High-resolution imaging and time-resolved proteomic profiling of COPII-dependent procollagen packaging.

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Collagen is the most abundant protein in our bodies. It is also one of the largest. This size presents a significant problem to cellular machinery involved in shipping procollagen from its site of synthesis (inside the endoplasmic reticulum of cells) to its site of action in the extracellular matrix. The machinery that controls the first step in this pathway normally generates transport carriers that are simply too small to accommodate procollagen. Nearly all cell types secrete one or more types of collagen and this process is absolutely essential during development to provide the extracellular matrix within which cells migrate to their final position and differentiate. It is also essential for those very differentiation processes. Later in life ongoing secretion of collagen is vital for tissues to maintain themselves by renewal and regeneration. Major pathologies are also associated with defects in collagen secretion including fibrosis. The field of stem cell biology increasingly relies on accurate reproduction of the "stem cell niche", of which the cohort of secreted collagens is a major component. The development and exploitation of engineered cells and tissue for therapeutic benefit will in many cases rely on a complete understanding of these processes. Consequently it is perhaps surprising how little we know about this process. The machinery that directs the first step in the secretion of small proteins (for which a 2013 Nobel Prize was awarded) has been very well characterised. This machinery generates small (60-80 nanometre) transport vesicles that are not sufficient to encapsulate the ~300 nanometre size of collagen molecules. While there have been some advances in this area, they either relate to specific collagen types (such as collagen VII) or are underdeveloped in terms of our understanding (such as modification of the core machinery using a small protein called ubiquitin). Our work aims to use a combination of cutting-edge imaging and proteomics methods to precisely define this machinery, and the nature of these transport carriers exactly at the point of procollagen export. Importantly we will focus on two major procollagen isotypes - I and II to provide insight into the pathway for these forms that are of major clinical interest. We have generated new tools to examine this process with unprecedented spatial and temporal precision. As such we anticipate making major advances in this important field.