Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process

Association of the Asn306Ser variant of the SP4 transcription factor and an intronic variant in the β-subunit of transducin with digenic disease

Purpose SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations. Methods 270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants. Results Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the α-, β- and γ-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the β-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant. Conclusions If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.PubMedWo

Sequence-Specific Binding of Recombinant Zbed4 to DNA: Insights into Zbed4 Participation in Gene Transcription and Its Association with Other Proteins

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process

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

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

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

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