Cells and tissues are highly dependent on the secretion of extracellular matrix proteins like collagen to fulfil their roles in multicellular organisms and enable complex processes such as organ and bone formation during early development. Due to its unique structure, extensive post-translational modifications, requirement for fast abundant protein secretion during wound-healing processes and size of about 300 nm in length, type I procollagen (the precursor molecule of type I collagen) imposes high demand on the secretory pathway. Defects in collagen transport and secretion can lead to osteogenesis imperfecta, Ehlers-Danlos Syndrome, and fibrosis. Transport of proteins from the endoplasmic reticulum to the Golgi is facilitated via the COPII vesicle coat complex. These are typically considered to be spherical 60 – 80 nm diameter structures that bud from the endoplasmic reticulum membrane, encapsulate cargo proteins, and subsequently merge with the Golgi apparatus or, the endoplasmic reticulum-Golgi intermediate compartment. Despite having identified many factors involved in COPII organisation and regulation a major question in the field remains – how are large secretory cargo proteins like procollagen transported in a COPII-dependent manner through the early secretory pathway? Hypotheses include the use of large COPII megacarriers and direct tunnel-like connections. Here, I have used a new engineered GFP-tagged procollagen reporter, combined with experiments in primary fibroblasts, to characterise procollagen transport. In addition, fibroblasts from patients were investigated to help classify the pathogenicity of novel/rare mutations in genes involved in collagen processing and transport. GFP-procollagen was observed to be transported to the Golgi in absence of large carriers. Super resolution microscopy revealed small procollagen puncta overlapping, but not surrounded by, COPII. These data are consistent with models where large cargo proteins, including fibrillar collagens, are transported in a progressive manner in close proximity to Golgi membranes. This does not require large carriers, nor discrete cargo encapsulation by COPII.
|Date of Award||26 Nov 2020|
- The University of Bristol
|Supervisor||David J Stephens (Supervisor)|
- procollagen transport