Biology subjects

Calverley, B. C.

Publications and source records attributed to Calverley, B. C..

3 recordsLinked to original sources

bioRxiv2024

Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.

cell biology↗

bioRxiv2021

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2- DOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in- line with reported proteinuria and liver damage in patients with COVID-19. We identified 35 drugs that reduced viral replication in Vero and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

immunology↗

bioRxiv2020

Dynamic protein quantification (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLucIferase tagging

The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery but remains a major technical challenge. Here, we describe dynamic protein quantitation (DyProQ) which is effective across microscopy and multiwell platforms. Using collagen as a test protein, CRISPR-Cas9-mediated introduction of nluc (encoding NanoLuciferase, NLuc) into the Col1a2 locus enabled simplification and miniaturisation of procollagen-I (PC-I) quantitation. We robustly assessed extracellular, intracellular, and subcellular PC-I levels, by correlating to known concentrations of recombinant NLuc in the presence of substrate. Loss of collagen causes tissue degeneration whereas excess collagen results in fibrosis (often with poor-outcome) and is evident in aggressive cancers; however, treatment options are extremely limited. Using collagen-DyProQ, we screened a library of 1,971 FDA-approved compounds and identified 10 candidates for repurposing in the treatment of fibrotic and 7 for degenerative diseases.

cell biology↗