Mechanisms of Neural Stem Cell Mechanoregulation

项目总结/摘要 编码在干细胞微环境的结构、力学和维度中的生物物理学线索是 现在被认为是自我更新和分化的重要调节剂。在过去的15年里，包括 R 01支持的两个时期，我们一直在探索这种调节的机制和翻译方面， 海马神经干细胞（NSC），在成年期产生新的神经元， 神经疾病和修复。我们对该领域的理解做出了一些重要贡献， 干细胞机械生物学，包括发现NSC谱系决定对 细胞外基质（ECM）力学在一个有限的时间窗口，在此期间，刚度线索是 由包括RhoAGT β、肌动蛋白/肌球蛋白、血管动蛋白、雅普和β-连环蛋白的信号网络处理。 我们的发现提出了两个重要的问题，普遍感兴趣的干细胞领域，这将作为 为我们的续约申请奠定基础。首先，什么样的分子机制控制神经干细胞的机械敏感性谱系 三维（3D）ECM中的承诺，以及这些机制与二维（2D）有何不同 ECM？我们将建立在我们令人兴奋的最近发现，转录因子Egr 1是一个关键的，3D特异性的， NSC机械敏感谱系定型的调节因子。第二，干细胞如何动态整合 在几分钟到几小时的时间尺度上的机械输入，以触发功能上重要的信号事件？这里 我们将利用我们的初步研究，其中我们已经探测了机械敏感信号事件的时间， 使用错配的DNA交联粘弹性水凝胶和允许定时激活的光遗传学试剂 RhoA和Cdc 42的激活。我们有两个具体目标：在目标1中，我们将通过以下方式研究机制： 其中Egr 1使用2D和3D的组合控制3D矩阵中的机械敏感谱系定型， 工程生物材料，候选人为基础的分子研究，并筛选，以确定Egr 1目标相关 神经发生在目标2中，我们将研究2D ECM的刚度提示如何在几分钟内触发， 小时时间尺度在数小时至数天内积分以控制谱系承诺。这一目标的工作建立在 我们观察到RhoA的定时光遗传学刺激和粘性（损失）性质并入RhoA中， 弹性ECM均抑制NSC神经发生。我们将确定关键的监管时间尺度， 干扰，并确定它们是否通过共同的机制影响血统承诺。整合 目的，我们将研究三维ECM中机械敏感谱系承诺的时间依赖性，并询问是否 RhoA刺激和应力松弛通过Egr 1抑制神经发生。我们的工作将加速 菲尔德对干细胞如何感知并作用于机械信号以指导命运决定的理解， 具有很高的基础和翻译价值。我们还将结合几种创新方法，包括 光遗传学、粘弹性ECM、基因组编辑和测序/筛选技术。我们希望我们 这些研究将提供一个可应用于其他ECM和干细胞系统的智能路线图。

PROJECT SUMMARY/ABSTRACT Biophysical cues encoded in the structure, mechanics, and dimensionality of the stem cell microenvironment are now appreciated as important regulators of self-renewal and differentiation. For the past 15+ years, including two periods of R01 support, we have been exploring mechanistic and translational aspects of this regulation in hippocampal neural stem cells (NSCs), which generate new neurons into adulthood and contribute to neurological disease and repair. We have made several important contributions to the field’s understanding of stem cell mechanobiology, including the discovery that NSC lineage decisions are maximally sensitive to extracellular matrix (ECM) mechanics within a restricted temporal window, during which stiffness cues are processed by a signaling network that includes RhoA GTPase, actin/myosin, angiomotin, YAP, and β-catenin. Our discoveries raise two important questions of general interest to the stem cell field, which will serve as the foundation for our renewal application. First, what molecular mechanisms govern NSC mechanosensitive lineage commitment in three-dimensional (3D) ECMs, and how do these mechanisms differ from two-dimensional (2D) ECMs? We will build on our exciting recent discovery that the transcription factor Egr1 is a critical, 3D-specific regulator of NSC mechanosensitive lineage commitment. Second, how do stem cells dynamically integrate mechanical inputs on the time scale of minutes to hours to trigger functionally important signaling events? Here we will leverage our preliminary studies in which we have probed the timing of mechanosensitive signaling events with mismatched DNA-crosslinked viscoelastic hydrogels and optogenetic reagents that allow timed activation of RhoA and Cdc42 activation. We have two specific aims: In Aim 1, we will investigate mechanisms through which Egr1 controls mechanosensitive lineage commitment in 3D matrices using a combination of 2D and 3D engineered biomaterials, candidate-based molecular studies, and screens to identify Egr1 targets relevant to neurogenesis. In Aim 2, we will investigate how stiffness cues from 2D ECMs are triggered on the minutes-to- hours time scale are integrated over hours to days to control lineage commitment. Work in this aim builds on our observation that timed optogenetic stimulation of RhoA and incorporation of viscous (loss) properties into elastic ECMs both suppress NSC neurogenesis. We will identify critical regulatory time scales for both perturbations and determine if they influence lineage commitment through common mechanisms. To integrate aims, we will investigate the time-dependence of mechanosensitive lineage commitment in 3D ECMs and ask if RhoA stimulation and stress relaxation act through Egr1 to suppress neurogenesis. Our work will accelerate the field’s understanding of how stem cells sense and act upon mechanical signals to guide fate decisions, a problem of high fundamental and translational value. We will also marry several innovative approaches, including optogenetics, viscoelastic ECMs, genome editing, and sequencing/screening technologies. We expect that our studies will provide an intellectual roadmap that can be applied to other ECM and stem cell systems.