Trafficking of procollagen in osteoblasts

在多细胞生物中，细胞的分布、功能和存活依赖于细胞外基质（ECM）。合成和沉积ECM的细胞，主要是胶原蛋白，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，其分泌的胶原蛋白是其他成纤维细胞的100倍。为了完成这一任务，成骨细胞内必须进行大量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。我们的目的是检查早期胶原（前胶原）通过膜室传递到细胞表面，并在细胞外沉积。我们的目标是了解前胶原运输背后的分子机制，特别是哪些Rab gtpase，指定了成骨细胞中前胶原的输送和分泌。Rab gtp酶作为分子开关，决定囊泡在细胞中的传递位置。这些蛋白从未在成骨细胞中研究过，我们将首先使用质谱法结合蛋白质组学和基因表达谱微阵列分析来筛选哪些Rab gtpase在分化成骨细胞中表达。然后通过在成骨细胞中表达荧光嵌合蛋白和活共聚焦成像来监测Rabs和前胶原蛋白的表达。候选Rab蛋白将通过突变Rab构建体的表达下调，前胶原运输将使用免疫荧光可视化。我们还将使用扫描电镜和透射电镜观察突变Rab表达对成骨细胞前胶原加工的影响。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产生中的关键作用，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.