mNeonGreen-collagen1α2: a tool for real time, high resolution, visualisation of collagen I trafficking

Abstract

Collagen I is a fibrillar protein of the extracellular matrix, where it provides strength and support to a number of connective tissues. Collagen I is produced by the cells of connective tissues as a short procollagen peptide, which self-assembles into fibrils upon secretion. Despite its important role in the skin, there are still gaps in our understanding of the intracellular trafficking and extracellular fibril assembly of collagen I. The highly dynamic nature of collagen I trafficking makes light microscopy the best tool to further investigate this process.
In this thesis, an mNeonGreen-collagen1α2 fusion protein will permit live imaging of collagen I in real time and with a higher resolution than previously achieved within the field. Following design of the construct, the expression of mNeonGreen-collagen1α2 was characterised within three immortalised cell lines of interest and extensively controlled for. mNeonGreen-collagen1α2 successfully permitted imaging all stages of collagen I trafficking: from synthesis, through the cell secretory pathway, and fibril assembly in the extracellular matrix. mNeonGreen-collagen1α2 was also used to investigate biological and cosmetic questions within the field. Overall showing that mNeonGreen-collagen1α2 is a robust, replicable and biologically relevant tool to study collagen I trafficking in real time with high resolution.