THE PRIMARY CILIUM OF CONECTIVE TISSUE CELLS: INCIDENCE AND ORIENTATION

描述(申请人提供)：现在公认的是，在身体的大多数细胞上，存在一种修饰的、不活动的初级纤毛，作为感知细胞外环境的天线，无论是管状器官的管腔还是结缔组织的细胞外基质。初级纤毛最终传递刺激，导致基因表达，控制细胞的基本生物学反应。在过去的五年中，人们对初级纤毛作为全身上皮细胞的感觉器的作用有了显著的了解，最大的突破来自于发现了初级纤毛的某些异常，这些异常与特定的疾病有关，如幼儿的多囊肾病。 为了在理解初级纤毛在结缔组织细胞中的功能作用方面取得平行的进展，必须开发一种方法学，以便在体外和体内容易地检测初级纤毛的发生率和定位。在上皮细胞中，每个细胞的初级纤毛突出到器官的管腔或单层培养的表面。在这个表面位置，它的存在和方向可以通过实验操作后的光学显微镜技术来分析。原生纤毛存在于软骨细胞、腱细胞和其他结缔组织细胞中，但对其功能的实验分析仍然是一个重大的挑战，因为1)纤毛投射到细胞外基质中，以及2)许多结缔组织细胞在长期单层培养中不保持表型稳定。这项建议的目标是开发基于多光子显微镜成像的快速通用分析方法，以检查关节软骨、生长板、肌腱和椎间盘中初级纤毛的发生率、方向和分子结构。这一方法论将使结缔组织中初级纤毛的功能的探索与上皮组织中的初级纤毛的功能相媲美。 这一方法学的建立将为通过分析纤毛在体内探索结缔组织细胞初级纤毛的功能打开可能性：1)在患有软骨发育不良的小鼠模型中，被认为与细胞极性和组织结构的发育异常有关；2)在发育过程中，结缔组织的细胞和基质组织的各向异性正在建立；以及3)在活体动物实验中，涉及与特定关节相关的生物力学环境的操纵。快速评估初级纤毛的发生率和方向的能力将提供一种目前在分子水平上分析体外实验结果所缺乏的方法，以检验结缔组织细胞初级纤毛作为机械传感器的假设。

DESCRIPTION (provided by applicant): It is now well established that on most cells of the body there exists a modified, non-motile primary cilium that acts as an antenna sensing the extracellular environment, be it the lumen of a tubular organ or the extracellular matrix of connective tissues. The primary cilium ultimately transduces stimuli that result in gene expression controlling fundamental biological responses of the cell. In the past five years there has been a significant increase in the understanding to the role of the primary cilium as a sensory organelle in epithelial cells throughout the body, with the greatest breakthroughs coming from discovering certain abnormalities of the primary cilium that are linked to specific diseases, such as polycystic kidney disease in young children. For parallel advancements to be made in understanding the functional role of the primary cilium in cells of connective tissues, methodology must be developed that allows easy detection of primary ciliary incidence and orientation, in vitro and in vivo. In epithelia, the primary cilium of each cell projects into the lumen of the organ or to the surface of a monolayer culture. In this superficial position its presence and orientation can be analyzed by light microscopical techniques following experimental manipulation. Primary cilia have been described as present in chondrocytes, tenocytes, and other connective tissue cells, but experimental analysis of their function remains a significant challenge because 1) the cilium projects into the extracellular matrix, and 2) many connective tissue cells do not maintain phenotypic stability in long-term monolayer culture. The goal of this proposal is to develop rapid versatile analytical methods based on imaging using multiphoton microscopy to examine the incidence, orientation and molecular structure of the primary cilium in articular cartilage, growth plate, tendon and intervertebral disc. This methodology will put the exploration of the function of the primary cilium in connective tissues on par with that of its counterpart in epithelial tissues. Establishment of this methodology will open the potential for exploring the function of the primary cilium of connective tissue cells in vivo through analysis of the cilium 1) in mouse models with chondrodysplasias thought to be linked to abnormalities in development of cellular polarity and tissue organization; 2) during development when anisotropy of cell and matrix organization of connective tissues is being established; and 3) in experiments in living animals involving manipulation of the biomechanical environment associated with a specific joint. An ability to assess rapidly the incidence and orientation of the primary cilium will provide a methodology that currently is missing for analysis of results from in vitro experimentation at the molecular level to test the hypothesis that the primary cilium of connective tissue cells functions as a mechanosensor.