Although light microscopy has been used to examine the early trafficking of collagen within the cell, much of our understanding of the detailed organisation of cell deposited collagen is from static electron microscopy studies. To understand the dynamics of live cell collagen deposition and fibril organisation, we generated a bright photostable mNGCol1α2 fusion protein and employed a range of microscopy techniques to follow its intracellular transport and elucidate extracellular fibril formation. Our findings reveal the dynamics of fibril growth and the dynamic nature of collagen network interactions at the cellular level. Notably we observed molecular events that build network organisation, including fibril bundling, bifurcation, directionality along existing fibrils, and looping/intertwining behaviours. Strikingly, mNGCol1α2 fluorescence intensity maxima can mark a fibril before another growing collagen fibril intersects at this location. Real-time, high-resolution imaging of collagen has enabled fibrillogenesis and organisational dynamics to be visualised together in an actively secreting cellular system. We also show that the N-terminal protease site is not an absolute requirement for collagen fibril incorporation. This approach paves the way for assessing the dynamic organisation and assembly of collagen into the extracellular matrix in skin models and other tissues during health, ageing and disease.

Collagen is the main component of the skin extracellular matrix and plays an integral role in supporting and maintaining the structure and biomechanical properties of the skin. There is increased interest to understand how collagen quality is controlled by the skin's fibroblast cells, particularly in aging, and to quantify how product formulations improve the appearance of aging skin. In this work, we have developed a new in vitro tool to visualize collagen in skin fibroblasts at high speed and high resolution using live cell imaging and super resolution microscopy. We have developed a bright mNeonGreen-collagen reporter that is efficiently expressed in fibroblasts and other cell types. The reporter molecule assembles into higher order structures, and is transported through the secretory pathway. We have used the system to track and calculate the in vitro movement of collagen through the cell. mNeonGreen-collagen can be harnessed to quantify the in vitro effects of peptides (eg, palmitoyl pentapeptide 4 as a well-established signaling peptide) and other antiaging materials on collagen quality control and secretion. The new tool will underpin our further understanding of the cell biology of collagen and the extracellular matrix in the skin.