CAREER: Biomechanics of Polymerization Motors and Cell Motility

许多细胞的运动和形状变化取决于细胞内部产生和控制机械力的能力。定向的细胞运动是维持健康和在体内传播疾病的基础。细胞骨架蛋白肌动蛋白聚合成细丝网络--就像充电时线性压电致动器的扩张--产生的指向力延长了细胞的前沿，并在引导细胞运动方面发挥了基础性作用。这份职业提案关注的是细胞运动性中的一个关键生物力学问题：肌动蛋白聚合，有时被描述为聚合马达，是如何产生力量的？回答这个问题将有助于揭示细胞运动的机械机制及其在疾病中的作用。未来机械测量技术的发展可能导致针对特定机械过程的药物发现的新策略，以及基于生物构件的新型传感器和执行器。拟议的研究计划通过(I)仪器开发、(Ii)体外实验和(Iii)细胞运动性建模和测量来研究聚合马达的生物力学。原子力显微镜是一种越来越受欢迎的细胞生物力学研究工具，但商业仪器不适合在生物相关的时间尺度上测量聚合力。这项工作开发了一种差分力显微镜技术，可以直接测量肌动蛋白聚合速率和失速力。使用基于细菌病原体单核细胞增多性李斯特菌的体外运动系统，实验将确定成核面几何形状和辅助蛋白浓度对聚合动力学的影响。最后，实验结果将与现有的基于肌动蛋白的运动模型的预测进行比较，并用于研究正常细胞和癌细胞的细胞运动速度和力的差异。拟议的教育计划通过(I)课程开发、(Ii)机械性能数据库和(Iii)动手实验，为当前和未来的细胞运动力学研究人员提供学习机会。PI将在加州大学伯克利分校生物工程系开发和教授一门关于分子力学的新课程，该课程强调蛋白质的机械特性在细胞运动和疾病传播中的作用。将建立一个基于网络的蛋白质和其他大分子的机械性能数据库，为该领域的研究人员提供目前已知的机械性能的有组织的摘要，用于运动学模型和比较。为了激励下一代科学家和工程师，PI将与本科生团体合作，为加利福尼亚州奥克兰的小学生创建动手显微镜实验，通过单细胞有机体的显微镜活动向他们介绍细胞运动。

0348758FletcherMany cell movements and shape changes depend on the cell's ability to internally generate and control mechanical forces. Directed cell movement is fundamental to both maintenance of health and spread of diseases within the body. Polymerization of the cytoskeletal protein actin into networks of filaments - like the expansion of a linear piezoelectric actuator upon charging - creates directed forces that extend the cell's leading edge and play a fundamental role in guiding cell motility. This CAREER proposal focused on a key biomechanical question in cell motility: how does actin polymerization, sometimes described as a polymerization motor, generate force? Answering this question will help reveal the mechanical mechanisms underlying cell motility and its role in disease.. Future development of mechanical measurement techniques for studying force-generating cellular "subsystems" may lead to new strategies for drug discovery that target specific mechanical processes and novel sensors and actuators based on biological building blocks. The proposed research plan investigates the biomechanics of polymerization motors through (i) instrument development, (ii) in vitro experimentation, and (iii) cell motility modeling and measurement. Atomic force microscopy is an increasingly popular tool for biomechanical studies of cells, but commercial instruments are not appropriate for measuring polymerization forces over biologically relevant timescales. This work develops a differential force microscopy technique to directly measure actin polymerization rates and stall forces. Using an in vitro motility system based on the bacterial pathogen Listeria monocytogenes, experiments will determine the effect of nucleation surface geometry and accessory protein concentration on polymerization dynamics. Finally, results from the experiments will be compared to predictions from existing models of actin-based motility and used to investigate differences in the cell motility speeds and forces of normal and cancerous cells. The proposed education plan provides learning opportunities for current and future researchers in the mechanics of cell movement through (i) course development, (ii) a mechanical property database, and (iii) hands-on experiments. The PI will develop and teach a new course on the mechanics of molecules in the Bioengineering Department at UC Berkeley that emphasizes the role of mechanical properties of proteins in cell movements and the spread of disease. A web-based database of the mechanical properties of proteins and other macromolecules will be created to provide researchers in the field with an organized summary of the currently known mechanical properties for use in motility models and comparisons. To motivate the next generation of scientists and engineers, the PI will work with undergraduate student groups to create hands-on microscopy experiments for Oakland, CA elementary school children introducing them to cell movements through microscopy activities with single-celled organisms.