Molecular determinants of cell mechanics

细胞力学的分子决定因素

• 批准号: RGPIN-2020-04608
• 负责人: Hendricks, Adam
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744452
• 关键词: Molecular; determinants; cell; mechanics

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.

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，它们赋予细胞形状和结构。这些聚合物的组织随着机械和生化信号的变化而变化，使细胞能够迅速调整其力学，从高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来成像细胞骨架组织，控制蛋白质活性，并在纳米和皮牛顿尺度上测量与细胞生物学相关的细胞内力。为了量化活细胞的粘弹性，我们使用光学镊子在活细胞的细胞质中操纵微米大小的珠子。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。目的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纤维组成：微管、肌动蛋白和中间纤维。这些细胞骨架细丝相互作用支配着细胞力学。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的重要作用。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。微管刚性是由翻译后修饰和微管相关蛋白沿微管晶格结合而调节的。我们将使用新的光遗传学工具来调节微管翻译后修饰，并以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力学的分子机制。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）进行区隔。LLPS是许多细胞过程的核心，包括基因表达、神经变性、纺锤体组装和内吞作用。我们将(1)利用光遗传学触发细胞质中无膜细胞器的形成，并测量其对细胞力学特性的影响；(2)使用光镊探测相分离无膜细胞器的粘弹性行为，以揭示多种蛋白质之间微弱的多价相互作用如何控制组装和拆卸。总之，这个程序将提供一个机制上的理解，如何改变在单一的聚合物和相分离凝聚物的力学调整细胞的粘弹性特性，以响应化学，机械和发育线索。