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Perinuclear localization of huntingtin as a consequence of its binding to microtubules through an interaction with ß-tubulin: relevance to Huntington's disease

CNRS — UPR 2228, Régulation de la Transcription et Maladies Génétiques, Université René Descartes, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France

View larger version (29K): [in a new window]   Fig. 1. Co-purification of huntingtin and ß-tubulin from lymphoblast and brain extracts. A whole lymphoblast extract from a patient with juvenile Huntington's disease and a rat brain extract were incubated in the presence of either the MAB 2166 anti-huntingtin (A) or the 3F3G2 anti-ß-tubulin (B) antibody coupled to magnetic beads. After several washes, the bound material was eluted. Equal amounts of the whole extract (WE) and of the immunopurified material (IP) were then examined by immunoblotting, using either an anti-huntingtin, an anti-ß-tubulin or an anti--tubulin antibody. In both lymphoblasts and brain the anti-huntingtin antibody purified specifically huntingtin and ß-tubulin. Reciprocally the anti-ß-tubulin antibody purified ß-tubulin and huntingtin. Neither ß-tubulin nor huntingtin were immunopurified when the corresponding specific antibodies were omitted from beads.

View larger version (45K): [in a new window]   Fig. 2. Dissociation of the /ß tubulin heterodimer by the anti-ß-tubulin antibody. Tubulin heterodimer purified from mouse brain by chromatography on phosphocellulose columns (Tubulin PC) and consisting of equal amounts of - and ß-tubulin was submitted to immunopurification by an anti-ß-tubulin antibody (3F3G2), as described for Fig. 1. The anti-ß-tubulin antibody purified almost exclusively ß-tubulin because it caused dissociation of the heterodimer.

View larger version (30K): [in a new window]   Fig. 3. Huntingtin coassembles with purified microtubules. A rat brain homogenate was clarified by high-speed centrifugation and the soluble tubulin present in the supernatant was allowed to polymerize in the presence of paclitaxel and GTP. Microtubules were then sedimented through a sucrose cushion. The pellet (P) containing microtubules and the supernatant (S) were examined by immunoblotting using monoclonal antibodies directed against either ß-tubulin, huntingtin, MAP-2, actin or caspase-1. Huntingtin and MAP-2 cosediment with microtubules, whereas actin and caspase-1, two proteins not known to be associated with microtubules, remained virtually entirely in the supernatant. In the absence of paclitaxel, no microtubules were formed and both huntingtin and MAP-2 remained in the supernatant.

View larger version (69K): [in a new window]   Fig. 4. Association of huntingtin with microtubules in 3T3 cells. 3T3 cells were double stained with an antibody raised against the first 17 amino acid residues of huntingtin (green) and the monoclonal 3F3G2 anti-ß-tubulin antibody (red). Nuclei were counterstained with Hoechst (blue). (A) Before treatment with nocodazole huntingtin and tubulin immunoreactivity largely overlaped. (B) After addition of nocodazole to the culture medium, microtubules were disrupted and both huntingtin and tubulin acquired a patchy and diffuse distribution. Arrows show fragments of microtubules to which huntingtin has remained attached. Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 5. Perinuclear distribution of huntingtin. 3T3 cells were stained simultaneously with the anti-huntingtin (green) and the anti-ß-tubulin (red) antibodies used in Fig. 4. Thin sections of the cells were then examined by confocal microscopy. In these little-exposed photomicrographs, huntingtin staining is mostly perinuclear, where it overlaps with the denser part of the microtubular network.

View larger version (39K): [in a new window]   Fig. 6. Huntingtin is found at the centrosome in a fraction of the cell population. (A) 3T3 cells were stained with both the anti-huntingtin antibody directed against the first 17 residues of the protein (green) and a monoclonal anti--tubulin (red) antibody, and examined with an epifluorescence microscope. In a large fraction of the cells the centrosome is clearly labeled by both antibodies (arrows). The centrosome of the lower cell of the upper panel is not stained by the anti-huntingtin antibody. (B) Cells were examined by confocal microscopy and double-labeled pixels were displayed in white. The centrosome shows clear colocalization of huntingtin and -tubulin.