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Latest Publications
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
NEW PUBLICATION!
Fine-scale molecular architecture is critical for nervous system and other biological functions. Methods to visualize these nanoscale structures would benefit from enhanced accessibility, 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 utilized the retina. We show that RAIN-STORM imaging is versatile and provide 3D nanoscopic data for over 20 synpase, 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 rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa
Michael A. Robichaux, Vy Nguyen, Fung Chan, lavanya 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 pigmentosa (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-T (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 slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant 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 adaptative 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.
