High resolution imaging of extracellular matrix formation in vertebrates

了解细胞外基质的生物学是所有多细胞动物发育和健康的基础。胶原蛋白是人体内最丰富的蛋白质，是支撑细胞和维持组织完整性的重要蛋白质支架。它是软骨和骨骼的重要组成部分。随着年龄的增长，皮肤弹性丧失，伤口愈合不良，骨关节炎和骨折的易感性增加变得普遍，其根本原因通常是受影响组织中胶原蛋白质量的降低。这些疾病中有许多都没有有效的治疗方法。相反，胶原蛋白的异常积累会导致纤维化，这是一种疤痕，与45%的死亡（包括癌症和心血管疾病）有关。最近的数据已经确定了昼夜节律在调节前胶原蛋白的合成和分泌中的关键作用。这已被证明直接影响早期分泌途径机制。这对我们理解这条通路是如何工作的，昼夜节律对基质形成的影响，以及对长期健康的影响有着深远的影响，例如，我们知道随着年龄的增长，生物钟会变得迟钝。在这里，我们开发了一个项目来定义关键细胞外基质蛋白的合成和分泌如何与功能性细胞外基质的形成相关联。我们从基质蛋白本身以及指导其合成和组装的机制的角度来研究这一点。最近的数据已经定义了这些过程的新调节器，包括昼夜节律。在这个项目中，我们建议使用基因组工程来敲除关键途径成分，然后定义ECM形成的结果。我们将针对早期分泌途径的蛋白质以及生物钟的关键驱动和调节因子。由这些细胞形成的基质将使用高分辨率成像技术进行分析，包括超分辨率光学显微镜、透射和罐头电子显微镜以及高速原子力显微镜。这些方法的整合为各种成像方法的培训提供了一个绝佳的机会，从技术实施到数据分析。该项目将主要在布里斯托尔的大卫·斯蒂芬斯实验室进行。WolfsonBioimaging Facility为大部分成像提供了核心技术平台。在迈克·艾伦教授的指导下，普利茅斯海洋实验室的高速原子力显微镜将进一步加强这一研究。

Understanding the biology of the extracellular matrix is fundamental to the development and health of all multicellularanimals. Collagen is the most abundant protein in the human body, forming a vital protein scaffold to support cellsand maintain tissue integrity. It is a critical component of cartilage and bone. As we age, loss of skin elasticity, poorwound healing, and an increased susceptibility to osteoarthritis and bone fractures become prevalent and theunderlying cause is usually a reduction in the quality of collagen in the affected tissues. There are no effectivetreatments for many of these diseases. Conversely, abnormal accumulation of collagen causes fibrosis, a type ofscarring, which is associated with 45% of all deaths (including those from cancer and cardiovascular disease). Recentdata have defined a key role for the circadian rhythm in regulating the synthesis and secretion of procollagen. This hasbeen shown to impact directly on the early secretory pathway machinery. This has profound implications for ourunderstanding of how this pathway works, the impacts of circadian rhythm on matrix formation, and the consequencesfor long term health, for example where we know the circadian clock becomes dampened as we age.Here, we have developed a project to define how the synthesis and secretion of key extracellular matrix proteins islinked to the formation of a functional extracellular matrix. We study this both from the perspective of the matrixproteins themselves as well as the machinery that directs its synthesis and assembly. Recent data have defined newregulators of these processes including the circadian rhythm. In this project we propose to use genome engineeringto knockout key pathway components to then define the outcomes on ECM formation. We will target proteins of theearly secretory pathway as well as key drivers and regulators of the circadian clock. The matrix formed by these cellswill then be analyzed using high resolution imaging technologies including super-resolution light microscopy,transmission and canning electron microscopy, and high-speed atomic force microscopy. The integration of theseapproaches presents a fantastic opportunity for training in diverse imaging methods, from technical implementationthrough to data analysis. The project will be based primarily in Bristol in the lab of David Stephens. The WolfsonBioimaging Facility provides the core technology platform for much of the imaging. This will be augmented by use ofthe high-speed atomic force microscopy at Plymouth Marine Laboratory under the direction of Professor Mike Allen.