Coupling Force, Tension and Cell Plasma Membrane Plasticity at the Nanoscale Functional Roles of Caveolin Nanodomains

施加在细胞质膜上的机械力直接影响着许多重要的细胞和组织过程，包括细胞的黏附、迁移和侵袭。在这些过程中，膜弯曲蛋白小窝蛋白1(CAV1)及其在PM形成的不同类型的纳米结构域正在成为哺乳动物细胞的关键机械转导枢纽和张力缓冲结构。然而，CAV1纳米结构域的结构可塑性及其与PM的局部力学和拉伸状态的功能耦合仍然是个谜。因此，细胞膜在纳米尺度上适应不同力的一些基本机制仍然不清楚。该项目旨在了解CAP1纳米尺度结构域如何对机械提示做出反应来调制PM结构，并定义支配PM可塑性、维持膜张力和适当的细胞对力反应的核心物理原理。该项目将通过集成超分辨率(SR)显微成像、光学力传感、用于细胞机械生物学的生物材料工程、定量生物物理和建模的多学科方法来实施。它将(I)定量地定义细胞PM处CAV1纳米尺度结构域的纳米尺度组织，(Ii)建立它们对特定PM力的响应的可塑性，(Iii)确定它们如何局部调节PM张力，以及(Iv)提供它们作为关键PM张力调节器的功能的物理模型。由于其独创性和多学科性质，该项目的参与者从事高度跨学科的研究。它将为他们提供与目前细胞生物学、物理学和工程研究之间的融合相匹配的技能。拟议的活动还通过(I)使用成像探头、细胞培养和显微成像的实践实验、(Ii)外联项目和(Iii)教育活动，为本科和高中传统上代表性不足的学生提供了一个整合现代技术和交叉授粉科学的平台。经济困难的本科生和高中生将通过南加州大学现有的项目和扩展到大洛杉矶地区的高中来招募。特别是，在项目过程中，将向高中生提供由PI和Co-PI组织的为期8周的暑期实习，以及南加州大学的数学、工程、科学成就(MESA)计划。通过积极参与该项目所获得的经验和知识将吸引这些年轻一代的学者进入生物物理领域，并将为他们提供坚实的科学基础。细胞粘附力对CAV1纳米结构域三维可塑性的功能影响及其与亚膜肌动蛋白纤维和焦点粘连(FA)的空间耦合将首先通过相关3D SR显微镜、稳健的空间相关性分析和微图案化细胞粘附几何的调节来建立。这将为细胞对特定的机械约束做出反应时，对CAP1纳米结构域的动态平衡PM功能提供新的理解。然后，通过结合光学力传感器测量、SR显微镜和细胞微图案化，CAV1纳米结构域的可塑性将与在FA处沿着PM发展的细胞外和细胞内的皮牛顿力进行定量关联。这将为CAP1作为细胞表面外力/内力的机械传递者的作用提供新的线索。利用曲率和非曲率偶联PM受体的量子点跟踪以及活细胞中CAV1纳米结构域的3D SR显微镜，将建立CAP1纳米结构域作为PM张力局部调节器的功能角色。这将揭示它们如何参与局部PM张力和基材上的细胞力产生之间的自适应耦合。最后，描述作为局部膜张力函数的CAV1纳米结构域的可塑性的物理模型将被构思和定量测试，以定义管理细胞PM对力的适应的一些核心物理原理。除了提供新的光学工具和原创方法来研究PM纳米结构在细胞中的功能外，这项工作还将通过提供对PM可塑性和对纳米级力的适应的物理原理的机械理解，为生命系统的物理学带来新的见解。该项目由物理学部的生命系统物理学项目和分子和细胞生物科学部的细胞动力学和功能项目共同支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Mechanical forces exerted at the cell plasma membrane (PM) direct many important cellular and tissue processes, including cell adhesion, migration and invasiveness. In these processes, the membrane curving protein caveolin 1 (cav1) and the different types of nano-scale domains it forms at the PM are emerging as critical mechano-transducing hubs and tension buffering structures of mammalian cells. Yet, the structural plasticity of cav1 nano-scale domains and their functional coupling to the local mechanical and tension states of the PM are still enigmatic. Consequently, some of the fundamental mechanisms by which the cell membrane adapts to varying forces at the nano-scale remain undefined. This project aims at understanding how cav1 nano-scale domains modulate the PM structure in response to mechanical cues and at defining the core physical principles that govern PM plasticity, maintenance of membrane tension and proper cell response to forces. This project will be implemented through a multidisciplinary approach that integrates super-resolution (SR) microscopy imaging, optical force sensing, engineering of bio-materials for cell mechano-biology, quantitative biophysics and modeling. It will (i) define, quantitatively, the nano-scale organization of cav1 nano-scale domains at the cell PM, (ii) establish their plasticity in response to specific PM forces, (iii) determine how they locally regulate PM tension and (iv) provide physical models of their functions as key PM tension modulators. By its original and multidisciplinary nature, the project engages its participants in highly interdisciplinary research. It will provide them with skills that match the current convergence between cell biology, physics and engineering research. The proposed activities also provide a platform integrating modern technology and cross-pollination science for traditionally underrepresented students at the undergraduate and high school levels, through (i) hands-on experimentation with imaging probes, cell culture and microscopy imaging, (ii) outreach projects and (iii) educational activities. Economically disadvantaged undergraduate and high school students will be recruited via established programs at USC and through outreach to high schools in the Greater Los Angeles Area. In particular, an 8-weeks Summer Internship organized by the PI and Co-PI together with the Mathematics, Engineering, Science Achievement (MESA) program at USC will be offered to high-school students over the course of the project. Experience and knowledge gained by actively taking part in the project will attract this younger generation of scholars to the field of Biophysics and will provide them with strong scientific foundationsThe functional influence of cellular adhesion forces on the 3D plasticity of cav1 nano-domains and their spatial coupling to sub-membranous actin fibers and focal adhesions (FAs) will first be established by correlative 3D SR microscopy, robust spatial correlation analyses and modulation of adhesion geometry for micro-patterned cells. This will provide new understanding of the homeostatic PM functions of cav1 nano-domains as cells respond to specific mechanical constraints. The plasticity of cav1 nano-domains will then be quantitatively correlated with extra- and intracellular picoNewton forces developed along the PM at FAs by combining optical force sensor measurements, SR microscopy and cell micropatterning. This will shed new light on the role of cav1 as a mechano-transducer of extra/intracellular forces at the cell surface. Using quantum dot tracking of curvature- and non-curvature-coupled PM receptors together with 3D SR microscopy of cav1 nano-domains in live cells, the functional roles of cav1 nano-domains as local modulators of PM tension will then be established. This will reveal how they participate in adaptive coupling between local PM tension and cellular force generation on substrates. Finally, physical models describing the plasticity of cav1 nano-domains as a function of local membrane tension will be conceived and tested quantitatively to define some of the core physical principles that govern the adaptation of the cell PM to forces. Beyond offering new optical tools and original methodologies to study the function of PM nano-structures in cells, this work will bring novel insights into the physics of living systems by providing a mechanistic understanding of the physical principles that dictate PM plasticity and adaptation to forces at the nano-scale. This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Cellular Dynamics and Function Program in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.