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First published online September 18, 2007
doi: 10.1242/10.1242/jcs.013102

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Centrosomal CAP350 protein stabilises microtubules associated with the Golgi complex

¹ Institut Curie, Section Recherche, UMR144-CNRS, 75248 Paris CEDEX, France
² School of Biological Sciences, University of East Anglia, Norwich, UK
³ Institut für Zellbiologie, Universitätsklinikum, Virchowstrasse, 45122 Essen, Germany

View larger version (40K): [in this window] [in a new window]   Fig. 1. CAP350 localisation during the cell cycle and after microtubule drug treatment. (A) Western blot analysis of Triton X-100-soluble (S) and insoluble (I) protein fractions from KE37 cells and a highly enriched centrosomal fraction (CTR). CAP350 is only detectable in the centrosomal fraction. (B) Western Blot analysis of total cell lysate obtained from HeLa cells after CAP350 SiRNA. The high molecular mass band, revealed by CAP350 antibody, is detectable in the control siRNA sample (GL2 lane), and absent after CAP350 RNAi (O1 and O2 lanes) indicating that our antibody is specific. (C-F) Double-labelling experiments on interphase cells using the CAP350 antibody and either -tubulin (C-D), -tubulin (E) or CTR433 a Golgi complex antibody (F). Note that a cloud of dots is evident around the centrosome with the CAP350 antibody (arrow). (D) 3x magnification of the boxed area in C, showing that the pericentrosomal dots are aligned with MTs. These dots are located in the Golgi complex area (F). (G) Metaphase cell double stained with anti-CAP350 and anti--tubulin antibodies. During metaphase, CAP350 still localised to the centrosome, but was also found along the spindle microtubules. (H) Cell in telophase, double stained with anti-CAP350 and anti--tubulin antibodies. At this stage, the CAP350 antibodies labelled the centrosome but also some dots around it, as well as the central spindle. (I) Nocodazole (NZ)-treated cells double labelled with CAP350 and -tubulin antibodies during interphase. The CAP350 antibody decorated the centrosome and the remaining microtubules. (J) Taxol-treated cells double labelled with CAP350 and -tubulin antibodies. CAP350 redistributed to the ends of the microtubule bundles. Bars, 20 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. CAP350 binds microtubules through an N-terminal domain and not through the CAP-Gly domain. (A) HeLa cell lysate in which tubulin was added or not (see Materials and Methods) and treated with either Nocodazole (NZ) to prevent tubulin polymerisation or with Taxol to stabilise the microtubules. After a 1-hour incubation the cell lysate was centrifuged through a glycerol cushion. Proteins from the supernatants (S) and pellets (P) were analysed by SDS-PAGE and western blotting using anti-tubulin, anti-p150 and anti-CAP350 antibodies. Note that a small amount of CAP350 and p150 specifically co-precipitated with microtubules in the absence of exogenous tubulin, which significantly increased after addition of tubulin in the lysate. (B) Different partial constructs of CAP350 as indicated on the figure were transcribed in reticulocyte lysate and analysed for their binding to pre-formed microtubules (+MTs). Control experiments were performed without pre-formed microtubules (–MTs). As in A, proteins from the supernatants (S) and pellets (P) were analysed by SDS-PAGE and exposed for different times using a phosphorimager. Only the N-terminal construct (1-983) sedimented with microtubules. This ability was observed with the two N-terminal constructs (C). Arrows indicate the mobility of the in vitro labelled CAP350 constructs.

View larger version (45K): [in this window] [in a new window]   Fig. 3. The N-terminal domain of CAP350 binds microtubules directly. (A,B) The fragment (403-725) produced in bacteria as a GST fusion protein and purified using the GST tag, as well as GST, were incubated for 1 hour with preformed MTs. The MTs were then centrifuged on a glycerol cushion and the proteins from the supernatants (S) and pellets (P) were analysed by SDS-PAGE. (A) Coomassie Blue staining. The arrow indicates either purified GST fusion protein or GST. Asterisks indicate tubulin. (B) Western blotting using anti-GST antibody. (C) Double immunofluorescence of the microtubule-binding domain (403-725) mutated in the two NLS in fusion with the 6xMyc tag using anti-myc (left and green in merged image) or -tubulin antibody (middle and red in merged image). As expected, it colocalised with the microtubule network. Bar, 20 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 4. CAP-Gly-containing domains are targeted to the Golgi-like network and CAP350 depletion induced Golgi complex scattering. Cells expressing either the Ser-rich CAP-Gly domain (984-2589) (A,B) or the C-terminus (1896-3117) (C) in fusion with a 6xMyc tag were fixed 24 hours after transfection with methanol after Triton X-100 extraction. Cells were subsequently stained with the anti-Myc antibody (green) and the Golgi antibody (red) CTR433, or A24 antibody which recognises the AKAP450 protein and stains the Golgi-like network. (A) The Ser-rich CAP-Gly domain is targeted to the centrosome when expressed at a low levels. (B) At higher expression levels, a Golgi-like network is decorated with the Myc antibody. (C) The C-terminal domain is also targeted to the Golgi-like network but no centrosome staining is observed. (D) Diagram representing the CAP350 protein with the Ser-rich regions in yellow and the CAP-Gly domain in blue. The different domains that were used for overexpression are shown below in green. The localisation of the overexpressed proteins are indicated for each construct in the table. Bar, 20 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 5. Overexpressed full-length CAP350 GFP localises to the centrosome and to a subset of pericentrosomal microtubules. (A-C) Full-length CAP350 GFP overexpressed in cells and stained for -tubulin. At low expression levels, GFP decorates the centrosome (A). When the expression level increases, a subset of microtubules close to the centrosome is covered with CAP350 (B) and extended further in the cell (C). (D) 2.5x magnification of the boxed area in C. Note that CAP350 GFP is not continuous on microtubules, and that where it is present the anti-tubulin antibody has no access to MTs (arrows). A microtubule well stained with the anti-tubulin is only partly decorated with the fusion protein (arrowhead). Bar, 20 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 6. CAP350 overexpression stabilises microtubules. (A) Representative cell expressing CAP350 GFP after 40 minutes of Nocodazole treatment, stained for acetylated tubulin. Note that in these cells the microtubule network is conspicuous whereas in the neighboring cell only the centrosome is observed (arrow). (B) Cell expressing CAP350 GFP stained for EB1. Cells expressing CAP350 GFP show a decrease of the number of EB1 spots compared with the non-transfected cells. Bars, 20 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 7. Microtubules are less stable in CAP350-depleted cells. Cells were transfected with GL2 (A) or CAP350 (B) duplexes for 48 hours and further treated with 5 10–6 M Nocodazole for 10 minutes and stained for CAP350 and tubulin. Note the depletion of CAP350 at the centrosome and the decrease in the microtubule number after CAP350 siRNA treatment. All images have been scaled identically for fluorescence intensity using Metamorph software to directly compare the decrease in siRNA CAP350 cells at the centrosome and in the microtubule pattern. Bar, 20 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 8. Effect of CAP350 depletion or overexpression on the Golgi complex. Cells transfected with the GL2 (A) or CAP350 (B) duplexes for 48 hours were fixed with methanol and stained for CAP350 and CTR433 a median Golgi marker. Cells transfected with the GL2 duplex present a classical Golgi complex. After depletion of CAP350 with the siRNA CAP350, the Golgi complex appeared scattered. (C-D) Representative cell expressing CAP350-GFP and stained for CTR433 (C) or A24, an antibody directed against AKAP450 (D). Note that the cell overexpressing CAP350-GFP presented a scattered Golgi complex in contrast to the untransfected cells. Interestingly, the Golgi-like network marker AKAP450 partially redistributed and colocalised with CAP350. Bars, 20 µm

View larger version (57K): [in this window] [in a new window]   Fig. 9. Microtubule and CAP350 deployment in cochlear supporting cells. (A) Schematic diagram of the microtubule organisation (red lines) in a typical inner pillar cell at P5 (5 days postnatal) showing the apical centrosome (green with centrioles/primary cilium in red) and the microtubule minus-ends (arrowhead) anchored at apical sites (blue peripheral ring; arrow). (B) Apical view of a 3D reconstruction (based on confocal optical sections) of the apex of two inner pillar cells (P5) showing microtubules (red) organised in a tube and their minus ends forming an apical peripheral ring. CAP350 (green) is concentrated at the centrioles, which are located above the middle of the tube of microtubules (see A). Note there is no accumulation of CAP350 at the peripheral ring where some 3000 microtubule minus ends are located (arrow). (C) Lateral view of a 3D of the apical region of three inner pillar cells (P5) showing the apico-basally aligned microtubules with their minus-ends at the apex. CAP350 is concentrated at the centrosome (located above the microtubule array) and diffuse in the cytoplasm surrounding the microtubules. Note the cytoplasmic diffuse CAP350 is not evident at the minus ends of the microtubules and shows no apparent association with the microtubule array. (D) Apical view of a 3D reconstruction (based on confocal optical sections) of the apex of an inner pillar cell (P5) showing ninein at the centrosome and concentrated in a peripheral ring. (E) Lateral-to-oblique view of a 3D image of the apical region of an inner pillar cell (P5) with ninein accumulated at the minus ends of the apico-basal microtubules (red) (arrowhead). Part of the apex is also apparent, revealing part of the peripheral ninein ring (arrow) and ninein concentrated at the two centrioles. Bars, 5 µm.

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