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

Summary 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.