Trafficking of procollagen in osteoblasts

在多细胞生物体中，细胞的分布、功能和存活依赖于细胞外基质（ECM）。合成和存款ECM（主要是胶原）的细胞是成纤维细胞。成骨细胞是成纤维细胞，其主要负责产生骨基质。成骨细胞一旦分化，分泌的胶原蛋白比其他ECM制造成纤维细胞多100倍。巨大的胶原蛋白细胞内运输必须发生在成骨细胞内完成这一任务，但这一领域仍然在很大程度上未被探索。我们的实验室是专门研究囊泡运输的蛋白质在活细胞中使用荧光显微镜。我们的目的是检查早期胶原（前胶原）通过内膜室传递到细胞表面，在细胞外沉积。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，指定成骨细胞中前胶原的递送和分泌。Rab GTP酶作为分子开关，决定囊泡在细胞中的递送位置。这些蛋白质从未在成骨细胞中研究过，我们将首先使用质谱结合蛋白质组学和基因表达谱的微阵列分析来筛选Rab GTP酶在分化的成骨细胞中表达。然后通过在成骨细胞中表达荧光嵌合蛋白和实时共聚焦成像，在体内监测表达的Rabs和前胶原。候选Rab蛋白将通过突变体Rab构建体的表达而下调，并且将使用免疫荧光观察前胶原运输。我们还将使用扫描和透射电子显微镜观察突变Rab表达对成骨细胞中前胶原加工的影响。这些研究将揭示成骨细胞功能的分子洞察力，并考虑到其在ECM生产中的关键作用，将引起细胞生物学家的广泛兴趣。

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.