Iterative Immunostaining and NEDD Denoising for Improved Signal-To-Noise Ratio in ExM-LSCM.

Expansion microscopy (ExM) has significantly reformed the field of super-resolution imaging, emerging as a powerful tool for visualizing complex cellular structures with nanoscale precision. Despite its capabilities, the epitope accessibility, labeling density, and precision of individual molecule detection pose challenges. We recently developed an iterative indirect immunofluorescence (IT-IF) method to improve the epitope labeling density, improving the signal and total intensity. In our protocol, we iteratively apply immunostaining steps before the expansion and exploit signal processing through noise estimation, denoising, and deblurring (NEDD) to aid in quantitative image analyses. Herein, we describe the steps of the iterative staining procedure and provide instructions on how to perform NEDD-based signal processing. Overall, IT-IF in ExM-laser scanning confocal microscopy (LSCM) represents a significant advancement in the field of cellular imaging, offering researchers a versatile tool for unraveling the structural complexity of biological systems at the molecular level with an increased signal-to-noise ratio and fluorescence intensity. Key features • Builds upon the method developed by Mäntylä et al. [1] and introduces the IT-IF method and signal-processing platform for several nanoscopy imaging applications. • Retains signal-to-noise ratio and significantly enhances the fluorescence intensity of ExM-LSCM data. • Automatic estimation of noise, signal reconstruction, denoising, and deblurring for increased reliability in image quantifications. • Requires at least seven days to complete.

The Effect of Substrate Stiffness on Elastic Force Transmission in the Epithelial Monolayers over Short Timescales.

Purpose: The importance of mechanical forces and microenvironment in guiding cellular behavior has been widely accepted. Together with the extracellular matrix (ECM), epithelial cells form a highly connected mechanical system subjected to various mechanical cues from their environment, such as ECM stiffness, and tensile and compressive forces. ECM stiffness has been linked to many pathologies, including tumor formation. However, our understanding of the effect of ECM stiffness and its heterogeneities on rapid force transduction in multicellular systems has not been fully addressed. Methods: We used experimental and computational methods. Epithelial cells were cultured on elastic hydrogels with fluorescent nanoparticles. Single cells were moved by a micromanipulator, and epithelium and substrate deformation were recorded. We developed a computational model to replicate our experiments and quantify the force distribution in the epithelium. Our model further enabled simulations with local stiffness gradients. Results: We found that substrate stiffness affects the force transduction and the cellular deformation following an external force. Also, our results indicate that the heterogeneities, e.g., gradients, in the stiffness can substantially influence the strain redistribution in the cell monolayers. Furthermore, we found that the cells' apico-basal elasticity provides a level of mechanical isolation between the apical cell-cell junctions and the basal focal adhesions. Conclusions: Our simulation results show that increased ECM stiffness, e.g., due to a tumor, can mechanically isolate cells and modulate rapid mechanical signaling between cells over distances. Furthermore, the developed model has the potential to facilitate future studies on the interactions between epithelial monolayers and elastic substrates. Supplementary Information: The online version of this article (10.1007/s12195-023-00772-0) contains supplementary material, which is available to authorized users.

Light-Induced Nanoscale Deformation in Azobenzene Thin Film Triggers Rapid Intracellular Ca2+ Increase via Mechanosensitive Cation Channels.

Epithelial cells are in continuous dynamic biochemical and physical interaction with their extracellular environment. Ultimately, this interplay guides fundamental physiological processes. In these interactions, cells generate fast local and global transients of Ca2+ ions, which act as key intracellular messengers. However, the mechanical triggers initiating these responses have remained unclear. Light-responsive materials offer intriguing possibilities to dynamically modify the physical niche of the cells. Here, a light-sensitive azobenzene-based glassy material that can be micropatterned with visible light to undergo spatiotemporally controlled deformations is used. Real-time monitoring of consequential rapid intracellular Ca2+ signals reveals that the mechanosensitive cation channel Piezo1 has a major role in generating the Ca2+ transients after nanoscale mechanical deformation of the cell culture substrate. Furthermore, the studies indicate that Piezo1 preferably responds to shear deformation at the cell-material interphase rather than to absolute topographical change of the substrate. Finally, the experimentally verified computational model suggests that Na+ entering alongside Ca2+ through the mechanosensitive cation channels modulates the duration of Ca2+ transients, influencing differently the directly stimulated cells and their neighbors. This highlights the complexity of mechanical signaling in multicellular systems. These results give mechanistic understanding on how cells respond to rapid nanoscale material dynamics and deformations.

Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina.

Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional-printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization-a prerequisite for studying intranuclear structural coregulation of cell function and fate.

Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients.

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell-derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches.

The retinal pigment epithelium displays electrical excitability and lateral signal spreading.

BACKGROUND: The non-neuronal retinal pigment epithelium (RPE) functions in intimate association with retinal photoreceptors, performing a multitude of tasks critical for maintaining retinal homeostasis and collaborating with retinal glial cells to provide metabolic support and ionic buffering. Accordingly, the RPE has recently been shown to display dynamic properties mediated by an array of ion channels usually more characteristic of astrocytes and excitable cells. The recent discovery of canonical voltage-activated Na+ channels in the RPE and their importance for phagocytosis of photoreceptor outer segments raises a question about their electrogenic function. Here, we performed a detailed electrophysiological analysis related to the functioning of these channels in human embryonic stem cell (hESC)-derived RPE. RESULTS: Our studies examining the electrical properties of the hESC-RPE revealed that its membrane mainly displays passive properties in a broad voltage range, with the exception of depolarization-induced spikes caused by voltage-activated Na+ current (INa). Spike amplitude depended on the availability of INa and spike kinetics on the membrane time constant, and the spikes could be largely suppressed by TTX. Membrane resistance fluctuated rapidly and strongly, repeatedly changing over the course of recordings and causing closely correlated fluctuations in resting membrane potential. In a minority of cells, we found delayed secondary INa-like inward currents characterized by comparatively small amplitudes and slow kinetics, which produced secondary depolarizing spikes. Up to three consecutive delayed inward current waves were detected. These currents could be rapidly and reversibly augmented by applying L-type Ca2+ channel blocker nifedipine to diminish influx of calcium and thus increase gap junctional conductance. CONCLUSIONS: This work shows, for the first time, that INa and INa-mediated voltage spikes can spread laterally through gap junctions in the monolayer of cells that are traditionally considered non-excitable. Our findings support a potential role of the RPE that goes beyond giving homeostatic support to the retina.

Pneumatic equiaxial compression device for mechanical manipulation of epithelial cell packing and physiology.

It is well established that mechanical cues, e.g., tensile- compressive- or shear forces, are important co-regulators of cell and tissue physiology. To understand the mechanistic effects these cues have on cells, technologies allowing precise mechanical manipulation of the studied cells are required. As the significance of cell density i.e., packing on cellular behavior is beginning to unravel, we sought to design an equiaxial cell compression device based on our previously published cell stretching system. We focused on improving the suitability for microscopy and the user-friendliness of the system. By introducing a hinge structure to the substrate stretch generating vacuum chamber, we managed to decrease the z-displacement of the cell culture substrate, thus reducing the focal plane drift. The vacuum battery, the mini-incubator, as well as the custom-made vacuum pressure controller make the experimental setup more flexible and portable. Furthermore, we improved the efficiency and repeatability of manufacture of the device by designing a mold that can be used to cast the body of the device. We also compared several different silicone membranes, and chose SILPURAN® due to its best microscopy imaging properties. Here, we show that the device can produce a maximum 8.5% radial pre-strain which leads to a 15% equiaxial areal compression as the pre-strain is released. When tested with epithelial cells, upon compression, we saw a decrease in cell cross-sectional area and an increase in cell layer height. Additionally, before compression the cells had two distinct cell populations with different cross-sectional areas that merged into a more uniform population due to compression. In addition to these morphological changes, we detected an alteration in the nucleo-cytoplasmic distribution of YAP1, suggesting that the cellular packing is enough to induce mechanical signaling in the epithelium.

Heterogeneity of Potassium Channels in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium.

Human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) is extensively used in RPE research, disease modeling, and transplantation therapies. For successful outcomes, a thorough evaluation of their physiological authenticity is a necessity. Essential determinants of this are the different ion channels of the RPE, yet studies evaluating this machinery in hPSC-RPE are scarce. We examined the functionality and localization of potassium (K+) channels in the human embryonic stem cell (hESC)-derived RPE. We observed a heterogeneous pattern of voltage-gated K+ (KV) and inwardly rectifying K+ (Kir) channels. Delayed rectifier currents were recorded from most of the cells, and immunostainings showed the presence of KV1.3 channel. Sustained M-currents were also present in the hESC-RPE, and based on immunostaining, these currents were carried by KCNQ1-KCNQ5 channel types. Some cells expressed transient A-type currents characteristic of native human fetal RPE (hfRPE) and cultured primary RPE and carried by KV1.4 and KV4.2 channels. Of the highly important Kir channels, we found that Kir7.1 is present both at the apical and basolateral membranes of the hESC- and fresh native mouse RPE. Kir currents, however, were recorded only from 14% of the hESC-RPE cells with relatively low amplitudes. Compared to previous studies, our data suggest that in the hESC-RPE, the characteristics of the delayed rectifier and M-currents resemble native adult RPE, while A-type and Kir currents resemble native hfRPE or cultured primary RPE. Overall, the channelome of the RPE is a sensitive indicator of maturity and functionality affecting its therapeutic utility.

Gap junctions and connexin hemichannels both contribute to the electrical properties of retinal pigment epithelium.

Gap junctions are intercellular channels that permit the transfer of ions and small molecules between adjacent cells. These cellular junctions are particularly dense in the retinal pigment epithelium (RPE), and their contribution to many retinal diseases has been recognized. While gap junctions have been implicated in several aspects of RPE physiology, their role in shaping the electrical properties of these cells has not been characterized in mammals. The role of gap junctions in the electrical properties of the RPE is particularly important considering the growing appreciation of RPE as excitable cells containing various voltage-gated channels. We used a whole-cell patch clamp to measure the electrical characteristics and connectivity between RPE cells, both in cultures derived from human embryonic stem cells and in the intact RPE monolayers from mouse eyes. We found that the pharmacological blockade of gap junctions eliminated electrical coupling between RPE cells, and that the blockade of gap junctions or Cx43 hemichannels significantly increased their input resistance. These results demonstrate that gap junctions function in the RPE not only as a means of molecular transport but also as a regulator of electrical excitability.

Cell maturation influences the ability of hESC-RPE to tolerate cellular stress.

BACKGROUND: Transplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina. The transplanted cells must tolerate cellular stress caused by various sources such as retinal inflammation and regain their functions rapidly after the transplantation. We have previously shown the maturation level of the cultured human embryonic stem cell-derived RPE (hESC-RPE) cells to influence for example their calcium (Ca2+) signaling properties. Yet, no comparison of the ability of hESC-RPE at different maturity levels to tolerate cellular stress has been reported. METHODS: Here, we analyzed the ability of the hESC-RPE populations with early (3 weeks) and late (12 weeks) maturation status to tolerate cellular stress caused by chemical cell stressors protease inhibitor (MG132) or hydrogen peroxide (H2O2). After the treatments, the functionality of the RPE cells was studied by transepithelial resistance, immunostainings of key RPE proteins, phagocytosis, mitochondrial membrane potential, Ca2+ signaling, and cytokine secretion. RESULTS: The hESC-RPE population with late maturation status consistently showed improved tolerance to cellular stress in comparison to the population with early maturity. After the treatments, the early maturation status of hESC-RPE monolayer showed impaired barrier properties. The hESC-RPE with early maturity status also exhibited reduced phagocytic and Ca2+ signaling properties, especially after MG132 treatment. CONCLUSIONS: Our results suggest that due to better tolerance to cellular stress, the late maturation status of hESC-RPE population is superior compared to monolayers with early maturation status in the transplantation therapy settings.

Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE.

Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.

Azobenzene-based sinusoidal surface topography drives focal adhesion confinement and guides collective migration of epithelial cells.

Surface topography is a key parameter in regulating the morphology and behavior of single cells. At multicellular level, coordinated cell displacements drive many biological events such as embryonic morphogenesis. However, the effect of surface topography on collective migration of epithelium has not been studied in detail. Mastering the connection between surface features and collective cellular behaviour is highly important for novel approaches in tissue engineering and repair. Herein, we used photopatterned microtopographies on azobenzene-containing materials and showed that smooth topographical cues with proper period and orientation can efficiently orchestrate cell alignment in growing epithelium. Furthermore, the experimental system allowed us to investigate how the orientation of the topographical features can alter the speed of wound closure in vitro. Our findings indicate that the extracellular microenvironment topography coordinates their focal adhesion distribution and alignment. These topographic cues are able to guide the collective migration of multicellular systems, even when cell-cell junctions are disrupted.

Analysis of ATP-Induced Ca2+ Responses at Single Cell Level in Retinal Pigment Epithelium Monolayers.

Calcium is one of the most important second messengers in cells and thus involved in a variety of physiological processes. In retinal pigment epithelium (RPE), Ca2+ and its ATP-dependent signaling pathways play important roles in the retina maintenance functions. Changes in intracellular Ca2+ concentration can be measured from living cells by Ca2+ imaging. Combining these measurements with quantitative analysis of Ca2+ response properties enables studies of signaling pathways affecting RPE functions. However, robust tools for response analysis from large cell populations are lacking. We developed MATLAB-based analysis tools for single cell level Ca2+ response data recorded from large fields of intact RPE monolayers. The analysis revealed significant heterogeneity in ATP-induced Ca2+ responses inside cell populations regarding magnitude and response kinetics. Further analysis including response grouping and parameter correlations allowed us to characterize the populations at the level of single cells.

Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium.

BACKGROUND: Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. RESULTS: Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Nav subtypes. Importantly, we show that the Nav channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process. CONCLUSIONS: Taken together, our study shows that Nav channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.

Viability of Mouse Retinal Explant Cultures Assessed by Preservation of Functionality and Morphology.

Purpose: Retinal explant cultures provide simplified systems where the functions of the retina and the effects of ocular therapies can be studied in an isolated environment. The purpose of this study was to provide insight into long-term preservation of retinal tissue in culture conditions, enable a deeper understanding of the interdependence of retinal morphology and function, and ensure the reliability of the explant technique for prolonged experiments. Methods: Retinal explants from adult mice were cultured as organotypic culture at the air-medium interface for 14 days in vitro (DIV). Retinal functionality was assessed by multielectrode array technique and morphology by immunohistochemical methods at several time points during culture. Results: Retinal explants retained viability for 14 DIV, although with diminishing neuronal activity, progressing neuronal loss, and increasing reactive gliosis. We recorded spontaneous retinal ganglion cell (RGC) activity up to 14 DIV with temporally changing distribution of RGC firing rates. Light responsiveness was measurable from RGCs for 7 DIV and from photoreceptors for 2 DIV. Apoptotic cells were detected beginning at 3 DIV with their density peaking at 7 DIV. The number of RGCs gradually decreased by 70% during 14 DIV. The change was accompanied by the loss of RGC functionality, resulting in 84% loss of electrically active RGCs. Conclusions: Retinal explants provide a valuable tool for studies of retinal functions and development of ocular therapies. However, critical for long-term use, retinal functionality was lost before structural loss, emphasizing a need for both functional and morphologic readouts to determine the overall state of the cultured retina.

Structural dynamics of tight junctions modulate the properties of the epithelial barrier.

Tight junctions are dynamic structures that are crucial in establishing the diffusion and electrical barrier of epithelial monolayers. Dysfunctions in the tight junctions can impede this barrier function and lead to many pathological conditions. Unfortunately, detailed understanding of the non-specific permeation pathway through the tight junctions, the so-called leak pathway, is lacking. We created computational models of the leak pathway to describe the two main barrier measures, molecular permeability and transepithelial electric resistance while using common structural dynamics. Our results showed that the proposed alternatives for the leak pathway, the bicellular strand opening dynamics and the tricellular pores, contribute together with distinct degrees, depending on the epithelium. The models can also capture changes in the tight junction barrier caused by changes in tight junction protein composition. In addition, we observed that the molecular permeability was markedly more sensitive to changes in the tight junction structure and strand dynamics compared with transepithelial electric resistance. The results highlight that our model creates a good methodological framework to integrate knowledge on the tight junction structure as well as to provide insights and tools to advance tight junction research.

Functional Voltage-Gated Calcium Channels Are Present in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) performs important functions for the maintenance of photoreceptors and vision. Malfunctions within the RPE are implicated in several retinal diseases for which transplantations of stem cell-derived RPE are promising treatment options. Their success, however, is largely dependent on the functionality of the transplanted cells. This requires correct cellular physiology, which is highly influenced by the various ion channels of RPE, including voltage-gated Ca2+ (CaV ) channels. This study investigated the localization and functionality of CaV channels in human embryonic stem cell (hESC)-derived RPE. Whole-cell patch-clamp recordings from these cells revealed slowly inactivating L-type currents comparable to freshly isolated mouse RPE. Some hESC-RPE cells also carried fast transient T-type resembling currents. These findings were confirmed by immunostainings from both hESC- and mouse RPE that showed the presence of the L-type Ca2+ channels CaV 1.2 and CaV 1.3 as well as the T-type Ca2+ channels CaV 3.1 and CaV 3.2. The localization of the major subtype, CaV 1.3, changed during hESC-RPE maturation co-localizing with pericentrin to the base of the primary cilium before reaching more homogeneous membrane localization comparable to mouse RPE. Based on functional assessment, the L-type Ca2+ channels participated in the regulation of vascular endothelial growth factor secretion as well as in the phagocytosis of photoreceptor outer segments in hESC-RPE. Overall, this study demonstrates that a functional machinery of voltage-gated Ca2+ channels is present in mature hESC-RPE, which is promising for the success of transplantation therapies. Stem Cells Translational Medicine 2019;8:179&15.

Effect of Rhodopsin Phosphorylation on Dark Adaptation in Mouse Rods.

UNLABELLED: Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that is activated when its 11-cis-retinal moiety is photoisomerized to all-trans retinal. This step initiates a cascade of reactions by which rods signal changes in light intensity. Like other GPCRs, rhodopsin is deactivated through receptor phosphorylation and arrestin binding. Full recovery of receptor sensitivity is then achieved when rhodopsin is regenerated through a series of steps that return the receptor to its ground state. Here, we show that dephosphorylation of the opsin moiety of rhodopsin is an extremely slow but requisite step in the restoration of the visual pigment to its ground state. We make use of a novel observation: isolated mouse retinae kept in standard media for routine physiologic recordings display blunted dephosphorylation of rhodopsin. Isoelectric focusing followed by Western blot analysis of bleached isolated retinae showed little dephosphorylation of rhodopsin for up to 4 h in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometeric determinations of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single-cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. We propose a model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires >3 h in complete darkness. SIGNIFICANCE STATEMENT: G-protein-coupled receptors (GPCRs) constitute the largest superfamily of proteins that compose ∼4% of the mammalian genome whose members share a common membrane topology. Signaling by GPCRs regulate a wide variety of physiological processes, including taste, smell, hearing, vision, and cardiovascular, endocrine, and reproductive homeostasis. An important feature of GPCR signaling is its timely termination. This normally occurs when, after their activation, GPCRs are rapidly phosphorylated by specific receptor kinases and subsequently bound by cognate arrestins. Recovery of receptor sensitivity to the ground state then requires dephosphorylation of the receptor and unbinding of arrestin, processes that are poorly understood. Here we investigate in mouse rod photoreceptors the relationship between rhodopsin dephosphorylation and recovery of visual sensitivity.

Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods.

Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(-/-) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo.

Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium.

In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.