Browsing by Author "Robinson, Phyllis R."
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Item Characterization of visual pigments, oil droplets, lens and cornea in the whooping crane Grus americana(The Company of Biologists Ltd, 2014-10-29) Porter, Megan L.; Kingston, Alexandra C. N.; McCready, Robert; Cameron, Evan G.; Hofmann, Christopher M.; Suarez, Lauren; Olsen, Glenn H.; Cronin, Thomas W.; Robinson, Phyllis R.Vision has been investigated in many species of birds, but few studies have considered the visual systems of large birds and the particular implications of large eyes and long-life spans on visual system capabilities. To address these issues we investigated the visual system of the whooping crane Grus americana (Gruiformes, Gruidae), which is one of only two North American crane species. It is a large, long-lived bird in which UV sensitivity might be reduced by chromatic aberration and entrance of UV radiation into the eye could be detrimental to retinal tissues. To investigate the whooping crane visual system we used microspectrophotometry to determine the absorbance spectra of retinal oil droplets and to investigate whether the ocular media (i.e. the lens and cornea) absorb UV radiation. In vitro expression and reconstitution was used to determine the absorbance spectra of rod and cone visual pigments. The rod visual pigments had wavelengths of peak absorbance (λmax) at 500 nm, whereas the cone visual pigment λmax values were determined to be 404 nm (SWS1), 450 nm (SWS2), 499 nm (RH2) and 561 nm (LWS), similar to other characterized bird visual pigment absorbance values. The oil droplet cut-off wavelength (λcut) values similarly fell within ranges recorded in other avian species: 576 nm (R-type), 522 nm (Ytype), 506 nm (P-type) and 448 nm (C-type). We confirm that G. americana has a violet-sensitive visual system; however, as a consequence of the λmax of the SWS1 visual pigment (404 nm), it might also have some UV sensitivity.Item Melanopsin Carboxy-terminus Phosphorylation Plasticity and Bulk Negative Charge, not Strict Site Specificity, Achieves Phototransduction Deactivation(2020-01-11) Valdez-Lopez, Juan C.; Gulati, Sahil; Ortiz, Elelbin A.; Palczewski, Krzysztof; Robinson, Phyllis R.Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin’s Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and β-arrestin binding are centered around these critical phosphorylation sites (S388-S395). This degree of functional versatility could help explain the diverse ipRGC light responses as well as non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.Item Photochemistry of retinal chromophore in mouse melanopsin(National Academy of Sciences, 2008-06-25) Walker, Marquis T.; Brown, R. Lane; Cronin, Thomas W.; Robinson, Phyllis R.In mammals, melanopsin is exclusively expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), which play an important role in circadian photoentrainment and other nonimage-forming functions. These ipRGCs reside in the inner retina, far removed from the pigment epithelium, which synthesizes the 11-cis retinal chromophore used by rod and cone photoreceptors to regenerate opsin for light detection. There has been considerable interest in the identification of the melanopsin chromophore and in understanding the process of photopigment regeneration in photoreceptors that are not in proximity to the classical visual cycle. We have devised an immuno-magnetic purification protocol that allows melanopsin-expressing retinal ganglion cells to be isolated and collected from multiple mouse retinas. Using this technique, we have demonstrated that native melanopsin in vivo exclusively binds 11-cis retinal in the dark and that illumination causes isomerization to the all-trans isoform. Furthermore, spectral analysis of the melanopsin photoproduct shows the formation of a protonated metarhodopsin with a maximum absorbance between 520 and 540 nm. These results indicate that even if melanopsin functions as a bistable photopigment with photo-regenerative activity native melanopsin must also use some other light-independent retinoid regeneration mechanism to return to the dark state, where all of the retinal is observed to be in the 11-cis form.Item Protein Phosphatase 2A and Clathrin-Mediated Endocytosis Facilitate Robust Melanopsin Light Responses and Resensitization(ARVO, 2020-10) Valdez-Lopez, Juan C.; Gebreegziabher, Meheret; Bailey, Robin J.; Flores, Jair; Awotunde, Olanike; Burnett, Thomas; Robinson, Phyllis R.Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the visual pigment melanopsin regulate non-image-forming visual tasks, such as circadian photoentrainment and pupil constriction, as well as contrast detection for image formation. Sustained ipRGC function throughout the day is, therefore, of great importance. Melanopsin is a bistable rhabdomeric-type (R-type) visual pigment, which is thought to use light to regenerate its chromophore from all-trans-retinal back to 11-cis-retinal and does not depend on constant chromophore supply to the extent required by visual pigment in rod and cone photoreceptors. Like the majority of photopigments and G-protein-coupled receptors (GPCRs), melanopsin deactivation requires C-terminal phosphorylation and subsequent β-arrestin binding. We hypothesize that melanopsin utilizes canonical GPCR resensitization mechanisms, including dephosphorylation and endocytosis, during the light, and together, they provide a mechanism for prolonged light responses. Methods: Here, we examined expression of protein phosphatases from a variety of subfamilies by RT-PCR and immunohistochemical analyses of the mouse retina. The expression of protein phosphatase 2A (PP2A) in ipRGCs was assessed. We also examine the role of phosphatase and endocytic activity in sustaining melanopsin signaling using transiently-transfected HEK293 cells. Results: Our analyses suggest that melanopsin-mediated light responses can be rapidly and extensively enhanced by PP2A activity. Light-activated melanopsin undergoes endocytosis in a clathrin-dependent manner. This endocytic activity enhances light responses upon repeated stimulation, implicating a role for endocytic activity in resensitization. Conclusions: Thus, we propose that melanopsin phototransduction is maintained by utilizing canonical GPCR resensitization mechanisms rather than reliance on chromophore replenishment from supporting cells.Item Spectral tuning and deactivation kinetics of marine mammal melanopsins(PLOS, 2021-10-15) Fasick, Jeffry I.; Algrain, Haya; Samuels, Courtland; Mahadevan, Padmanabhan; Schweikert, Lorian E.; Naffaa, Zaid J.; Robinson, Phyllis R.In mammals, the photopigment melanopsin (Opn4) is found in a subset of retinal ganglion cells that serve light detection for circadian photoentrainment and pupil constriction (i.e., mydriasis). For a given species, the efficiency of photoentrainment and length of time that mydriasis occurs is determined by the spectral sensitivity and deactivation kinetics of melanopsin, respectively, and to date, neither of these properties have been described in marine mammals. Previous work has indicated that the absorbance maxima (λmax) of marine mammal rhodopsins (Rh1) have diversified to match the available light spectra at foraging depths. However, similar to the melanopsin λmax of terrestrial mammals (~480 nm), the melanopsins of marine mammals may be conserved, with λmax values tuned to the spectrum of solar irradiance at the water’s surface. Here, we investigated the Opn4 pigments of 17 marine mammal species inhabiting diverse photic environments including the Infraorder Cetacea, as well as the Orders Sirenia and Carnivora. Both genomic and cDNA sequences were used to deduce amino acid sequences to identify substitutions most likely involved in spectral tuning and deactivation kinetics of the Opn4 pigments. Our results show that there appears to be no amino acid substitutions in marine mammal Opn4 opsins that would result in any significant change in λmax values relative to their terrestrial counterparts. We also found some marine mammal species to lack several phosphorylation sites in the carboxyl terminal domain of their Opn4 pigments that result in significantly slower deactivation kinetics, and thus longer mydriasis, compared to terrestrial controls. This finding was restricted to cetacean species previously found to lack cone photoreceptor opsins, a condition known as rod monochromacy. These results suggest that the rod monochromat whales rely on extended pupillary constriction to prevent photobleaching of the highly photosensitive all-rod retina when moving between photopic and scotopic conditions.