High resolution imaging of extracellular matrix formation in vertebrates

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Understanding the biology of the extracellular matrix is fundamental to the development and health of all multicellularanimals. Collagen is the most abundant protein in the human body, forming a vital protein scaffold to support cellsand maintain tissue integrity. It is a critical component of cartilage and bone. As we age, loss of skin elasticity, poorwound healing, and an increased susceptibility to osteoarthritis and bone fractures become prevalent and theunderlying cause is usually a reduction in the quality of collagen in the affected tissues. There are no effectivetreatments for many of these diseases. Conversely, abnormal accumulation of collagen causes fibrosis, a type ofscarring, which is associated with 45% of all deaths (including those from cancer and cardiovascular disease). Recentdata have defined a key role for the circadian rhythm in regulating the synthesis and secretion of procollagen. This hasbeen shown to impact directly on the early secretory pathway machinery. This has profound implications for ourunderstanding of how this pathway works, the impacts of circadian rhythm on matrix formation, and the consequencesfor long term health, for example where we know the circadian clock becomes dampened as we age.Here, we have developed a project to define how the synthesis and secretion of key extracellular matrix proteins islinked to the formation of a functional extracellular matrix. We study this both from the perspective of the matrixproteins themselves as well as the machinery that directs its synthesis and assembly. Recent data have defined newregulators of these processes including the circadian rhythm. In this project we propose to use genome engineeringto knockout key pathway components to then define the outcomes on ECM formation. We will target proteins of theearly secretory pathway as well as key drivers and regulators of the circadian clock. The matrix formed by these cellswill then be analyzed using high resolution imaging technologies including super-resolution light microscopy,transmission and canning electron microscopy, and high-speed atomic force microscopy. The integration of theseapproaches presents a fantastic opportunity for training in diverse imaging methods, from technical implementationthrough to data analysis. The project will be based primarily in Bristol in the lab of David Stephens. The WolfsonBioimaging Facility provides the core technology platform for much of the imaging. This will be augmented by use ofthe high-speed atomic force microscopy at Plymouth Marine Laboratory under the direction of Professor Mike Allen.