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Cadherin-Dependent Regulation of Satellite Cell Function

卫星细胞功能的钙粘蛋白依赖性调节

基本信息

批准号：
10451802
负责人：
Robert S. Krauss
金额：
$ 54.72万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-15 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10451802
关键词：
Adult Axon Basal lamina Binding Proteins Biological Cadherins Cell Communication Cell physiology Cells Cellular Morphology Cellular biology Cytoskeleton Engraftment F-Actin Fluorescent Probes Genetic Genetic study Guanosine Triphosphate Phosphohydrolases Homeostasis Image In Vitro Injury Knowledge Maintenance Methodology Methods Microscopy Microtubules Minus End of the Microtubule Molecular Molecular Analysis Morphology Mus Muscle Muscle satellite cell Myoblasts Myopathy Natural regeneration Nature Neurons Play Preparation Procedures Process Proliferating Property Protocols documentation Regenerative Medicine Regenerative capacity Regulation Research Role Scanning Signal Transduction Signaling Protein Skeletal Muscle Structure Surface System Techniques Testing Therapeutic Therapeutic Agents Time Tissues base cell motility in vivo muscle aging muscle regeneration novel novel marker regeneration following injury repaired response satellite cell stem cell biology stem cells

项目摘要

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.

骨骼肌具有非凡的再生能力。这种能力来源于常驻肌干这些细胞被称为卫星细胞（SC）。在成人肌肉稳态期间，SC是静止的。一旦受伤， “激活”以产生用于肌肉修复的成肌细胞。SC位于肌纤维及其周围，丁基膜;这个小生境促进SC静止。适当调节静止是必要的，因为长- 长期SC功能和SC的成功植入。因此，了解利基如何促进静止对于利用SC用于治疗目的至关重要。然而，支持SC的机制静止和静止到激活（Q-to-A）的转变仍然知之甚少。在未受伤在体内肌肉中，SC具有长的突起。很少有人知道这些结构，因为它们是失去了在ESTA- 用于体外研究的SC的分离。我们已经开发出一种单一的肌纤维分离程序，可以保持- 的预测。QSC投射富含微管（MT），并被皮质F-肌动蛋白环绕，类似于神经元轴突我们的研究结果表明，SC投射是能动的，可能允许QSC扫描肌纤维表面的损伤和/或调节静止与激活的信号。撤回项目- 在SC激活时发生，并且是Q到A转变中的非常早期的步骤，先于其他已知的早期阶段。步钙粘蛋白是重要的影响因素，它参与了细胞的静止和Q-到-A的转换用于预测的维护。我们假设SC静止是一种动态的状态，其特征在于：由钙粘蛋白和细胞骨架调节的运动投射。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GT3，Rac 1; MT负端结合蛋白，CAMSAP 3;和 RhoA全球环境基金，LFC。我们将结合小鼠遗传学研究和细胞生物学研究来验证我们的假设。用单一肌纤维制备物进行研究。提出了以下目标：1）确定Rac 1的作用， CAMSAP 3和LFC对SC静止和Q到A转换的影响，我们将构建条件性缺乏CAMSAP 3和LFC的小鼠，这些因子在成体SC中的表达，并评估体内和单个肌纤维上的SC稳态、功能和结构准备与我们的新方法;和2）调查细胞骨架动力学收回SC的预测， Q-to-A过渡，我们将调整活成像协议，以我们的单肌纤维准备。SC-1小鼠 F-actin和MT动力学的荧光探针的特异性表达将与延时显微镜一起使用，内窥镜干扰钙粘蛋白、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。在肌肉治疗中利用SC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，但它们知之甚少。的所提出的目的是高度新颖的，并且涉及遗传和细胞生物学分析的协同组合。它们有望为SC生物学提供基础性的新信息。因此，预计它们将对SC作为再生医学中的治疗剂的潜在用途具有显著影响。