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First published online 13 June 2007
doi: 10.1242/jcs.008979

Journal of Cell Science 120, 2248-2258 (2007)
Published by The Company of Biologists 2007
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Climp-63-mediated binding of microtubules to the ER affects the lateral mobility of translocon complexes

Andrei V. Nikonov1, Hans-Peter Hauri2, Brett Lauring3 and Gert Kreibich1,*

1 Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
2 Biozentrum, University of Basel, CH-4056, Basel, Switzerland
3 Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA


Figure 1
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  		Fig. 1. DRM-tagged wild-type spastin (wtSPG4) severs microtubules in COS-1 cells. Cos-1 cells seeded on glass coverslips were transfected with plasmids encoding the fusion proteins DRM-wtSPG4 or DRM-NK#1. After fixation, cells were immunolabeled with anti-{alpha}-tubulin monoclonal antibody and secondary antibody conjugated to Alexa Fluor-633. Images were collected with a LSM510 microscope equipped with a Plan-Apochromate 100x/1.4 oil DIC lens at scan-zoom set at `1', line average set at `4' and pinhole set to optical slice of 0.5 µm. The HeNe2 laser was used to visualize microtubules (a,d) and the HeNe1 laser was used to visualize the DRM-tagged constructs (b,e). The channel representing immuno-labeled microtubules was pseudo-colored in green. Most of microtubules are severed in the cells expressing DRM-wtSPG4 (a-c). The DRM-wtSPG4 fusion protein has a characteristic punctate distribution. Expression of the biologically inactive DRM-NK#1 construct leads to the decoration of intact microtubules by the fusion protein (d-f). Bar, 20 µm.

 

Figure 2
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  		Fig. 2. DRM-tagged CLIMP-63 related constructs expressed in M3/18 cells have the molecular mass expected. (A) Schematic representation of CLIMP-63 related constructs tagged with the red fluorescent protein (DRM) of Discosoma sp. CLIMP-63 is a type II transmembrane protein. It has a relatively short N-terminal cytosolic domain (hatched box), which is capable of binding to microtubules. A larger C-terminal lumenal domain (open box) can form oligomers. The transmembrane domain is represented by a black box. Monomeric DRM used as a tag is represented as a gray oval. (B) The cells transfected with recombinant plasmids encoding p63-DRM (lane 1), {Delta}Lp63-DRM (lane 2), DRM-{Delta}Cp63 (lane 3), {Delta}TLp63-DRM (lane 4) or the cloning vector encoding DRM (lane 5) were subjected to western blot analysis using a commercial M3/18 directed against the red fluorescent protein. All expressed fusion proteins had the expected molecular mass.

 

Figure 3
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  		Fig. 3. Intracellular localization of DRM-tagged CLIMP-63 related constructs in M3/18 cells. M3/18 cells grown at 39.5°C were transfected with recombinant plasmids encoding the corresponding fusion proteins. Images were collected with an LSM510 microscope equipped with a Plan-Apochromate 100x/1.4 oil DIC lens at scan-zoom set at `1' and line average set at `4'. Due to low fluorescent intensity of stably expressed GFP-Dad1 the pinholes were set wide-open. Expression of GFP-Dad1 was visualized using the argon laser (a,d,g,j,m). The HeNe1 laser was used to visualize DRM-tagged constructs (b,e,h,k,n). To assess colocalization of two proteins, the images of both channels were merged (c,f,i,l,o). Expression of p63-DRM and {Delta}Lp63-DRM fusion constructs revealed that these fusion proteins are localizing to the ER (a-c and d-f, respectively). No changes in the ER morphology were observed. Expression of the soluble {Delta}TLp63-DRM and DRM revealed that these proteins are localizing to the cytosol (j-l and m-o, respectively). The morphology of the ER is not altered. Expression of the DRM-{Delta}Cp63 construct lead to some accumulation in a perinuclear area, while the morphology of the ER appeared was not affected (g-i). {Delta}TLp63-DRM apparently had cytosolic localization (j-l) similar to that of red fluorescent tag, DRM (m-o). Bar, 20 µm.

 

Figure 4
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  		Fig. 4. siRNA knock-down of CLIMP-63 expression led to an increased lateral mobility of the TCs in M3/18 cells. (A) Western blot analysis of M3/18 cells transfected with siRNA#1 (lane 1), siRNA#2 (lane 2), siRNA#3 (lane 3), mock transfected (lane 4) or untreated (lane 5). The samples were collected 4 days after transfection. Staining of the western blot with anti-CLIMP-63 pAb shows that siRNA#1 and siRNA#2 are able to silence the expression of CLIMP-63 in M3/18 cells (lanes 1 and 2), wheras siRNA#3 had no effect on CLIMP-63 expression (lane 3) when compared with mock-transfected (lane 4) or untreated (lane 5) cells. The staining of the same blot with anti-calnexin or anti-ribophorin I pAbs revealed that expression levels of these proteins were not affected by the CLIMP-63 knock-down. (B) Immuno-fluorescent microscopy of mock-transfected cells (a-c) or cells transfected with siRNA#2 (d-f). The cells were collected on day 1,3 and 5 after transfection, fixed in 2% PFA in PBS and stained with anti-CLIMP-63 pAb followed by goat anti-rabbit IgG conjugated to Alexa Fluor-543. The images were collected with the LSM510 microscope equipped with a HeNe1 laser and a Plan-Apochromate 100x/1.4 oil DIC lens at scan-zoom set at `1' and line average set at `4'. The pinhole was set to obtain 0.5 µm thick optical slices. The immunofluorescence microscopy results support western blot data showing that siRNA#2 can effectively knock down of CLIMP-63 expression.Bar, 20 µm. (C) FRAP analysis of untreated M3/18 cells confirmed our previous findings that the lateral mobility of the TCs is very low (Deff=0.063 µm2/second). FRAP analysis of mock-transfected cells performed at the day 4 after transfection suggested that electro-shocking of the cells suspended in transfection buffer had no effect on the TC mobility (Deff=0.067 µm2/second, P(T<=t)=0.427). However, transfection of the cells with siRNA#2 led to an increased diffusion rate of the TCs (Deff=0.137 µm2/second, P(T<= t)<0.001). Knock-down of CLIMP-63 with siRNA had no effect on the lateral mobility of the ER membrane protein LBR-GFP.

 

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