Trafficking of procollagen in osteoblasts

成骨细胞中前胶原的运输

• 批准号: 298538-2009
• 负责人: Harrison, Rene
• 金额: $ 2.33万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=548362
• 关键词: Trafficking; procollagen; osteoblasts

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.

细胞的分布、功能和生存依赖于多细胞生物中的细胞外基质(ECM)。合成和沉积细胞外基质(主要是胶原)的细胞是成纤维细胞。成骨细胞是主要负责形成骨基质的成纤维细胞。成骨细胞一旦分化，分泌的胶原蛋白比其他制造细胞外基质的成纤维细胞多一百倍。为了完成这一任务，成骨细胞内必须发生巨大的胶原蛋白细胞内运输，然而这一领域在很大程度上仍未被探索。我们的实验室专门使用荧光显微镜研究活细胞中蛋白质的囊泡运输。我们的目标是检查早期胶原(前胶原)通过内膜间隔输送到细胞表面，在那里它被沉积在细胞外。我们的目标是了解前胶原运输背后的分子机制，特别是Rab GTP酶在成骨细胞中指定前胶原的输送和分泌。RAB GTP酶起到分子开关的作用，决定囊泡在细胞内的传递位置。这些蛋白质在成骨细胞中从未被研究过，我们将首先利用质谱学结合蛋白质组学和基因表达谱芯片分析筛选哪些Rab GTP酶在分化的成骨细胞中表达。然后，将通过在成骨细胞中表达荧光嵌合蛋白和实时共聚焦成像来在体内监测表达的RABS和前胶原。候选的Rab蛋白将被突变的Rab结构表达下调，前胶原蛋白的运输将使用免疫荧光进行可视化。我们还将使用扫描和透射电子显微镜来观察突变的Rab表达对成骨细胞中的前胶原蛋白加工的影响。这些研究将揭示成骨细胞功能的分子洞察力，并鉴于其在细胞外基质产生中的关键作用，将引起细胞生物学家的广泛兴趣。