Cadherin-Dependent Regulation of Satellite Cell Function

骨骼肌具有显著的再生能力。这种能力来源于常驻肌干。 细胞，称为卫星细胞(SC)。在成年肌肉动态期，干细胞处于静止状态。一旦受伤，他们就会 “激活”以产生成肌细胞，用于肌肉修复。SCs定位于肌纤维及其周围之间。 丁基膜；这一利基促进SC静止。对静止的适当调节对于长时间- 术语SC功能和SC的成功植入。因此，了解利基市场如何促进 静息是利用干细胞进行治疗的关键。然而，支撑SC的机制 静止和静止到激活(Q-to-A)的转变仍然知之甚少。在安然无恙 在活体肌肉中，干细胞有很长的投射。人们对这些结构知之甚少，因为它们是在制备过程中丢失的。 用于体外研究的干细胞移植。我们已经开发出一种单一的肌纤维分离程序，可以维持- 投影之差。QSC投射富含微管(MT)，皮质F-肌动蛋白环状，类似 神经元轴突。我们的发现表明SC投影是动态的，潜在地允许QSC扫描 肌纤维表面的损伤和/或调节静止和激活的信号。撤回项目- Ttions发生在SC激活时，并且是Q-A转换的非常早期的步骤，先于其他已知的早期 台阶。钙粘附素是重要的因素，它是静止和调节Q-A转换所必需的。 用于维护投影。我们假设SC静止是一种动态的状态，其特征是 受钙粘附素和细胞骨架调节的运动性投射。我们已经确定了新的因素作为候选 这一过程的调节因子包括GTPase rac1、MT负端结合蛋白CAMSAP3和 罗亚全球环境基金，利物浦。我们将结合小鼠的遗传学研究和细胞生物学来验证我们的假设。 关于单肌纤维制剂的研究。提出了以下目标：1)确定rac1的角色， CAMSAP3和LFC对SC静止和Q-A转换的影响，我们将构建条件缺失的小鼠 成年干细胞中的这些因素，并评估体内和单个肌纤维上的干细胞的动态平衡、功能和结构 用我们的新方法准备；和2)研究细胞骨架动力学的收缩SC投射在 在Q-to-A转换中，我们将使实时成像协议适用于我们的单一肌纤维制剂。患有SC-的小鼠 用于F-肌动蛋白和MT动力学的荧光探针的特定表达将用于延时显微 镜检术。干扰钙粘附素、细胞骨架和特定信号蛋白对这一初始步骤的影响 将确定SC激活的比例。在肌肉疗法中利用干细胞的能力需要详细的分子 以及SC静止和Q-to-A转换的细胞生物学知识，但对它们知之甚少。这个 提议的目标非常新颖，涉及遗传分析和细胞生物学分析的协同组合。 他们有望提供有关干细胞生物学的基本新信息。因此，预计他们将 对干细胞在再生医学中作为治疗剂的潜在使用有重大影响。

Skeletal muscle has remarkable capacity for regeneration. This capability derives from resident muscle stem cells, called satellite cells (SCs). During adult muscle homeostasis, SCs are quiescent. Upon injury, they are “activated” to generate myoblasts for muscle repair. SCs are localized between myofibers and their surround- ding basal lamina; this niche promotes SC quiescence. Proper regulation of quiescence is necessary for long- term SC function and for successful engraftment of SCs. Therefore, knowledge of how the niche promotes quiescence is critical to harnessing SCs for therapeutic purposes. However, the mechanisms that underlie SC quiescence and the quiescence-to-activation (Q-to-A) transition remain poorly understood. In uninjured muscles in vivo, SCs have long projections. Little is known of these structures, as they are lost during prepara- tion of SCs for study in vitro. We have developed a single myofiber isolation procedure that allows mainten- ance of projections. QSC projections are microtubule (MT)-rich and ringed with cortical F-actin, resembling neuronal axons. Our findings indicate that SC projections are motile, potentially allowing QSCs to scan the surface of the myofiber for damage and/or signals that regulate quiescence vs. activation. Retraction of project- tions occurs upon SC activation and is a very early step in the Q-to-A transition, preceding other known early steps. Cadherins, which are required for quiescence and regulate the Q-to-A transition, are important factors for maintenance of projections. We hypothesize that SC quiescence is a dynamic state, characterized by motile projections regulated by cadherins and the cytoskeleton. We have identified novel factors as candidate regulators of this process, including the GTPase, Rac1; the MT minus-end binding protein, CAMSAP3; and the RhoA GEF, LFC. We will test our hypotheses with a combination of genetic studies in mice and cell biological studies with single myofiber preparations. The following aims are proposed: 1) to determine the roles of Rac1, CAMSAP3, and LFC on SC quiescence and Q-to-A transition, we will construct mice conditionally lacking these factors in adult SCs and assess SC homeostasis, function, and structure in vivo and on single myofibers prepared with our new methods; and 2) to investigate cytoskeletal dynamics of retracting SC projections during the Q-to-A transition, we will adapt live imaging protocols to our single myofiber preparations. Mice with SC- specific expression of fluorescent probes for F-actin and MT dynamics will be employed with time-lapse micro- scopy. The effects of perturbing cadherins, the cytoskeleton, and specific signaling proteins on this initial step of SC activation will be determined. The ability to exploit SCs in muscle therapies requires detailed molecular and cell biological knowledge of SC quiescence and the Q-to-A transition, but they are poorly understood. The proposed aims are highly novel and involve a synergistic combination of genetic and cell biological analyses. They are expected to provide fundamental new information on SC biology. They are therefore anticipated to have a significant impact on the potential use of SCs as therapeutic agents in regenerative medicine.