Abstract
Purpose :
Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the visual pigment melanopsin, involved in modulating circadian photoentrainment, pupil constriction, and contrast sensitivity. It has been observed that ipRGCs can sustain a light response ranging from minutes to hours. We hypothesize that clathrin-mediated endocytosis contributes to melanopsin’s ability to sustain signaling during long light exposures by facilitating the visual pigment’s re-sensitization. We investigated this hypothesis using biochemical assays to test melanopsin localization in the cell. We analyzed the cellular localization of a phosphonull melanopsin mutant, which lacks C-terminal phosphorylation, and wildtype mouse melanopsin. We predict that wildtype melanopsin will colocalize with clathrin-coated pits through arrestin-mediated binding, while the mutant melanopsin will not.
Methods :
We transiently expressed C-terminal 1D4-tagged mouse melanopsin, wildtype and phosphonull, in HEK293 cells. These cells were dark-adapted overnight prior to exposure to varying light intervals, fixed, and incubated with α-1D4 and α-clathrin antibodies. Cells were visualized with confocal microscopy. Cellular localization of melanopsin and clathrin were analyzed using pixel intensity of fluorescence in the cell using the Plot Profile tool in ImageJ. Membrane fractions were isolated and used for Western blot to analyze the membrane population of melanopsin prior and after light exposure. Lastly, transfected cells were used for in vitro calcium imaging with or without chloroquine and chloropromazine, which are chemical inhibitors of clathrin-mediated endocytosis.
Results :
Confocal microscopy revealed wild-type and phosphonull melanopsin localization at the membrane in the dark, however only wild-type melanopsin colocalizes with clathrin at both the membrane and cytoplasm after light exposure. Preliminary in vitro calcium imaging suggests a reduction of light response when clathrin-mediated endocytosis is inhibited. Preliminary confocal microscopy of the treated cells also shows inhibited cytoplasmic trafficking of melanopsin after light exposure.
Conclusions :
This investigation indicates light-stimulated melanopsin undergoes clathrin-mediated endocytosis, in contrast to rod and cone visual pigments that lack this behavior. Additionally, melanopsin endocytosis is important for robust light responses, implicating this in melanopsin re-sensitization.
This is a 2020 ARVO Annual Meeting abstract.