Publications by authors named "Laura J Robles"

5 Publications

  • Page 1 of 1

Rho signaling mediates cytoskeletal re-arrangements in octopus photoreceptors.

Am Malacol Bull 2008 Dec;26(1-2):19-26

Department of Biology, California State University, Dominguez Hills, 1000 East Victoria Street, Carson, California 90747, U.S.A.,

Light sensitive rhabdoms in the octopus retina increase in cross-sectional area in the dark and shrink in the light. Growth in the dark is due to the formation of microvilli in an avillar region of the photoreceptor cell membrane and lengthening of rhabdomere microvilli already present. Diminution in the light is the result of the disassembly and shortening of the same microvilli. Each microvillus contains an actin filament core that must be assembled or disassembled in the dark or light, respectively. To understand the regulation of the construction and breakdown of rhabdomere microvilli in the light and dark, we used centrifugation to separate the rhabdom membranes followed by Western blotting and Rho pull-down assays to investigate the role of Rho GTPases in this process. Western blotting showed a difference in the distribution of Rho in rhabdom membrane and supernatant fractions. In the light, Rho was mostly present in the supernatant but in the dark it was found in the fraction enriched with rhabdom membranes. Complementing these results, pull-down assays showed that Rho is activated in the dark but in the light, Rho is mostly inactive. We believe that in the dark, activated Rho binds to the rhabdom membrane and initiates signaling pathways, leading to growth of rhabdomere microvilli. In the light, Rho is present in the soluble fraction, is inactivated, and is likely bound to a Rho GDI. Receptors involved in the activation of Rho in the dark are undetermined and may involve rhodopsin or another membrane protein.
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December 2008

Analysis of the 3' untranslated regions of alpha-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas.

Mol Vis 2008 Aug 4;14:1446-55. Epub 2008 Aug 4.

Department of Biology, California State University, Dominguez Hills, Carson, CA 90747, USA.

Purpose: We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)-dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in alpha-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas.

Methods: 3'-RACE and sequencing were used to isolate and analyze the 3'-UTRs of alpha-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB.

Results: The following CPE-like sequence was detected in the 3'-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3'-UTRs of alpha-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas.

Conclusions: The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3'-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of alpha-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas.
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August 2008

Rho GTPases regulate rhabdom morphology in octopus photoreceptors.

Vis Neurosci 2005 May-Jun;22(3):295-304

Department of Biology, California State University, Dominguez Hills, Carson, 90747, USA.

In the cephalopod retina, light/dark adaptation is accompanied by a decrease/increase in rhabdom size and redistribution of rhodopsin and retinochrome. Rearrangements in the actin cytoskeleton probably govern changes in rhabdom size by regulating the degradation/formation of rhabdomere microvilli. Photopigment movements may be directed by microtubules present in the outer segment core cytoplasm. We believe that rhodopsin activation by light stimulates Rho and Rac signaling pathways, affecting these cytoskeletal systems and their possible functions in controlling rhabdom morphology and protein movements. In this study, we localized cytoskeletal and signaling proteins in octopus photoreceptors to determine their concurrence between the lighting conditions. We used toxin B from Clostridium difficile to inhibit the activity of Rho/Rac and observed its effect on the location of signaling proteins and actin and tubulin. In both lighting conditions, we found Rho in specific sets of juxtaposed rhabdomeres in embryonic and adult retinas. In the light, Rho and actin were localized along the length of the rhabdomere, but, in the dark, both proteins were absent from a space beneath the inner limiting membrane. Rac colocalized with tubulin in the outer segment core cytoplasm and, like Rho, the two proteins were also absent beneath the inner limiting membrane in the dark. The distribution of actin and Rho was affected by toxin B and, in dark-adapted retinas, actin and Rho distribution was similar to that observed in the light. Our results suggest that the Rho/Rac GTPases are candidates for the regulation of rhabdomere size and protein movements in light-dark-adapted octopus photoreceptors.
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October 2005

S-crystallin and arginine kinase bind F-actin in light- and dark-adapted octopus retinas.

Curr Eye Res 2004 May;28(5):343-50

Department of Chemistry, California State University, Dominguez Hills, Carson, CA, USA.

Purpose: Rhabdomere microvilli dramatically reorganize in conditions of light and dark. This reorganization involves remodeling of the microvillus actin cytoskeleton. We are using the rhabdomeric retina of Octopus bimaculoides to identify actin-binding proteins that may be involved in this remodeling.

Methods: Octopus were light-/dark-adapted, retinas separated into dorsal and ventral halves, and homogenized. Actin-binding proteins were recognized using F-actin overlay blot assays and selected proteins from the overlays were identified using N-terminal sequencing methods or mass spectroscopy. Protein concentrations were quantified and compared by statistical analysis.

Results: Total protein gels of light-/dark-adapted, ventral/dorsal halves were almost identical except for a protein band at 26 kD. The relative amount of this protein in the dark was almost double that found in the light. The levels of other proteins did not vary significantly between the light and dark. F-actin overlays also showed matching patterns of actin-binding proteins except for the 26 kD protein. Although the 26 kD protein from light-adapted retinas transferred to the blotting membranes, it did not bind F-actin while the 26 kD protein on overlays from dark-adapted retinas always demonstrated F-actin binding. Besides the 26 kD protein, other proteins at 200 kD, 80 kD, 40 kD appeared on the overlays. These proteins and the 26 kD protein were sequenced and identified as hemocynanin, transitional ER ATPase, arginine kinase and S-crystallin, respectively.

Conclusions: The amount of S-crystallin present in the octopus retina is significantly greater in dark-adapted retinas and it binds to F-actin. In the light, the level of S-crystallin is greatly reduced and there is no apparent F-actin binding. No other studies, to our knowledge, show that S-crystallin binds to the actin cytoskeleton or that its expression is regulated by light. Arginine kinase may provide energy for cytoskeletal remodeling as it may in other neural tissues.
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May 2004

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

J Neurocytol 2002 Feb;31(2):161-74

Biology Department, California State University, Dominguez Hills, Carson, CA 90747, USA.

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.
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February 2002