COLLAGEN FORMATION IN DEVELOPING MOUSE TENDON

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Kadler实验室专注于了解间充质细胞如何沉积由胶原原纤维的空间组织阵列组成的细胞外基质。我们最近报道了一种新的蛋白质分泌途径，即“纤维定位器途径”，胚胎肌腱成纤维细胞利用该途径将平行束的胶原原纤维沉积到细胞间通道。该通路的关键特征是在分泌通路的后期，前胶原（胶原的前体）在细胞内分裂成胶原，早期胶原原纤维在高尔基向质膜载体（GPCs）中组装，原纤维通过称为纤维定位器的特殊质膜结构出口。今年2月，dr。David Holmes， Toby Starborg和Karl Kadler参观了三维实验室，使用他们的电子断层扫描仪器来确定单个纤维支架和含有早期胶原原纤维的gpc的结构。在我们为期6天的访问中，我们收集了26个倾斜系列，从正交双轴倾斜系列生成层析图。我们能够收集到如此出色的数据，在很大程度上要归功于Eileen O'Toole博士的大力支持和科学投入，她操作显微镜并指导如何使用断层扫描软件。这些数据集包含了胶原原纤维束（包括纤维间丝）三维结构的新信息。这些数据集中的每一个都可能导致2005年和2006年的单独研究文章。这些数据是我们研究小组独有的。它们为我们提供了一个无与伦比的机会来发现细胞介导的细胞外基质合成的机制，以及细胞骨架在决定细胞形状和纤维支架组装中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Kadler laboratory is focused on understanding how mesenchymal cells deposit an extracellular matrix comprising spatially organized arrays of collagen fibrils. We recently reported on a novel protein secretion pathway, the "fibripositor pathway"that is utilized by embryonic tendon fibroblasts to deposit parallel bundles of collagen fibrils to intercellular channels. The key features of the pathway are intracellular cleavage of procollagen (the precursor of collagen) to collagen in the late secretory pathway, the assembly of early collagen fibrils in Golgi-to-plasma membrane carriers (GPCs) and export of the fibrils through specialized plasma membrane structures called fibripositors. In February of this year, Drs. David Holmes, Toby Starborg and Karl Kadler visited the 3-D lab to use their electron tomography instrument to determine the structure of single fibripositors and the GPCs that contain early collagen fibrils. During our 6-day visit we collected 26 tilt series to generate tomograms from orthogonal dual axis tilt series. Our ability to collect such excellent data was in large part attributable to the excellent support and scientific input we obtained from Dr. Eileen O'Toole, who operated the microscope and provided instruction on how to use the tomography software. These data sets contain new information on the 3D structure of collagen fibril bundles including interfibrillar filaments. Each of these data sets is likely to lead to separate research articles in 2005 and 2006. These data are unique to our research group. They provide us with an unrivalled opportunity to discover the mechanism of cell-mediated synthesis of the extracellular matrix, and the role of the cytoskeleton in determining cell shape and fibripositor assembly.