A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina.

Daily mitochondrial dynamics in cone photoreceptors.

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover.

Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca uptake in photoreceptor mitochondria.

Rods and cones use intracellular Ca to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu zebrafish and a rod photoreceptor-specific Mcu mouse.

Increasing Ca in photoreceptor mitochondria alters metabolites, accelerates photoresponse recovery, and reveals adaptations to mitochondrial stress.

Photoreceptors are specialized neurons that rely on Ca to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca homeostasis is disrupted. Ca homeostasis is maintained partly by mitochondrial Ca uptake through the mitochondrial Ca uniporter (MCU), which can influence cytosolic Ca signals, stimulate energy production, and trigger apoptosis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu.

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs.

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology.

Mitochondria Maintain Distinct Ca Pools in Cone Photoreceptors.

Ca ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca domains are maintained by Ca uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment.

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption.

Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin.

Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina.

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain.

Conformation of receptor-bound visual arrestin.

Arrestin-1 (visual arrestin) binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate G-protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin-1 and double electron-electron resonance was used to monitor interspin distance changes upon P-Rh* binding. The results indicate that the relative position of the N and C domains remains largely unchanged, contrary to expectations of a "clam-shell" model.

Progressive reduction of its expression in rods reveals two pools of arrestin-1 in the outer segment with different roles in photoresponse recovery.

Light-induced rhodopsin signaling is turned off with sub-second kinetics by rhodopsin phosphorylation followed by arrestin-1 binding. To test the availability of the arrestin-1 pool in dark-adapted outer segment (OS) for rhodopsin shutoff, we measured photoresponse recovery rates of mice with arrestin-1 content in the OS of 2.5%, 5%, 60%, and 100% of wild type (WT) level by two-flash ERG with the first (desensitizing) flash at 160, 400, 1000, and 2500 photons/rod.

Robust self-association is a common feature of mammalian visual arrestin-1.

Arrestin-1 binds light-activated phosphorhodopsin and ensures rapid signal termination. Its deficiency in humans and mice results in prolonged signaling and rod degeneration. However, most of the biochemical studies were performed on bovine arrestin-1, which was shown to self-associate forming dimers and tetramers, although only the monomer binds rhodopsin.

Arrestin mobilizes signaling proteins to the cytoskeleton and redirects their activity.

Arrestins regulate the activity and subcellular localization of G protein-coupled receptors and other signaling molecules. Here, we demonstrate that arrestins bind microtubules (MTs) in vitro and in vivo. The MT-binding site on arrestins overlaps significantly with the receptor-binding site, but the conformations of MT-bound and receptor-bound arrestin are different.