Compartment Of Photoreceptors Research Articles

Multimodal imaging of acute fovealitis with angular sign of Henle fiber layer hyperreflectivity (ASHH): Report of a case and review of the literature

Acute retinal pigment epitheliitis (ARPE) is a rare and transient macular disorder that was first described by Krill and Deutman in 1972. Later, Gerardo Ledesma-Gil and Spaide described a novel disease named acute fovealitis in 2022. According to them, this entity was different from ARPE on the basis of distinct features found in high-resolution optical coherence tomography (OCT) imaging. Angular sign of Henle fiber layer hyperreflectivity (ASHH) is a newly described terminology that is caused by an acute insult to photoreceptor compartments. In this article, we report a case that has both the ophthalmoscopic and OCT features of acute fovealitis as described in the literature, with the presence of ASHH in the acute stage, which resolves on its own rapidly along with the symptoms without any intervention.

Retinal Layer Separation (ReLayS) method enables the molecular analysis of photoreceptor segments and cell bodies, as well as the inner retina

Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method—a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases.

Photoreceptor Compartment-Specific TULP1 Interactomes

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High-Resolution Imaging Mass Spectrometry.

The human retina provides vision at light levels ranging from starlight to sunlight. Its supporting tissues regulate plasma-delivered lipophilic essentials for vision, including retinoids. The macula is an anatomic specialization for high-acuity and color vision that is also vulnerable to prevalent blinding diseases. The retina's exquisite architecture comprises numerous cell types that are aligned horizontally, yielding structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. MALDI imaging mass spectrometry (IMS) is now capable of uniting low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach fortified with accurate multi-image registration was used to localize lipids in human retina tissue at laminar, cellular, and subcellular levels. Multimodal imaging results indicate differences in distributions and abundances of lipid species across and within single cell types. Of note are distinct localizations of signals within specific layers of the macula. For example, phosphatidylethanolamine and phosphatidylinositol lipids were localized to central RPE cells, whereas specific plasmalogen lipids were localized to cells of the perifoveal RPE and Henle fiber layer. Subcellular compartments of photoreceptors were distinguished by PE(20:0_22:5) in the outer nuclear layer, PE(18:0_22:6) in outer and inner segments, and cardiolipin CL(70:5) in the mitochondria-rich inner segments. Several lipids, differing by a single double bond, have markedly different distributions between the central fovea and the ganglion cell and inner nuclear layers. A lipid atlas, initiated in this study, can serve as a reference database for future examination of diseased tissues.

Phosphatidylinositol 5-phosphate 4-kinase regulates early endosomal dynamics during clathrin-mediated endocytosis.

ABSTRACTEndocytic turnover is essential for the regulation of the protein composition and function of the plasma membrane, and thus affects the plasma membrane levels of many receptors. In Drosophila melanogaster photoreceptors, photon absorption by the G-protein-coupled receptor (GPCR) rhodopsin 1 (Rh1; also known as NinaE) triggers its endocytosis through clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5-positive compartment. The Rh1-trafficking defect in PIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting PIP4K in the PI3P-enriched early endosomal compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5-positive compartment in cultured Drosophila cells. Collectively, our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME.

Integrative subcellular proteomic analysis allows accurate prediction of human disease-causing genes.

Proteomic profiling on subcellular fractions provides invaluable information regarding both protein abundance and subcellular localization. When integrated with other data sets, it can greatly enhance our ability to predict gene function genome-wide. In this study, we performed a comprehensive proteomic analysis on the light-sensing compartment of photoreceptors called the outer segment (OS). By comparing with the protein profile obtained from the retina tissue depleted of OS, an enrichment score for each protein is calculated to quantify protein subcellular localization, and 84% accuracy is achieved compared with experimental data. By integrating the protein OS enrichment score, the protein abundance, and the retina transcriptome, the probability of a gene playing an essential function in photoreceptor cells is derived with high specificity and sensitivity. As a result, a list of genes that will likely result in human retinal disease when mutated was identified and validated by previous literature and/or animal model studies. Therefore, this new methodology demonstrates the synergy of combining subcellular fractionation proteomics with other omics data sets and is generally applicable to other tissues and diseases.

Pericentrin interacts with KASH domain-containing protein Syne-2

Objective Pericentrin, a highly conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Mutations in the human PCNT gene are associated with a range of diseases including primordial dwarfism and ciliopathies. In the mouse retina Pericentrin colocalizes with several proteins responsible for transport processes at the connecting cilium between the two photoreceptor compartments. In order to get more insights on the function of Pericentrin in the retina we try to identify new ciliary as well as centrosomal interaction partners.

Protein partners of dynamin-1 in the retina.

Dynamin proteins are involved in vesicle generation, providing mechanical force to excise newly formed vesicles from membranes of cellular compartments. In the brain, dynamin-1, dynamin-2, and dynamin-3 have been well studied; however, their function in the retina remains elusive. A retina-specific splice variant of dynamin-1 interacts with the photoreceptor-specific protein Tubby-like protein 1 (Tulp1), which when mutated causes an early onset form of autosomal recessive retinitis pigmentosa. Here, we investigated the role of the dynamins in the retina, using immunohistochemistry to localize dynamin-1, dynamin-2, and dynamin-3 and immunoprecipitation followed by mass spectrometry to explore dynamin-1 interacting proteins in mouse retina. Dynamin-2 is primarily confined to the inner segment compartment of photoreceptors, suggesting a role in outer segment protein transport. Dynamin-3 is present in the terminals of photoreceptors and dendrites of second-order neurons but is most pronounced in the inner plexiform layer where second-order neurons relay signals from photoreceptors. Dynamin-1 appears to be the dominant isoform in the retina and is present throughout the retina and in multiple compartments of the photoreceptor cell. This suggests that it may function in multiple cellular pathways. Surprisingly, dynamin-1 expression and localization did not appear to be disrupted in tulp1−/− mice. Immunoprecipitation experiments reveal that dynamin-1 associates primarily with proteins involved in cytoskeletal-based membrane dynamics. This finding is confirmed by western blot analysis. Results further implicate dynamin-1 in vesicular protein transport processes relevant to synaptic and post-Golgi pathways and indicate a possible role in photoreceptor stability.

Proteomic Profiling of a Layered Tissue Reveals Unique Glycolytic Specializations of Photoreceptor Cells

The retina is a highly ordered tissue whose outermost layers are formed by subcellular compartments of photoreceptors generating light-evoked electrical responses. We studied protein distributions among individual photoreceptor compartments by separating the entire photoreceptor layer of a flat-mounted frozen retina into a series of thin tangential cryosections and analyzing protein compositions of each section by label-free quantitative mass spectrometry. Based on 5038 confidently identified peptides assigned to 896 protein database entries, we generated a quantitative proteomic database (a "map") correlating the distribution profiles of identified proteins with the profiles of marker proteins representing individual compartments of photoreceptors and adjacent cells. We evaluated the applicability of several common peptide-to-protein quantification algorithms in the context of our database and found that the highest reliability was obtained by summing the intensities of all peptides representing a given protein, using at least the 5-6 most intense peptides when applicable. We used this proteome map to investigate the distribution of glycolytic enzymes, critical in fulfilling the extremely high metabolic demands of photoreceptor cells, and obtained two major findings. First, unlike the majority of neurons rich in hexokinase I, but similar to other highly metabolically active cells, photoreceptors express hexokinase II. Hexokinase II has a very high catalytic activity when associated with mitochondria, and indeed we found it colocalized with mitochondria in photoreceptors. Second, photoreceptors contain very little triosephosphate isomerase, an enzyme converting dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. This may serve as a functional adaptation because dihydroxyacetone phosphate is a major precursor in phospholipid biosynthesis, a process particularly active in photoreceptors because of the constant renewal of their light-sensitive membrane disc stacks. Overall, our approach for proteomic profiling of very small tissue amounts at a resolution of a few microns, combining cryosectioning and liquid chromatography-tandem MS, can be applied for quantitative investigation of proteomes where spatial resolution is paramount.

3D-Frap of PAGFP Reveals Inhomogeneity in Cytoplasmic Structures between the Major Rod Photoreceptor Compartments

Introduction: Diffusion of signaling proteins is thought to be essential for photoreceptor physiology, allowing, for example, regulation of the photoresponse through dynamic sequestration of key transduction proteins in either of the two major photoreceptor compartments, the inner and outer segments (IS and OS, respectively). The influence of the physical nature of the cytoplasm in these compartments on protein mobility is not known, but is essential for understanding photoreceptor function. We have thus developed a novel approach to quantifying protein mobility in 3D in live photoreceptors.Methods: Transgenic Xenopus laevis expressing the photoactivatable variant of GFP, PAGFP, exclusively in rod photoreceptors were generated using established methods. Live retinal slices were imaged with a custom-built multiphoton/confocal microscope. A source of photoactivated PAGFP was generated within selected compartments using multiphoton excitation at 820nm and its dissipation was monitored with 488nm confocal scanning. Images were compared with the output of a 3D diffusion model to estimate effective radial and axial diffusion coefficients.Results: PAGFP diffusion in the IS was isotropic and faster than in the OS, DIS = 5.2 μm2 s−1. In the OS PAGFP diffusion was anisotropic, with faster radial diffusion, DOS-radial = 3.5 μm2 s−1, and slower axial diffusion, DOS-axial = 0.19 μm2 s−1.Conclusion: PAGFP diffusion in both compartments was substantially retarded relative to aqueous solution, Daq,PAGFP ∼ 90 μm2 s−1, and cultured Chinese hamster ovary (CHO) cell cytoplasm, DCHO,PAGFP ∼ 20 μm2 s−1. Moreover, axial diffusion of PAGFP in the OS was hindered to a larger extent than expected from the geometry of disc membranes that span the compartment orthogonal to the cylinder axis. These results suggest that the photoreceptor cytoplasm possess higher density of cytoskeleton and/or macromolecules.

Analysis of Protein Expression and Compartmentalization in Retinal Neurons Using Serial Tangential Sectioning of the Retina

The progress in understanding visual signal transduction in vertebrate photoreceptors, arguably the best studied G protein-mediated signal transduction cascade in modern biology, was facilitated by the unique anatomy of rod photoreceptors. Held only by thin connected cilia, rod outer segments can be readily separated from the rest of the retina simply by shaking, and then purified by gradient centrifugation. The availability of such an efficient procedure of rod outer segment purification not only previously facilitated the identification of many principal visual signaling proteins located in this cellular compartment, but it is also currently being exploited in proteomics studies. In this paper, we describe a simple and inexpensive technique that allows for the quantitative analysis of protein expression within different subcellular compartments of photoreceptors, and could also be used for studying protein expression in the secondary retinal neurons. This technique is based on the Western blot analysis of the protein content of serial sections obtained by tangential sectioning of flat-mounted frozen retinas from mouse and rat, and it could serve as a way to validate proteomic data, similar to the way the quantitative RT-PCR technique is used for validation of gene-microarray data. To demonstrate the utility of this technique, we have determined the expression profiles in normal mouse retina of several signaling, energy-producing, and chaperone proteins, which were recently identified in bovine rod photoreceptors by mass spectrometry.

The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton

In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells.

Arrestin Translocation Is Induced at a Critical Threshold of Visual Signaling and Is Superstoichiometric to Bleached Rhodopsin

Light induces massive translocation of major signaling proteins between the subcellular compartments of photoreceptors. Among them is visual arrestin responsible for quenching photoactivated rhodopsin, which moves into photoreceptor outer segments during illumination. Here, for the first time, we determined the light dependency of arrestin translocation, which revealed two key features of this phenomenon. First, arrestin translocation is triggered when the light intensity approaches a critical threshold corresponding to the upper limits of the normal range of rod responsiveness. Second, the amount of arrestin entering rod outer segments under these conditions is superstoichiometric to the amount of photoactivated rhodopsin, exceeding it by at least 30-fold. We further showed that it is not the absolute amount of excited rhodopsin but rather the extent of downstream cascade activity that triggers translocation. Finally, we demonstrated that the total amount of arrestin in the rod cell is nearly 10-fold higher than previously thought and therefore sufficient to inactivate the entire pool of rhodopsin at any level of illumination. Thus, arrestin movement to the outer segment leads to an increase in the free arrestin concentration and thereby may serve as a powerful mechanism of light adaptation.

The dynamic range and domain-specific signals of intracellular calcium in photoreceptors

Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca2+ signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca2+ gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca2+ concentration within synaptic terminals of ∼2 μM and lower (∼750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca2+ enters the two compartments via both local L-type Ca2+ channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca2+ channels and plasma membrane Ca2+ ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca2+ homeostasis is controlled in a region-specific manner by direct Ca2+ entry and diffusion as well as Ca2+ extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca2+ signaling in photoreceptors is approximately 40-fold, from ∼50 nM in the light to ∼2 μM in darkness.

Transducin Activation State Controls Its Light-dependent Translocation in Rod Photoreceptors

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.

Identification of RanBP2- and kinesin-mediated transport pathways with restricted neuronal and subcellular localization.

Ran-binding proteins, karyopherins, and RanGTPase mediate and impart directionality to nucleocytoplasmic transport processes. This biological process remains elusive in neurons. RanBP2 has been localized at the nuclear pore complexes and is very abundant in the neuroretina. RanBP2 mediates the assembly of a large complex comprising RanGTPase, CRM1/exportin-1, importin-beta, KIF5-motor proteins, components of the 19S cap of the 26S proteasome, ubc9 and opsin. Here, we show RanBP2 is abundant in the ellipsoid compartment of photoreceptors and RanGTPase-positive particles in cytoplasmic tracks extending away from the nuclear envelope of subpopulations of ganglion cells, suggesting RanBP2's release from nuclear pore complexes. KIF5C and KIF5B are specifically expressed in a subset of neuroretinal cells and differentially localize with RanBP2 and importin-beta in distinct compartments. The C-terminal domains of KIF5B and KIF5C, but not KIF5A, associate directly with importin-beta in a RanGTPase-dependent fashion in vivo and in vitro, indicating importin-beta is an endogenous cargo for a subset of KIF5s in retinal neurons. The KIF5 transport pathway is absent from the myoid region of a topographically distinct subclass of blue cones and the distribution of kinesin-light chains is largely distinct from its KIF5 partners. Altogether, the results identify the existence of neuronal- and subtype-specific kinesin-mediated transport pathways of importin-beta-bound cargoes to and/or from RanBP2 and indicate RanBP2 itself may also constitute a scaffold carrier for some of its associated partners. The implications of these findings in protein kinesis and pathogenesis of degenerative neuropathies are discussed.