Susan Bolch

Light-dependent translocation of arrestin in rod photoreceptors is signaled through a phospholipase C cascade and requires ATP

Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation. Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation. In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light. Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input.

Interaction of arrestin with enolase1 in photoreceptors.

PURPOSE Arrestin is in disequilibrium in photoreceptors, translocating between inner and outer segments in response to light. The purpose of this project was to identify the cellular component with which arrestin associates in the dark-adapted retina. METHODS Retinas were cross-linked with 2.5 mM dithiobis(succinimidylpropionate) (DSP), and arrestin-containing complexes purified by anion-exchange chromatography. Tandem mass spectrometric analysis was used to identify the protein components in the complex. Enolase localization in photoreceptors was assessed by immunohistochemistry. Confirmation of interacting components was performed using immunoprecipitation and surface plasmon resonance (SPR). Enolase activity was also assessed in the presence of arrestin1. RESULTS In retinas treated with DSP, arrestin cross-linked in a 125-kDa complex. The principal components of this complex were arrestin1 and enolase1. Both arrestin1 and -4 were pulled down with enolase1 when enolase1 was immunoprecipitated. In the dark-adapted retina, enolase1 co-localized with arrestin1 in the inner segments and outer nuclear layer, but remained in the inner segments when arrestin1 translocated in response to light adaptation. SPR of purified arrestin1 and enolase1 demonstrated direct binding between arrestin1 and enolase1. Arrestin1 modulated the catalytic activity of enolase1, slowing it by as much as 24%. CONCLUSIONS The results show that in the dark-adapted retina, arrestin1 and -4 interact with enolase1. The SPR data show that the interaction between arrestin1 and enolase1 was direct, not requiring a third element to form the complex. Arrestin1 slowed the catalytic activity of enolase1, suggesting that light-driven translocation of arrestin1 may modulate the metabolic activity of photoreceptors.

Light-dependent phosphorylation of Bardet–Biedl syndrome 5 in photoreceptor cells modulates its interaction with arrestin1

Arrestins are dynamic proteins that move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the light conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that Bardet–Biedl syndrome 5 (BBS5) is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions. Our principal findings in this study are threefold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.

Activation of Phospholipase C Mimics the Phase Shifting Effects of Light on Melatonin Rhythms in Retinal Photoreceptors

Many aspects of retinal photoreceptor function and physiology are regulated by the circadian clocks in these cells. It is well established that light is the primary stimulus that entrains these clocks; yet, the biochemical cascade(s) mediating light’s effects on these clocks remains unknown. This deficiency represents a significant gap in our fundamental understanding of photoreceptor signaling cascades and their functions. In this study, we utilized re-aggregated spheroid cultures prepared from embryonic chick retina to determine if activation of phospholipase C in photoreceptors in the absence of light can phase shift the melatonin secretion rhythms of these cells in a manner similar to that induced by light. We show that spheroid cultures rhythmically secrete melatonin and that these melatonin rhythms can be dynamically phase shifted by exposing the cultures to an appropriately timed light pulse. Importantly, we show that activation of phospholipase C using m-3M3FBS in the absence of light induces a phase delay in photoreceptor melatonin rhythms that mirrors that induced by light. The implication of this finding is that the light signaling cascade that entrains photoreceptor melatonin rhythms involves activation of phospholipase C.