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First published online 10 June 2003
doi: 10.1242/jcs.00619

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Intracellular clusterin causes juxtanuclear aggregate formation and mitochondrial alteration

¹ Information et Programmation Cellulaire, UMR6026 CNRS-Université de Rennes 1, Campus de Beaulieu, Bat. 13, 35042 Rennes Cedex, France
² CNRS-UPRES-A 8087, Laboratoire de génétique moléculaire et physiologique de l'EPHE, Université de Versailles/Saint-Quentin, Bâtiment Fermat, 45 avenue des Etats-Unis, 78035 Versailles Cedex, France

View larger version (68K): [in a new window]   Fig. 1. Subcellular distribution of various domains of clusterin. (A) Schematic representation of the different GFP chimeras between GFP and various domains of rat clusterin: full-length clusterin deleted from secretory signal peptide (SP-clu, amino acids 22-447), -subunit (-clu, amino acids 22-226), ß-subunit (ß-clu, amino acids 227-447). (B) COS-7 cells were observed in fluorescence microscopy 36 hours after transfection with the different expressing vectors. Cells transfected with GFP--clu were counterstained with DAPI in order to visualize the nucleus. Protein expression patterns shown are representative of patterns observed in the majority of transfected cells obtained from several experiments. Bar, 10 µm. (C) Juxtanuclear aggregation was also detected by clusterin immunostaining when SP-clu and -clu domains are expressed without GFP fusion. Cells were counterstained with DAPI. Bar, 10 µm. (D) Morphology of clusterin immunostained aggregates in tissue sections of aged rat brain closely resembles those observed in COS-7 cells overexpressing GFP--clu (right). Bar, 10 µm. Reproduced with permission from Blackwell Publishers/Blackwell Science Ltd/Polity Press (Senut et al., 1992).

View larger version (100K): [in a new window]   Fig. 2. GFP--clu juxtanuclear inclusions are immunoreactive for different aggresome markers. 36 hours after transfection with GFP--clu, COS-7 cells were fixed, permeabilized and incubated with antibodies against -tubulin, vimentin, the MSS1 subunit of 19S complex of proteasome and Hsp70, coupled with rhodamine-conjugated secondary antibodies. Nucleus was counterstained with DAPI. For all of these markers, intense immunostaining co-localized with the clusterin aggresome in a very reproducible manner. Arrowhead indicates centriole position after -tubulin labeling. Bar, 10 µm.

View larger version (86K): [in a new window]   Fig. 3. GFP--clu aggregates share functional features with aggresomes. Perinuclear relocalization of peripheral GFP--clu aggregates can be prevented by nocodazole, a microtubule-disrupting agent and by the chaperone Hsp70. Addition of 10 µg ml-1 nocodazole during 12 hours in culture medium led to the scattering of GFP--clu into multiple dots (C,D), without formation of massive juxtanuclear aggregates observed in controls (A,B). COS-7 cells overexpressing GFP--clu and Hsp70 in a fourfold excess presented a strong size reduction of juxtanuclear aggregates and an increase of the diffuse cytoplasmic distribution (E,F). Photographs are highly representative of the effect of the treatments observed in a large population of cells. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 4. Ubiquitination of clusterin. (A) 48 hours after transfection with GFP--clu or GFP-SP-clu, COS-7 cells were fixed, permeabilized and incubated with antibody against ubiquitin, coupled with secondary rhodamine-conjugated antibody. Diffused and aggregated GFP fluorescence closely co-localized with ubiquitin and this co-localization was systematically observed. Bar, 10 µm. (B) COS-7 cells were co-transfected with GFP-SP-clu, pCMV-ß-gal and expression plasmids encoding ubiquitin, wild-type (Ubi) or mutated (K48R-Ubi), or the empty vector (Control). 36 hours after transfection, extracts from cells treated (+) or not (-) with MG-132 (10 µM for 6 hours) were prepared by 100,000 g ultracentrifugation. Supernatants and pellets were analysed by western blotting with a clusterin antibody after normalization of cell extracts with ß-galactosidase. In addition to the expected molecular weight of GFP-SP-clu between 70 kDa and 80 kDa, high molecular weight forms (hmw) appeared after MG-132 treatment. These forms were no longer visible when using the polyubiquitin chain terminator K48R mutant ubiquitin and probably correspond to ubiquitinated clusterin.

View larger version (73K): [in a new window]   Fig. 5. Formation of clusterin aggresome induced relocalization of mitochondria. (A) COS-7 cells transfected with GFP--clu were incubated for 15 minutes with 100 nM MitoTracker RedTM, fixed, counterstained with DAPI and then analysed by fluorescence microscopy. In cells presenting clusterin aggresomes, mitochondria collapsed and massively relocalized at the juxtanuclear aggresome. The same results were obtained with immunostaining using a COX6C antibody coupled with rhodamine-conjugated secondary antibody. Bar, 10 µm for MitoTracker Red and COX6C labeling. (B) The co-localization of mitochondria and GFP--clu is visible only at the level of large GFP--clu aggregates. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 6. Effect of Bcl-2 expression and caspase inhibition on survival of HeLa cells transfected with different GFP-clusterin chimeras. The expression of Bcl-2 was induced or not in cells containing a tetracycline-regulated inducible Bcl-2 gene and transfected with one of the indicated constructs. (A) 24 hours after transfection, fluorescent transfected cells were analysed by fluorimetry based on their size and density. The proportion of small cells with highly condensed cytoplasm (left in cytograms) was increased after transfection with GFP-SP-clu, GFP--clu and GFP-ß-clu compared with GFP alone. This proportion was lower in cells overexpressing Bcl-2 after tetracycline removal (right) (percentages correspond to only one experiment). (B) Proportion of variation of small GFP-positive transfected cells after Bcl-2 induction (black bars) or not (white bars) were calculated from three independent transfection experiments. Each bar represents the mean±s.d. (n=3). *P<0.05 significantly different from control values; NS, not significantly different in Student's t-test.

View larger version (37K): [in a new window]   Fig. 7. Effects of Bcl-2 and ZVAD on mitochondrial events associated with clusterin-induced apoptosis of HeLa cells by multiparametric analysis of HeLa/Bcl-2 cells by flow cytometry. Each cell is represented by a dot. The data are relative to GFP-SP-clu-, GFP--clu-, GFP-ß-clu- and GFP-transfected cells, overexpressing (b,e,h,k) or not (a,c,d,f,g,i,j,l) Bcl-2 and in the presence (c,f,i,l) or not (a,b,d,e,g,h,j,k) of ZVAD. For each sample, the mitochondrial membrane potential as measured by MitoTracker RedTM fluorescence was reported in cytogram (MitoTracker Red versus size). Only GFP-positive cells with normal size were considered in multiparametric cytograms. Cells can be separated into two populations based on their MitoTracker Red fluorescence: one with high fluorescence (top) and the other with lower fluorescence (bottom). The proportion of cells in each population is specified in each panel of the cytograms (percentages correspond to only one experiment). The expression of GFP-clusterin constructs induced a loss of mitochondrial membrane potential, but cell size was not affected. (B) Proportion of variation of normally sized GFP-positive and MitoTracker-negative cells after Bcl-2 induction (black bars), ZVAD treatment (gray bars) or no Bcl-2 induction and no caspase inhibition (white bars). Values were calculated from three independent transfection experiments. Each bar represents the mean value±s.d. (n=3). *P<0.05 significantly different from control values; NS, not significantly different in Student's t-test.

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