Super-resolution microscopy reveals a Rab6a-dependent trafficking hub for rhodopsin at the mammalian rod photoreceptor Golgi
Maryam Hekmatara, Samantha L Thompson, Kristen N Haggerty, Sydney Hagen, Brooke A Brothers, Bali Daniels, Guillaume Luxardi, Ala Moshiri, Wen-Tao Deng, Michael A Robichaux
Rod photoreceptor stability is critical for retinal health and lifelong vision. Rhodopsin (Rho) trafficking is essential for rod homeostasis, as its mislocalization precedes rod cell death in inherited retinal disorders such as retinitis pigmentosa. Despite its importance, the molecular mechanisms of Rho trafficking in mammalian rods remain largely undefined. We investigated Rho's subcellular organization in the mammalian rod Golgi complex. We utilized STORM and structured illumination microscopy super-resolution imaging to map Golgi proteins with Rho in mouse and macaque rods. Our analysis found that a large proportion of Rho in this subcellular region colocalizes with Rab6a in the trans-Golgi. To functionally test this interaction, we utilized a dominant-negative Rab6a mutant in HEK293T cells and mouse rods. The mutant significantly inhibits Rho secretion in cell culture, causing intracellular retention. In mouse rods, the mutant similarly causes significant trans-Golgi Rho retention; however, a majority of Rho protein still escaped the Golgi and reached the outer segment. Together, these findings uncover critical new subcellular details about Rho organization at the Golgi and establish a role for Rab6a as a regulator of Rho protein release from the trans-Golgi in mammalian rods. Our results provide critical insight into the protein trafficking mechanisms essential for long-term photoreceptor health.
P23H rhodopsin accumulation causes transient disruptions to synaptic protein levels in rod photoreceptors in a model of retinitis pigmentosa
Samantha L Thompson, Sophie M Crowder, Maryam Hekmatara, Emily R Sechrest, Wen-Tao Deng , Michael A Robichaux
Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segment (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. However, given the importance of protein trafficking to the OS, little is known about the trafficking of rod synaptic proteins. Furthermore, the subcellular impact of Rho mislocalization on rod synapses (i.e. 'spherules') has not been investigated. In this study, we used super-resolution and electron microscopies, along with proteomics, to perform a subcellular analysis of Rho synaptic mislocalization in P23H-Rho-RFP mutant mice. We discovered that mutant P23H-Rho-RFP protein mislocalized in distinct accumulations within the spherule cytoplasm, which we confirmed with adeno-associated virus overexpression. Additionally, we found specific synaptic protein abundance differences in P23H-Rho-RFP mice. Interestingly, in P23H knock-in mice with no RFP tag, we detected no synaptic protein abundance changes. In rd10 mutant rods, Rho mislocalized along the spherule plasma membrane, and there were synaptic protein abundance differences at postnatal day 20. Our findings demonstrate that some rod photoreceptor synaptic proteins are sensitive to Rho mislocalization.
Super-resolution mapping of rod photoreceptors identifies rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway
Kristen N. Haggerty, Shannon C. Eshelman, Lauren A. Sexton, Emmanuel Frimpong, Leah M. Rodgers, Melina A. Agosto, Michael A. Robichaux
Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient phototransduction and vision. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in mammalian rod inner segments remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecular localization analysis of rhodopsin in the inner segment of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane, at the surface, in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors.
Rapid 3D-STORM imaging of diverse molecular targets in tissue
Nicholas E Albrecht, Danye Jiang, viktor Akhanov, Robert Hobson, Colenso M Speer, Michael A Robichaux,
Melanie A Samuel
Fine-scale molecular architecture is critical for nervous system and other biological functions. Methods to visualize these nanoscale structure would benefit from enhanced accessibilty, throughput, and tissue compatibility. Here, we report RAIN-STORM, a rapid and scalable nanoscopic imaging optimization approach that improved three-dimensional visualization for subcellular targets in tissue at depth. RAIN-STORM uses conventional tissue samples and readily available reagents and is suitable for commercial instrumentation. To illustrate the efficacy of RAIN-STORM, we utilize the retina. We show that RAIN-STORM imaging is versatile and provide 3D nanoscopic data for over 20 synapse, neuron, glia, and vasculature targets. Sample preparation is also rapid, with a 1-day turnaround from tissue to image, and parameters are suitable for multiple tissue sources. Finally, we show that this method can be applied to clinical samples to reveal nanoscale features of human cells and synapse. RAIN-STORM thus paves the way for high-throughput studies of nanoscopic targets in tissue.
Subcellular localization of mutant P23H rhodospin in an RFP fusion knockin mouse model of retinitis pigmentosa
Michael A. Robichaux, Vy Nguyen, Fung Chan, Iavanya Kailasam, Feng He, John H. Wilson, Theodore G. Wensel
The P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal dominant retinitis pigmentsa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has not yet been elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowlly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA levels for markers for ER stress and the unfolded protein response revealed little to no increase. mRNA levels for both the mutant and human rhodopsin allele and the WT mouse rhodopsin levels were reduced, but protein levels revealed selective degradation of the mutant protein. The results suggest the mutant rods undergo an adaptive process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.
Superresolution microscopy reveals photoreceptor-specific subciliary localion and function of ciliopathy-associated protein, CEP290
Valencia L. Potter, Abigail R. Moye, Michael A. Robichaux, and Theodore G. Wensel
Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multi-organ ciliopathies or as retina-specific disease. The precise locaition and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used superresolution fluorescnce microscopy and electron microscopy (TEM) to localize CEP290 in the CC and in primary cilia of cultured cells with sub-diffraction resolution, and to determine effects of CEP290 deficiency in three mutant models. Radially, CEP290 localizes in close proximity to the mircotubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in hRPE-1 cells. We found Y-shaped links, ciliary cub-structures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliar components in early stages of degeneration. There were fewer cilia and no normal outer segment in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.
Structure and dynamics of photoreceptor sensory cilia
Theodore G Wensel, Valenicia L Potter, Abigail Moye, Zhixian Zhang, Michael A Robichaux
The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optomized spatial and temporal resolution. Most prominent are thier unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the pirmary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of material moves on a daily basis to sustain the continual turnover of outer segment discs and the energetic demands of phototransduction. After decades of study, the details of the fine structures and distribution of molecular components of these structures are still incompletely understoof, but recent advances in cellular imaging techniques and animal models of inherited ciliary defects are yielding important new insights. This knowledge informs our understanding both of the mechanisms of trafficking and assembly and of the pathophysiological mechanisms of human blinding ciliopathies.