COLLAGEN FORMATION IN DEVELOPING MOUSE TENDON

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。卡德勒实验室专注于了解间充质细胞如何沉积由空间组织的胶原纤维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“纤维载体途径”，胚胎肌腱成纤维细胞利用这种途径将平行的胶原束沉积到细胞间通道。该途径的关键特征是在分泌晚期将前胶原(胶原的前体)在细胞内裂解为胶原，早期胶原纤维在高尔基体到质膜载体(GPC)中组装，并通过称为纤维载体的特殊质膜结构输出纤维。今年2月，David Holmes博士、Toby Starborg博士和Karl Kadler博士访问了3D实验室，使用他们的电子断层扫描仪来确定单个纤维载体和含有早期胶原纤维的GPC的结构。在为期6天的访问中，我们收集了26个倾斜序列，从正交双轴倾斜序列生成断层图像。我们能够收集如此出色的数据，在很大程度上要归功于我们从艾琳·奥图尔博士那里获得的出色支持和科学投入，他操作着显微镜，并提供了如何使用断层成像软件的指导。这些数据集包含关于胶原原纤维束(包括纤维间细丝)的3D结构的新信息。这些数据集中的每一个都可能导致在2005年和2006年分别发表研究文章。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蛋白原组装中的作用。

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.