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First published online 5 October 2004
doi: 10.1242/jcs.01420

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Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line

Centro de Biología Molecular `Severo Ochoa', Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Ma drid, 28049, Spain

View larger version (40K): [in a new window]   Fig. 1. Gene expression of MAL, MAL2 and BENE in different cell lines and raft-association of these proteins in PC-3 cells. (A) Total RNA from the indicated cell lines was hybridized to cDNA probes specific to MAL, MAL2, BENE and -actin as indicated. (B) Cells were extracted with 1% Triton X-100 at 4°C and subjected to centrifugation to equilibrium in sucrose density gradients. Aliquots from each fraction were analyzed by immunoblotting with antibodies specific to MAL, BENE and MAL2. Antibodies to cav-1 (a protein normally associated to rafts) and to calnexin (an ER-associated protein) were used as controls for the fractionation procedure. Two isoforms of cav-1, and ß, are recognized by the anti-cav-1 antibody. The smear of bands between 30 and 40 kDa observed for MAL2 (*) represents glycosylated forms of this protein. Fractions 1-4 represent the 40% sucrose layer and contain the bulk of cellular membranes and cytosolic proteins and fractions 5-12 represent the 5-30% sucrose layer and contain rafts. The position of molecular weight markers in kDa is indicated on the right. (C) PC-3 cells stably expressing MAL-GFP or BENE-myc were extracted with 1% Triton X-100 at 4°C and subjected to centrifugation to equilibrium in sucrose density gradients. Aliquots from each fraction were analyzed by immunoblotting with anti-tag antibodies.

View larger version (101K): [in a new window]   Fig. 2. Distribution of MAL, MAL2 and BENE in PC-3 cells. PC-3 cells stably expressing MAL-GFP (A-C) or BENE-myc (D-I) were fixed and stained with cav-1 and MAL-GFP (A-C), cav-1 and MAL2 (D-F), and cav-1 and BENE (G-I) and analyzed using a confocal laser microscope. Cells have been outlined for better visualization. Bars, 10 µm.

View larger version (61K): [in a new window]   Fig. 3. PC-3 cells stably expressing BENE-myc or MAL-GFP. Cells were fixed and stained with MAL (A), MAL2 (B,D) and BENE (C) and analyzed with a confocal laser microscope. PC-3 cells stained with cav-1 were analyzed by immunofluorescence microscopy (E) and by phase-contrast microscopy (F). PC-3 cells stably expressing MAL-GFP were analyzed by immunofluorescence microscopy (G) and by phase-contrast microscopy (H). (I) Electron micrograph of PC-3 cells embedded in Epon. Cells in A-D have been outlined for better visualization. Bars A-H, 10 µm; I, 1 µm.

View larger version (111K): [in a new window]   Fig. 4. Confocal immunofluorescence studies of cav-1. (A-F) PC-3 cells stably expressing BENE-myc were fixed and double-labelled with antibodies to cav-1 and the GPI-anchored protein CD59, and cav-1 and transferrin receptor (Tfr). (G-I) In some experiments, PC-3 cells were previously incubated with BFA (2.5 µg/ml) for 30 minutes, fixed and then double-labelled with antibodies to cav-1 and transferrin receptor. Cells have been outlined for better visualization. Bars, 10 µm.

View larger version (105K): [in a new window]   Fig. 5. Confocal immunofluorescence studies of cav-1 and markers of different organelles in PC-3 cells. (A-L) PC-3 cells stably expressing BENE-myc were fixed and double-labelled with antibodies to cav-1 and: the early endosomal marker EEA1 (A-C); the lysosomal marker CD63 (D-F); the Golgi marker giantin (G-I); and lipid droplets stained with Nile red (J-L). Cells have been outlined for better visualization. Bars, 10 µm.

View larger version (94K): [in a new window]   Fig. 6. Cav-1 and MAL-GFP are released in prostasomes in PC-3 cells. (A) Whole-mount immunoelectron microscopy of prostasomes isolated from PC-3 cell culture supernatants labelled with anti-CD59 or anti-CD63 mAbs. Bars, 0.2 µm. (B) PC-3 cell-derived prostasomes were analyzed by western blotting using antibodies to CD59 or CD63. The position of molecular weight markers in kDa is indicated on the left. (C) Prostasomes were isolated from the culture supernatant of PC-3 cells stably expressing BENE-myc or MAL-GFP and analyzed by western blotting using antibodies to cav-1 or GFP. The position of the molecular weight markers in kDa is indicated. (D) Prostasomes isolated from PC-3 MAL-GFP cell culture supernatants were subjected to whole-mount immunoelectron microscopy and labelled with anti-GFP antibodies. Bar, 0.2 µm. (E) Prostasomes from PC-3 cells stably expressing BENE-myc were isolated as previously described and then floated on a continuous sucrose density gradient. Aliquots from each fraction (1-12) were run on SDS-PAGE and immunoblotted with anti-cav-1 or anti-CD59 antibodies. Prostasomes floated at a sucrose density of 1.11 g/ml.

View larger version (44K): [in a new window]   Fig. 7. Cav-1 and MAL-GFP secretion in prostasomes continues in the presence of BFA but it is reduced by wortmannin. (A-D) PC-3 cells stably expressing BENE-myc or MAL-GFP were incubated without or with BFA (2.5 µg/ml) (A,B) and without or with wortmannin (wort.) (100 nM) (C,D) in serum-free medium for 20 hours. Prostasomes were then isolated from the culture supernatant and analyzed by western blotting using antibodies to cav-1 and GFP. A and C show a representative experiment and a quantitative analysis of cav-1 secretion from three experiments. (E) PC-3 cells were treated with wortmannin (100 nM) for 1 hour and then fixed and double-labelled with antibodies to cav-1 and the endosomal marker Rab5. Bar, 10 µm.