Biology subjects

Arshavsky, V. Y.

Publications and source records attributed to Arshavsky, V. Y..

3 recordsLinked to original sources

bioRxiv2020

Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

biophysics↗

bioRxiv2020

Deletion of INPP5E in the murine retina impairs axoneme formation and prevents photoreceptor disc morphogenesis

INPP5E (pharbin) is a ubiquitously-expressed, farnesylated phosphatidylinositol polyphosphate 5-phosphatase which modulates the phosphoinositide composition of membranes. INPP5E resides in primary cilia, and mutations or loss of INPP5E are associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans, a syndromic ciliopathy affecting multiple tissues including brain, liver, kidney and retina. We show that, differing from other primary cilia, INPP5E is present in the wildtype photoreceptor inner segment and absent in the outer segment--a modified primary cilium dedicated to phototransduction. We generated Inpp5eF/F;Six3Cre (in short, retInpp5e-/-) mice which exhibit a rapidly progressing rod-cone degeneration nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. While P10 mutant outer segments contain phototransduction and structural proteins, they do not form axonemes and fail to elaborate disc membranes. Connecting cilia of retInpp5e-/- rods appear normal, although IFT-B/A particles accumulate at their distal ends suggesting disrupted intraflagellar transport. These results show that ablation of INPP5E does not impair the secretory pathway responsible for delivery of outer segment-specific proteins, but blocks axonemal extension and prevents disc morphogenesis.

neuroscience↗

bioRxiv2020

The GARP Domain of the Rod CNG Channel's β1-subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disc Rim

Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disc-shaped membranes. Disc membranes are separated from the surrounding plasma membrane and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane where it mediates the electrical response to light. We now show that the targeted delivery of the CNG channel to the outer segment requires pre-assembly of its constituent 1 and {beta}1 subunits and that CNG{beta}1 contains specific targeting information encoded within the glutamic acid-rich region of its N-terminal GARP domain. We also found that the GARP domain connects the CNG channel to photoreceptor disc rims likely through an interaction with peripherin-2 and demonstrated that this function is performed by a proline-enriched region adjacent to the GARP domain. Our data reveal fine functional specializations within the structural domains of the CNG channel and suggest that channel delivery to the outer segment is independent of peripherin-2 interactions. Significance StatementThe precise delivery and organization of signaling proteins in the ciliary outer segment organelle of photoreceptor cells is critical for light detection. We report that the CNG channel, mediating the electrical response to light in rods, contains a region within the N-terminus of its CNG{beta}1 subunit that encodes the outer segment targeting information for the entire channel. This targeting region is adjacent to a region that connects CNG{beta}1 to the rims of photoreceptor discs, likely determining the subcellular compartmentalization of the CNG channel into the outer segment plasma membrane.

cell biology↗