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

Background Collagen fibrils are the primary supporting scaffolds of vertebrate tissues, but the mechanism of assembly is unclear. Methods Here, using CRISPR-tagging of type I collagen, high-resolution light imaging, and SILAC labelling, we elucidated the cellular mechanism underlying the spatiotemporal assembly of collagen fibrils in cultured fibroblasts. Results Our findings reveal the multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiated the processes of collagen secretion and fibril assembly and identified the crucial involvement of endocytosis in the regulation of fibril formation. By employing Col1a1 knockout fibroblasts, we demonstrated the incorporation of exogenous collagen into the nucleation sites at the plasma membrane through these recycling mechanisms. Conclusions Our study sheds light on a complex and previously unidentified collagen assembly process and its regulation of health and disease. Mass spectrometry data were available via ProteomeXchange with the identifier PXD036794.