Molecular determinants of cell mechanics

我们的长期目标是确定细胞骨架如何控制细胞组织和力学。细胞骨架是一组自组装的动态聚合物，赋予细胞形状和结构。这些聚合物的组织响应于机械和生物化学信号而变化，使细胞能够快速调整其力学，从高度交联的弹性网络到弱交联的粘性网络。精确控制细胞的机械特性是细胞分裂、移动和分化形成组织和器官所必需的。 我们开发了单分子方法，用于成像细胞骨架组织，控制蛋白质活性，并测量与细胞生物学相关的纳米和皮牛顿尺度上的细胞内力。为了量化活细胞的粘弹性，我们使用光镊操纵活细胞细胞质中的微米级珠子。与此同时，我们使用单分子定位显微镜以纳米分辨率绘制细胞骨架的组织。 目标1.揭示微管细胞骨架如何控制细胞力学。细胞骨架由三种类型的细丝组成：微管、肌动蛋白和中间丝。这些细胞骨架丝相互作用以控制细胞力学。虽然该领域主要集中在肌动蛋白和中间丝，但最近的研究表明微管在控制心肌力学中起着重要作用。我们假设微管不仅在肌肉细胞中是细胞力学的关键调节器，而且这是许多细胞类型使用的一般机制。微管刚性受翻译后修饰和沿沿着微管晶格结合的微管相关蛋白调节。我们将使用新的光遗传学工具来调节微管翻译后修饰，并通过单分子分辨率量化对细胞骨架组织和力学的影响。目标2.剖析控制无膜细胞器组装和机械的分子机制。虽然许多细胞器通过膜与细胞质分离，但其他细胞器通过液-液相分离（LLPS）进行区室化。LLPS是许多细胞过程的中心，包括基因表达、神经变性、纺锤体组装和内吞作用。我们将（1）使用光遗传学触发细胞质中无膜细胞器的形成，并测量对细胞机械特性的影响，以及（2）使用光镊探测相分离的无膜细胞器的粘弹性行为，以揭示多个蛋白质之间的弱多价相互作用如何控制组装和拆卸。总之，该计划将提供对单一聚合物和相分离冷凝物的力学变化如何响应化学，机械和发育线索调整细胞的粘弹性的机械理解。

Our long-term objective is to determine how the cytoskeleton governs cellular organization and mechanics. The cytoskeleton is a set of self-assembling, dynamic polymers that give the cell its shape and structure. The organization of these polymers changes in response to mechanical and biochemical signals, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked, viscous networks. Precise control of the cell's mechanical properties is required for cells to divide, move, and differentiate to form tissues and organs. We develop single-molecule methods for imaging cytoskeletal organization, controlling protein activity, and measuring intracellular forces on the nanometer and pico-Newton scales relevant to cell biology. To quantify the viscoelasticity of living cells, we manipulate micron-sized beads in the cytoplasm of living cells using optical tweezers. In parallel, we use single-molecule localization microscopy to map the organization of the cytoskeleton with nanometer resolution. Aim 1. To uncover how the microtubule cytoskeleton controls cell mechanics. The cytoskeleton consists of three types of filaments: microtubules, actin, and intermediate filaments. These cytoskeletal filaments interact to govern cell mechanics. While the field has largely focused on actin and intermediate filaments, recent studies demonstrate an important role for microtubules in governing the mechanics of cardiac muscle. We hypothesize that microtubules are a key regulator of cell mechanics not only in muscle cells, but rather this is a general mechanism used by many cell types. Microtubule rigidity is modulated by post-translational modifications and microtubule-associated proteins that bind along the microtubule lattice. We will use new optogenetic tools to modulate microtubule post-translational modifications, and quantify the impact on cytoskeletal organization and mechanics with single-molecule resolution. Aim 2. To dissect the molecular mechanisms that govern the assembly and mechanics of membraneless organelles. While many cellular organelles are separated from the cytoplasm by a membrane, others compartmentalize through liquid-liquid phase separation (LLPS). LLPS is central to many cell processes including gene expression, neurodegeneration, spindle assembly, and endocytosis. We will (1) trigger the formation of membraneless organelles in the cytoplasm using optogenetics and measure the effect on cell mechanical properties, and (2) use optical tweezers to probe the viscoelastic behavior of phase-separated membraneless organelles to uncover how weak, multivalent interactions between multiple proteins control assembly and disassembly. Together, this program will provide a mechanistic understanding of how changes in the mechanics of single polymers and phase-separated condensates tune the viscoelastic properties of the cell in response to chemical, mechanical, and developmental cues.