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

## TL;DR

This study reveals how collagen fibrils form at the surface of cells, showing that they grow from an intracellular collagen pool and are not continuously secreted.

## Contribution

The study identifies a rhythmic, intracellular collagen trafficking mechanism and differentiates collagen secretion from fibril assembly.

## Key findings

- Collagen fibrils grow at the plasma membrane from an intracellular collagen pool.
- Endocytosis regulates collagen fibril formation at the plasma membrane.
- Collagen trafficking defects are observed in cells from individuals with mucopolysaccharidosis.

## Abstract

Collagen fibrils are the primary supporting scaffolds of vertebrate tissues, but the mechanism of assembly is unclear.

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.

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.

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.

Fibrous tissue such as tendons, ligaments, skin and even the heart, contain millimetre long fibres that resist pulling forces and help maintain the shape and form of the tissue during everyday activities. The fibres are made of collagen protein molecules – by analogy, the collagen molecules are the building bricks in a wall or the steel rods in reinforced concrete. Studies of collagen fibres provide insights into a wide-spectrum of diseases associated with ageing and tissue degeneration. The fibres were seen using electron microscopes over 80 years ago but how they are formed was unknown, with several theories being proposed. In this study we tagged collagen molecules with probes that can be seen with light microscopes and observed, in real time, cells producing collagen and assembling the molecules into the fibres. We could visualise collagen molecules being synthesised within cells and being trafficked to the outer membrane (the plasma membrane) where the fibrils grew out from the cells and into the extracellular space. Therefore, the fibres grow on the surfaces of cells. We discovered that cells do not produce a continuous stream of collagen fibres but instead produce an intracellular pool of collagen that is ‘emptied’ on a rhythmic basis to make individual fibres. We looked at cells from people with mucopolysaccharidosis (a lysosome storage disorder) and showed that these cells were partially defective in starting new fibrils. This hints to how cells make the fibres, which could help medical scientists produce collagen-trafficking strategies to treat disease.