No description available.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 95-101).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 74-81).

High temporal resolution of vision relies on the rapid kinetics of the photoresponse in the light-sensing photoreceptor neurons. It is well known that the rapid recovery of photoreceptor membrane potential at the end of light stimulation depends on timely deactivation of the visual transduction cascade within photoreceptors. Whether any extrinsic factor contributes to the termination speed of the photoresponse is unknown. In this thesis, using Drosophilaas a model system, I show that a feedback circuit mediated by both neurons and glia in the visual neuropile lamina is required for rapid repolarization of the photoreceptor at the end of the light response. In the first part of my thesis work, I provide evidence that lamina epithelial glia, the major glia in the visual neuropile, is involved in a retrograde regulation that is critical for rapid repolarization of the photoreceptor at the end of light stimulation. I identified the gene affected in a slrp (slow receptor potential) mutant that is defective in photoreceptor response termination, and found it needs to be expressed in both neurons and epithelial glia to rescue the mutant phenotype. The gene product SLRP, an ADAM (a disintegrin and metalloprotease) protein, is localized in a special structure of epithelial glia, gnarl, and is required for gnarl formation. This glial function of SLRP is independent of the metalloprotease activity. In the second part of my thesis work, I demonstrate that glutamatergic transmission from lamina intrinsic interneurons, the amacrine cells, to the epithelial glia is required for the rapid repolarization of photoreceptors at the end of the light response. From an RNAi-based screen, I identified a vesicular glutamate transporter (vGluT) in amacrine cells as an indispensable factor for the rapid repolarization of the photoreceptor, suggesting a critical role of glutamatergic transmission from amacrine cells in this retrograde regulation. Further, I found that loss of a glutamate-gated chloride channel GluCl phenocopies vGluT downregulation. Cell specific knockdown indicates that GluCl functions in both neurons and glia. In the lamina, a FLAG-tagged GluCl colocalized with the SLRP protein in the gnarl-like structures, and this localization pattern of GluCl depends on SLRP, suggesting that lamina epithelial glia receive glutamatergic input from amacrine cells through GluCl at the site of gnarl. Since the amacrine cell itself is innervated by photoreceptors, these observations suggest that a photoreceptor — amacrine cell — epithelial glia — photoreceptor feedback loop facilitates rapid repolarization of photoreceptors at the end of the light response. In summary, my thesis research has revealed a feedback regulation mechanism that helps to achieve rapid kinetics of photoreceptor response. This visual regulation contributes to the temporal resolution of the visual system, and may be important for vision during movement and for motion detection. In addition, this work may also advance our understanding of glial function, and change our concept about the effect of glutamatergic transmission.

High visual sensitivity is a common but important characteristic of animal eyes. It is especially critical for night vision. In animal eyes, photoreceptors are the first to receive the incoming rays of light and they convert the light signals to electrical signals before passing the information to interneurons in the eye and finally to the brain. To function in dim light conditions, photoreceptors have developed high sensitivities to light. It is reported that both mammalian rod photoreceptors and Drosophilaphotoreceptors can detect single photons. The high sensitivities of photoreceptors largely depend on a high content of rhodopsin, a light-stimulated G protein-coupled receptor (GPCR), in light sensory organelles, outer segments in mammals and rhabdomeres in Drosophila. Two shared characteristics, the tightly packed photoreceptive membrane and the high concentration of rhodopsin in the membrane, work together to enable the photoreceptors to achieve the high content of rhodopsin in photosensory organelles in both mammals and Drosophila. In this thesis, I have used the Drosophilaeye as a model system to study the molecular mechanisms required for the maintenance of these two characteristics. In the second chapter, I present a new molecular mechanism of preventing Gq-mediated rhabdomeral degeneration. A new gene named tadr (for torn and diminished rhabdomeres), when mutated, leads to visual sensitivity reduction and photoreceptor degeneration. Degeneration in the tadr mutant is characterized by shrunken and disrupted rhabdomeres. The TADR protein interacts in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppresses the tadr-induced degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 fail to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses reveal that, in the tadr mutant, the Gq protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of Gq level by introducing a hypomorphic allele of Gαq gene greatly inhibits the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the Gqsignaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans. In the third chapter, I present a Drosophila CUB- and LDLa-domain transmembrane protein CULD that counteracts the visual arrestin Arr1-mediated endocytosis to retain rhodopsin in rhabdomeral membrane. CULD is mostly localized in rhabdomeres, but is also detected in scarce rhodopsin endocytic vesicles that contain Arr1. An intracellular region of CULD interacts with Arr1 in vitro. In both culdmutant and knockdown flies, a large amount of rhodopsin is mislocalized in the cell body of photoreceptors through lightdependent, Arr1-mediated endocytosis, leading to reduction of photoreceptor sensitivity. Expressing a wild-type CULD protein in photoreceptors, but not a mutant variant lacking the Arr1-interacting site, rescues both the rhodopsin mislocalization and the low sensitivity phenotypes. Once rhodopsin has been internalized in adult mutant flies, it is reversed only by expression of CULD but not by blocking endocytosis, suggesting that CULD promotes recycling of endocytosed rhodopsin to the rhabdomere. Our results demonstrate an important role of CULD in the maintenance of membrane rhodopsin density and photoreceptor sensitivity. We propose that a common cellular function of CUB- and LDLa-domain proteins, in both mammals and invertebrates, is to concentrate receptors including GPCRs in particular regions of cell membrane. In summary, the work addressed in this thesis has identified new molecular mechavii nisms underlying the maintenance of visual sensitivity in Drosophila, either through preventing Gq-mediated rhabdomeral degeneration or through antagonizing arrestin-mediated rhodopsin endocytosis. This work has advanced our understanding of visual biology and the general regulatory mechanisms of GPCR signaling, and may provide valuable clues to pathologic studies of human retinal degeneration disorders.