Dynamics Control and Analysis of Biopolymer Networks

描述(由申请人提供)： 这个项目的目标是开发新的技术，利用全息激光镊子阵列来分析和控制生物聚合物网络结构，这是一种功能强大的新仪器，其潜力才刚刚开始探索。该工具将允许对动态生物聚合物网络中的力进行复杂的空间分布扰动和测量。虽然这些技术将在肌动蛋白上开发，但它们应该适用于研究形成生物分子的广泛网络中的力和前沿不稳定性。网络对扰动的响应应该揭示聚合前沿动力学与网络力学性质之间的相互关系。了解这种相互关系对于理解各种细胞骨架蛋白的聚合动力学如何影响细胞功能至关重要，例如，在与细胞流动性相关的形态变化中。了解和控制细胞的迁移率对人类健康的许多方面都有影响，从促进损伤后的神经再生到降低转移癌细胞的迁移率。 具体目标1：使用全息镊子阵列控制亚微米分辨率的肌动蛋白聚合前沿。通过加热或通过影响聚合的分子的局部取消，聚合前沿的空间扩展扰动将被引入镊子阵列。从聚合前沿对扰动的响应，研究人员可以提取特征长度尺度和时间尺度，并研究不稳定性。 具体目标2：开发使用全息镊子阵列测量力的空间分布的技术。皮科·牛顿力将用被捕获的珠子和被捕获的延伸物体来测量。他们将测量由肌动蛋白聚合前锋产生的力以及这些力之间的相关性。使用这项技术，他们将确定聚合力如何取决于前锋的形状、生长速度和其他参数。 具体目标3：用多个激光陷阱使含有肌动蛋白的囊泡变形，以探测网络性质和机械不稳定性。用多个激光镊子握住和变形膜将使我们能够探测并可能控制突起和不规则膜形状的发展，类似于在活细胞中观察到的片状脂膜或伪足。这项工作将为研究细胞运动性的新方法奠定技术和分析基础。

DESCRIPTION (provided by applicant): The goal of this project is to develop new techniques to analyze and control biopolymer network microstructures with a holographic laser tweezer array, a powerful new instrument whose potential is just beginning to be explored. This tool will permit complex spatially distributed perturbations and measurements of forces in dynamic biopolymer networks. While the techniques will be developed on actin, they should be suitable to investigate forces and front instabilities in a broad range of network forming biomolecules. The response of the networks to perturbations should reveal the interrelationship between the polymerization front dynamics and the mechanical properties of the network. Understanding this inter-relationship is crucial for understanding how the polymerization dynamics of various cytoskeletal proteins affect cell function, for example, in the morphological changes associated with cell mobility. Understanding and controlling cell mobility has implications for many aspects of human health, from enhancing nerve regeneration after injury to reducing the mobility of metastatic cancer cells. Specific Aim 1: Control actin polymerization fronts with submicron resolution using a holographic tweezer array. Spatially extended perturbations of polymerization fronts will be introduced with the tweezer array by thermal heating or by localized uncaging of molecules that affect the polymerization. From the response of the polymerization front to the perturbation the investigators can extract characteristic length scales and timescales, and investigate instabilities. Specific Aim 2: Develop technology to measure the spatial distribution of forces using the holographic tweezer array. Pico Newton forces will be measured with trapped beads and trapped extended objects. They will measure the forces generated by a front of polymerizing actin and the correlations between those forces. With this technique they will determine how the polymerization force depends on the shape of the front, growth velocity, and other parameters. Specific Aim 3: Deform actin-containing vesicles with multiple laser traps to probe network properties and mechanical instabilities. Holding and deforming the membrane with multiple laser tweezers will permit us to probe and possibly control the development of protrusions and of irregular membrane shapes similar to the lamellipodia or pseudopodia observed in living cells. This work will lay the technical and analytical foundations for a new approach to investigating cell motility.