Integrative Cell Biophysics

综合细胞生物物理学

• 批准号: 8351223
• 负责人: Ralph Nossal
• 金额: $ 59.54万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351223
• 关键词: Adaptor Signaling Protein; Affect; Atomic Force Microscopy; Binding; Biogenesis; Biological; Biological Models; Biophysics; Buffers; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Structures; Charge; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Coated vesicle; Collagen; Complement; Complex; Coupled; Coupling; Crowding; Cytoplasm; Data; Development; Dextrans; Diffusion; Electronics; Electrostatics; Endocytosis; Eukaryotic Cell; Film; Fluorescence; Free Energy; Glass; Indium; Intracellular Transport; Investigation; Journals; Label; Leg; Locomotion; Measurement; Measures; Mechanics; Mediating; Membrane; Methods; Modeling; Molecular; Movement; Neoplasm Metastasis; Nutrient; Pancreatic ribonuclease; Paper; Pathway interactions; Polymers; Procedures; Process; Property; Protein Binding; Proteins; Publishing; Quartz; Regulation; Role; Scheme; Signaling Molecule; Solutions; Source; Spectrum Analysis; Staging; Structure; Surface; Techniques; Time; Transport Process; Vesicle; Work; Wound Healing; base; biological systems; coated pit; dextran; embryo tissue; instrumentation; macromolecule; mathematical model; nanomechanical; photopolymerization; physical model; polyacrylamide; protein complex; protein structure; receptor internalization; receptor mediated endocytosis; response; simulation; ultraviolet

We employ advanced physical and mathematical methods to understand the biophysics of complex cellular processes. A major emphasis has been on the biogenesis of coated vesicles involved in clathrin mediated endocytosis (CME) and other intracellular transport processes. CME is the principal pathway for the regulation of receptors, and internalization of certain nutrients and signaling molecules, at the plasma membrane of eukaryotic cells. The early stage of receptor mediated endocytosis involves the formation of transient structures known as clathrin coated pits (CCPs) which, depending on the detailed energetics of protein binding and associated membrane transformations, either mature into clathrin coated vesicles (CCVs) or regress and vanish from the cell surface. The former are referred to as productive CCPs and the latter as abortive CCPs. We have posited a simple model for CCP dynamics and have carried out Monte Carlo simulations to investigate the time development of CCP size and explain the origin of abortive pits and features of their lifetime distribution. By fitting the results of the simulations to experimental data, we have been able to estimate values of the free energy changes involved in formation of the clathrin-associated protein complexes that comprise the coat, and have shown how the binding of cargo might modify the coat parameters and thereby facilitate CCV formation. A paper based on this work currently is being prepared. In a related study, we having been using quartz crystal microbalance-dissipation (QCM-D) instrumentation to investigate the mechanical properties of clathrin triskelia, clathrin coated vesicles (CCVs), and clathrin cages assembled with and without AP180 adaptor proteins (APs). We have also performed atomic force microscopy (AFM) measurements that complement these QCM-D measurements and facilitate interpretation of the QCM-D data. Using these methods, we find the apparent shear moduli of these structures to be approximately one to two orders of magnitude smaller than the Youngs modulus of a triskelion leg. The values of these shear moduli vary strongly with buffer properties and, to a lesser degree, also depend on properties of their substrate support. For example, we find that the shear modulus of CCVs attached to glass surfaces varies from 5 kPa to 12 kPa when buffer pH is changed over the range pH6.2 to pH7.5. This investigation is a continuation of our earlier work to establish the various mechanical properties of clathrin structures, which are important elements in our physical models. Moreover, pH modulation of the nanomechanical properties of clathrin lattices and related protein structures may be an essential aspect of vesicle transformations that are involved in cellular function. A paper based on this work currently is being prepared. A different project, also relating to mechanical aspects of cell response, involves establishing a reliable method for assessing the coupling between substrate properties and the locomotion of eukaryotic cells.. Our emphasis has been on developing collagen-coupled polymer films (e.g., polyacrylamide) whose formation can be controlled by photopolymerization in a quantitative and reproducible fashion. Our scheme uses an ultraviolet (UV) source of spatially-defined intensity to set up rigidity gradients in a film, The efficacy of the procedure has been confirmed by AFM. Our work currently is directed at examining the response of single cells (e.g., durotaxis). However, an extension will focus on understanding the effects of substrate rigidity on the collective movements of mechanically-interacting cells. Such work may have applications in studies of wound healing, cancer metastasis, and normal and aberrant development of embryonic tissues. Finally, model systems have been devised to examine the movement of charged macromolecules through crowded polymer solutions. These have been developed to mimic the diffusion of proteins within the interiors of biological cells. We measure the movement of labeled molecules by Fluorescence Correlation Spectroscopy (FCS). Using a small probe (RNase A) and dextrans that carry different electronic charge, we have shown that transient, charge-mediated binding can retard the movement of proteins to an extent similar to that due to molecular crowding. This work has been published in the Biophysical Journal.

我们采用先进的物理和数学方法来理解复杂细胞过程的生物物理学。一个主要的重点一直是生物起源的包被囊泡参与网格蛋白介导的内吞作用（CME）和其他细胞内运输过程。CME是调节真核细胞质膜上受体以及某些营养素和信号分子内化的主要途径。受体介导的内吞作用的早期阶段涉及称为网格蛋白包被的小凹（CCP）的瞬时结构的形成，其取决于蛋白质结合和相关膜转化的详细能量学，成熟为网格蛋白包被的囊泡（CCV）或从细胞表面退化和消失。 前者称为生产性CCP，后者称为流产性CCP。 我们提出了一个简单的CCP动力学模型，并进行了Monte Carlo模拟，调查CCP尺寸的时间发展，并解释了失败坑的起源和它们的寿命分布特征。 通过拟合的模拟结果的实验数据，我们已经能够估计的自由能变化的值参与形成的网格蛋白相关的蛋白质复合物，包括外套，并显示如何结合货物可能会修改的外套参数，从而促进CCV的形成。目前正在根据这项工作编写一份文件。 在一项相关研究中，我们一直使用石英晶体微天平-耗散（QCM-D）仪器来研究网格蛋白三聚体、网格蛋白包被囊泡（CCV）和组装有和没有AP 180衔接蛋白（AP）的网格蛋白笼的机械性能。 我们还进行了原子力显微镜（AFM）测量，补充这些QCM-D测量和促进QCM-D数据的解释。 使用这些方法，我们发现这些结构的表观剪切模量是约一至两个数量级小于杨氏模量的三重腿。 这些剪切模量的值随着缓冲性能而变化很大，并且在较小程度上也取决于其基底支撑的性能。 例如，我们发现当缓冲液pH在pH6.2至pH7.5的范围内变化时，附着于玻璃表面的CCV的剪切模量从5 kPa变化至12 kPa。这项调查是我们早期工作的延续，以建立网格蛋白结构，这是我们的物理模型中的重要元素的各种机械性能。此外，pH调节网格蛋白晶格和相关蛋白质结构的纳米力学性质可能是参与细胞功能的囊泡转化的一个重要方面。目前正在根据这项工作编写一份文件。 一个不同的项目，也涉及到细胞反应的机械方面，涉及建立一个可靠的方法来评估基板属性和真核细胞的运动之间的耦合。 我们的重点是开发胶原偶联聚合物膜（例如，聚丙烯酰胺），其形成可以通过光聚合以定量和可再现的方式控制。 我们的方案使用空间定义强度的紫外（UV）源在膜中建立刚性梯度，该过程的有效性已被AFM证实。我们目前的工作是针对检查单细胞的反应（例如，硬旋转）。 然而，扩展将集中在理解基板刚度的机械相互作用的细胞的集体运动的影响。这些工作可能在伤口愈合、癌症转移以及胚胎组织的正常和异常发育的研究中有应用。 最后，模型系统已被设计来检查通过拥挤的聚合物溶液的带电大分子的运动。 这些已经被开发来模拟生物细胞内部的蛋白质扩散。 我们通过荧光相关光谱（FCS）测量标记分子的运动。使用携带不同电子电荷的小探针（RNA酶A）和葡聚糖，我们已经证明，瞬时的电荷介导的结合可以在一定程度上延缓蛋白质的运动，其程度与分子拥挤相似。这项工作已发表在生物物理杂志上。