Retinoschisin secretion into the conditioned medium is decreased by addition of taxol.

<p>(A) Effects of taxol on cell morphology and microtubule organization. Weri-Rb1 cells were cultured with medium containing DMSO or the indicated concentrations of taxol for 72 h. Cells were then fixed and labeled with an α tubulin antibody (red). The upper and lower rows of pictures show transmitted light images and fluorescent light images, respectively. The microtubule cytoskeleton of cells shows bundles after treatment with 5 µM and 10 µM taxol. Bar, 5 µm. (B) Cells were treated with the indicated concentrations of taxol for 72 h and retinoschisin in the conditioned media was analyzed on Western blots using the RS24-37 antibody. Retinoschisin secretion into the medium decreases in a dose-dependent manner between 0.5 µM and 10 µM taxol. (C) Quantification of the intensity of the bands from the Western blot in (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± SEM of three different biological samples for each condition. *: p<0.05 was obtained for the differences between 0.5 µM–10 µM taxol-treated and DMSO-treated cells. (D) After 72 hr incubation in medium containing DMSO or the indicated concentrations of taxol, cells were collected and analyzed by Western blot using the RS24-37 antibody. The intensities of the bands corresponding to the dimer and monomer of retinoschisin at each taxol concentration were added and normalized to GAPDH. The normalized values were compared to that of cells treated with DMSO and shown as RS1/DMSO ratios. Retinoschisin accumulates inside the cells exposed to taxol at concentrations between 0.5 µM and 10.0 µM.</p

Confocal microscopy images of Weri-Rb1 cells.

<p>(A) Cells were double-stained using the rabbit RS24-37 retinoschisin antibody followed by Cy3-conjugated donkey-anti-rabbit IgG (RS1, red) and Alexa Fluor 488-phalloidin for F-actin (green). DAPI labeled the nuclei (blue). The right image corresponds to the merged RS1 and F-actin images. The white squares limit Areas 1, 2 and 3 that have been zoomed in below. (B) Cells were double-stained for retinoschisin with RS24-37 antibody followed by Alexa Fluor 488 goat anti-rabbit IgG (green) and alpha tubulin antibody followed by Alexa Fluor 568 goat anti-mouse IgG (red). Nuclei were labeled with DAPI (blue). A merged image of the cells labeled for RS1 and alpha-tubulin is at the right. Retinoschisin co-localized with the cytoskeleton proteins F-actin and alpha-tubulin as indicated by the arrowheads in the enlarged images (1–6). Bar, 5 µm.</p

Retinoschisin secretion into the conditioned medium is increased by the ROCK kinase inhibitor, Y-27632.

<p>(A) Effects of Y-27632 on cell morphology and F-actin cytoskeleton organization. Weri-Rb1 cells were cultured in medium alone (untreated control) or the indicated concentrations of Y-27632 for 72 h. Cells were then fixed and labeled with Alexa Fluor 488-phalloidin (green). The upper row of pictures shows transmitted light images and the lower row, fluorescent light images. Bar, 5 µm. (B) Retinoschisin from the conditioned media of cells treated as in (A) was detected on a Western blot using the RS24-37 antibody. As seen, retinoschisin secretion into the conditioned medium is increased at 20 µM Y-27632. (C) Quantification of the intensity of each band on the Western blot from (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± SEM of three different biological samples for each condition. *: p<0.05 was obtained for the difference between 20 µM Y-27632-treated and untreated cells. (D) The level of retinoschisin in the lysates of whole cells treated with the concentrations of Y-27632 used in (B) does not change. After 72 hr incubation in medium alone or medium containing the indicated concentrations of Y-27632, cells were collected and their retinoschisin was analyzed on a Western blot using the RS24-37 antibody. The band intensities of the dimer and monomer for each condition were added and normalized to GAPDH. The normalized values for treated and untreated cells were compared and are shown as RS1/control ratios.</p

DBcGMP enhances retinoschisin secretion and modifies F-actin localization.

<p>(A) Effects of DBcGMP on the F-actin cytoskeleton. Weri-Rb1 cells were cultured with medium alone (untreated control) or the indicated concentrations of DBcGMP for 72 h. Cells were then fixed and labeled with Alexa Fluor 488-phalloidin (green). Note the F-actin protrusions in cells treated with 1 mM and 2 mM DBcGMP. Bar, 5 µm. (B) Retinoschisin secretion into the medium increases with the three concentrations of DBcGMP used to treat the cells for 72 h, as seen on the Western blot of the media samples reacted with the RS24-37 antibody. (C) Quantification of the retinoschisin bands on the Western blot in (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± SEM of three different biological samples for each condition. Retinoschisin levels in the culture medium of cells treated with the different concentrations of DBcGMP are all higher than in the medium of untreated cells, but the results are statistically significant only for the cells treated with 1 and 2 mM DBcGMP. *: p<0.05 was obtained for the difference between DBcGMP-treated and untreated cells.</p

Retinoschisin secretion into the conditioned medium is decreased by addition of cytochalasin D.

<p>(A) Effects of cytochalasin D on cell morphology and F-actin cytoskeleton organization are dose dependent. Weri-Rb1 cells were cultured for 72 h in medium alone (untreated control) and medium containing DMSO or the indicated concentrations of cytochalasin D. Cells were then fixed, labeled with Alexa Fluor 488-phalloidin (green) and DAPI, and examined for changes in F-actin cytoskeleton organization. The upper row of pictures shows transmitted light images and the lower row, fluorescent light images. Bar, 5 µm. (B) Retinoschisin secretion into the conditioned medium decreases by treatment of cells with 2 µM cytochalasin D. Cells were treated as in (A) for 72 h. The medium for each condition was analyzed by Western blot using the RS24-37 antibody against retinoschisin. (C) Quantification of the intensity of each band on the Western blot in (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± standard error of the mean (SEM) of three biological samples analyzed for each condition. (*) indicates that a p<0.05 was obtained for the difference between 2 µM cytochalasin D- and DMSO-treated cells. (D) Retinoschisin secretion decreases in a dose-dependent manner by treatment of cells with cytochalasin D at concentrations between 0.5 µM and 2 µM. Retinoschisin secretion was examined in the media of cells treated with cytochalasin D concentrations in the range of 0.02 to 2 µM as in (B) and quantified as stated in (C). (E) Retinoschisin level in the whole cell lysate increases with 2 µM cytochalasin D treatment. After 72 hr incubation in medium alone, medium containing DMSO or the indicated concentrations of cytochalasin D (left and middle panels) the cells were collected and analyzed by Western blot. Both the monomer and dimer of retinoschisin were detected in the retinal lysate (right panel). The intensities of the dimer and monomer bands for each sample were added and normalized to GAPDH. The normalized results were compared to the values obtained for the cells incubated in medium containing only DMSO and are shown as RS1/DMSO ratios. (F) Retinoschisin accumulates within cells treated with 2 µM cytochalasin D. Cells were treated with DMSO or 2 µM cytochalasin D for 72 h. Cells were then fixed and incubated with RS24-37 antibody, followed by Alexa Fluor 488 goat anti-rabbit IgG. Nuclei were labeled with DAPI.</p

The effect of cytochalasin D on retinoschisin secretion is reversible.

<p>(A) F-actin cytoskeleton organization visualized with Alexa Fluor 488-phalloidin (green). Duplicate cell samples were treated with DMSO or 1 µM and 2 µM cytochalasin D (CyD) for 72 hr. One of the sets of cells was then fixed and labeled for F-actin (left panel). The conditioned medium of the duplicate set of samples was replaced by fresh medium without DMSO (DMSO→medium) or cytochalasin D (CyD 1 or 2 µM→medium) in it. Cells were incubated for another 72 hr in the fresh medium, fixed and labeled with Alexa Fluor 488-phalloidin. (B) Western blot of proteins from the medium of each sample described in (A). Retinoschisin was detected using the RS24-37 antibody. (C) Quantification of the intensity of each band on the Western blot in (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± SEM of three different biological samples for each condition. *: p<0.05 was obtained for the difference between 1 µM or 2 µM cytochalasin D- and DMSO-treated cells.</p

Summary diagram for the roles of the F-actin and microtubule cytoskeletons in retinoschisin secretion.

<p>Different pharmacological treatments of Weri-Rb1 cells showed that: The F-actin depolymerizing drug cytochalasin D causes fragmentation of F-actin and inhibits secretion of retinoschisin in a dose-dependent manner at concentrations higher than 0.5 µM. At 2 µM cytochalasin D, this inhibition of secretion results in a 2.1-fold accumulation of retinoschisin within the cultured cells. The natural toxin Jasplakinolide (1 µM) hyperpolymerizes F-actin and results in the reduction of retinoschisin secretion and its 2.9-fold accumulation within the cells. Inhibition of ROCK using 20 µM Y-27632 or treatment with 2 mM DBcGMP relaxes the F-actin cytoskeleton and almost doubles retinoschisin secretion. Hyperpolmerization of microtubules by taxol results in disorganized microtubule bundles that reduce retinoschisin secretion. The solid arrows indicate increased retinoschisin secretion and the dotted arrows, decreased secretion.</p

Retinoschisin secretion into the conditioned medium is decreased by addition of jasplakinolide.

<p>(A) Effects of jasplakinolide on cell morphology and F-actin cytoskeleton organization. Weri-Rb1 cells were cultured in medium alone (untreated control) and medium containing DMSO or the indicated concentrations of jasplakinolide for 72 h. Cells were then fixed and labeled with Alexa Fluor 488-phalloidin (green). The upper row of pictures shows transmitted light images and the lower row, fluorescent light images. Bar, 5 µm. (B) Western blot of proteins in the conditioned media of cells treated with DMSO or the indicated concentrations of jasplakinolide for 72 h using the RS24-37 antibody. As seen, retinoschisin secretion into the conditioned medium is decreased by 1 uM jasplakinolide. (C) Quantification of the intensity of each band in the Western blot in (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± SEM of three different biological samples for each condition. *: p<0.05 was obtained for the difference between 1 µM jasplakinolide- and DMSO-treated cells. (D) Restinoschisin accumulates inside cells treated with 1 µM jasplakinolide. After 72 hr incubation in medium alone, medium containing DMSO or the indicated concentrations of jasplakinolide, cells were collected and analyzed by Western blot. The intensities of the dimer and monomer bands for each condition were added and normalized to GAPDH. The normalized values were then compared to that of DMSO-treated cells and are shown as RS1/DMSO ratios. (E) Retinoschisin accumulates within cells treated with 1 µM jasplakinolide. Cells were treated with DMSO or 1 µM jasplakinolide for 72 h. Cells were then fixed and incubated with RS24-37 antibody followed by Alexa Fluor 488 goat anti-rabbit IgG. Nuclei were labeled with DAPI.</p

The effect of jasplakinolide on retinoschisin secretion is reversible.

<p>(A) The F-actin cytoskeleton was visualized by Alexa Fluor 488-phalloidin (green). Duplicate cell samples were treated with DMSO or 0.5 µM and 1.0 µM jasplakinolide (Jas) for 72 h and then one set was fixed and labeled for F-actin (left panel). The conditioned medium of the other set of samples was replaced by fresh medium without DMSO (DMSO→medium) or jasplakinolide (Jas 0.5 µM or 1.0 µM→medium) in it. Cells were incubated for another 72 h in the fresh medium, fixed, and labeled with Alexa Fluor 488-phalloidin. (B) Western blot of proteins from the medium of each sample described in (A). Retinoschisin was detected using the RS24-37 antibody. (C) Quantification of the intensity of each band on the Western blot in (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020707#s4" target="_blank">MATERIALS AND METHODS</a>). The data are means ± SEM of three different biological samples for each condition. *: p<0.05 was obtained for the difference between 0.5 µM or 1 µM jasplakinolide- and DMSO-treated cells.</p

Far-UV CD spectrum of Zbed4 (black curve) and curve obtained using the CONTINLL program (white curve).

<p>The CONTINLL-calculated curve conforms well to the experimental spectra of Zbed4. SELCON and CDSSTR-calculated curves (not shown) were essentially identical to that of CONTINLL.</p