Trafficking of procollagen in osteoblasts

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The distribution, function and survival of cells is dependent on the extracellular matrix (ECM) in multicellular organisms. The cells that synthesize and deposit ECM, primarily collagen, are the fibroblasts. Osteoblasts are fibroblasts which are chiefly responsible for creating the bone matrix. Osteoblasts once differentiated, secrete one hundred times more collagen then other ECM-making fibroblasts. Enormous collagen intracellular trafficking must occur within the osteoblast to accomplish this task, yet this area remains largely unexplored. Our lab is specialized in studying vesicle trafficking of proteins in living cells using fluorescent microscopy. We aim to examine early collagen (procollagen) delivery through the endomembrane compartments to the cell surface where it is deposited extracellularly. Our goal is to understand the molecular mechanism behind procollagen trafficking, particularly which Rab GTPases, specify delivery and secretion of procollagen in osteoblasts. Rab GTPases act as molecular switches and dictate where vesicles are delivered in the cell. These proteins have never been studied in osteoblasts and we will first screen which Rab GTPases are expressed in differentiated osteoblasts using mass spectrometry coupled with proteomics and microarray analysis for gene expression profiling. Expressed Rabs and procollagen will then be monitored in vivo by expressing fluorescent-chimeric proteins in osteoblasts and live confocal imaging. Candidate Rab proteins will be down-regulated by expression of mutant Rab constructs and procollagen trafficking will be visualized using immunofluorescence. We will also use scanning and transmission electron microscopy to see the effect of mutant Rab expression on procollagen processing in osteoblasts. These studies will reveal molecular insight into osteoblast function, and given its critical role in ECM production, will be of broad interest to cell biologists.