A quantitative in vitro collagen uptake assay.

Collagen remodelling is a vital process for embryonic development and homoeostatic maintenance of the adult body. Collagen remodelling is a complex process in fibroblasts, macrophages and other cells, whereby new collagen is secreted and polymerized into fibrils and old collagen is removed by proteolysis and endocytosis. Whereas the production of collagen is well-studied, the removal of collagen is less understood. In this protocol, we describe a method for the quantification of collagen uptake by cells. This protocol is based on the polymerisation of collagen type I-FITC conjugate in cell culture plate wells. Next, unpolymerized collagen is washed away and the cells are added in cell culture media. At this stage, they can be treated with inhibitors and/or stimulants if required. Afterwards, the cells are detached from the collagen using the protease accutase and the FITC signal is quantified using microscopy and/or flow cytometry.•Easy-to-use protocol for the quantitative measurement of collagen uptake in cells.•Cell detachment from collagen is quick and easy with accutase, even with strong adhering cells like macrophages.•Downstream applications can be a wide selection of analysis techniques like microscopy, RNA- and protein isolation, and flow cytometry.

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters.

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.