti-Ran (F) antibodies (green), respectively. Bar, 10 μm. (G) The ratio of the amount of pericentrin, γ-tubulin and Ran at spindle poles in Crm1-RNAi cells to that in control cells. The data is shown as the mean percentage plus the standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
