A complete collagen IV fluorophore knock-in toolkit reveals α-chain diversity in basement membrane

Abstract

The type IV collagen triple helix, composed of three ⍺-chains, is a core basement membrane (BM) component that assembles into a network within BMs. Endogenous tagging of all ⍺-chains with genetically encoded fluorophores has remained elusive, limiting our understanding of this crucial BM component. Through genome editing, we show that the C-termini of theC. eleganstype IV collagen ⍺-chains EMB-9 and LET-2 can be fused to a variety of fluorophores to create a strain toolkit with wild-type health. Using quantitative imaging, our results suggest a preference for LET-2-LET-2-EMB-9 trimer construction, but also tissue-specific flexibility in trimers assembled driven by differences in ⍺-chain expression levels. By taggingemb-9andlet-2mutants that model human Gould Syndrome, a complex multi-tissue disorder, we further discover defects in extracellular accumulation and turnover that might help explain disease pathology. Together, our findings identify a permissive tagging site that will allow diverse studies on type IV collagen regulation and function in animals. Summary Srinivasan et al., construct a collagen IV fluorophore knock-in toolkit inC. elegansusing a newly identified permissive genome editing site and reveal tissue-specific α-chain diversity and basement membrane turnover defects in collagen IV mutants modeling human COL4A1/A2 (Gould) syndrome.