Biology subjects

Miner, J. H.

Publications and source records attributed to Miner, J. H..

4 recordsLinked to original sources

bioRxiv2021

Comparative Analysis and Rational Design of dCas9-VP64 Variants for CRISPR Activation

CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on VP64 have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.

synthetic biology↗

bioRxiv2021

Aminoglycoside-induced premature termination codon readthrough of COL4A5 nonsense mutations that cause Alport syndrome

Alport syndrome (AS) is characterized by glomerular basement membrane (GBM) abnormalities leading to progressive glomerulosclerosis. Mutations in the COL4A3, COL4A4 or COL4A5 genes encoding type IV collagen 345 cause AS. Truncated 3, 4, and 5 chains lacking an intact COOH-terminal noncollagenous domain due to a premature termination codon (PTC) cannot assemble into heterotrimers or incorporate into the GBM. Therefore, achieving full-length protein expression is a potential therapy for AS caused by truncating nonsense mutations. Small molecule-based PTC readthrough (PTC-RT) therapy has been well studied in other genetic diseases, but whether PTC-RT is applicable to AS is unexplored. To investigate the feasibility of PTC-RT therapy in AS, we made a cDNA to express COL4A5 fused to a C-terminal NanoLuc luciferase (NLuc) to monitor full-length translation. Full-length COL4A5-NLuc produces luminescence, but mutants truncated due to a PTC do not. To screen for COL4A5 nonsense mutants susceptible to PTC-RT, we introduced 49 individual nonsense mutations found in AS patients into the COL4A5-NLuc cDNA. Luciferase assays revealed that 11 mutations (C29X, S36X, E130X, C1521X, R1563X, C1567X, W1594X, S1632X, R1683X, C1684X and K1689X) were susceptible to PTC-RT induced by G418, which is known to have high readthrough activity. Moreover, we found that some next-generation "designer" PTC-RT drugs induced RT, and RT enhancer compounds increased the efficacy of PTC-RT in a G418-susceptible PTC mutant. These results suggest that PTC-RT therapy is a feasible approach for some patients with AS. Our luciferase-based COL4A5 translation reporter system will contribute to further development of PTC-RT therapies in a personalized medicine approach to treating AS.

genetics↗

bioRxiv2021

3D Visualization of the Podocyte Actin Network using Integrated Membrane Extraction, Electron Microscopy, and Deep Learning

Although actin stress fibers are abundant in cultured cells, little is known about these structures in vivo. In podocytes of the kidney glomerulus, much evidence suggests that mechanobiological mechanisms underlie injury, with changes to actin stress fiber structures potentially responsible for pathological changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. We therefore developed the first visualization technique capable of resolving the three-dimensional (3D) podocyte actin network with unprecedented detail in healthy and injured podocytes, and applied this technique to reveal the changes in the actin network that occur upon podocyte injury. Using isolated glomeruli from healthy mice as well as from three different mouse injury models (Cd2ap-/-, Lamb2-/- and the Col4a3-/- model of Alport syndrome), we applied our novel imaging technique that integrates membrane-extraction, focused ion bean scanning electron microscopy (FIB-SEM), and deep learning image segmentation. In healthy glomeruli, we observed actin cables that link the interdigitating podocyte foot processes to newly described actin structures located at the periphery of the cell body. The actin cables within the foot processes formed a continuous, mesh-like, electron dense sheet that incorporated the slit diaphragms required for kidney filtration. After injury, the actin network was markedly different, having lost its organization and presenting instead as a disorganized assemblage of actin condensates juxtaposed to the glomerular basement membrane. The new visualization method enabled us, for the first time, to observe the detailed 3D organization of actin networks in both healthy and injured podocytes. Shared features of actin condensations across all three injury models further suggested common mechanobiological pathways that govern changes to podocyte morphology after injury.

cell biology↗

bioRxiv2020

Mapping the molecular and structural specialization of the skin basement membrane for inter-tissue interactions

Inter-tissue interaction is fundamental to multicellularity. Although the basement membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue interactions remains poorly understood, mainly because the molecular and structural details of the BM at distinct inter-tissue interfaces remain unclear. By combining quantitative transcriptomics and immunohistochemistry, we systematically identify the cellular origin, molecular identity and tissue distribution of extracellular matrix molecules in mouse hair follicles, and reveal that BM composition and architecture are exquisitely specialized for distinct inter-tissue interactions, including epidermal-fibroblast, epidermal-muscle and epidermal-nerve interactions. The epidermal-fibroblast interface, namely, hair germ-dermal papilla interface, makes asymmetrically organized side-specific heterogeneity in BM, defined by the newly characterized interface, hook and mesh BMs. One component of these BMs, laminin 5, is required for the topological and functional integrity of hair germ-dermal papilla interactions. Our study highlights the significance of BM heterogeneity in distinct inter-tissue interactions.

developmental biology↗