Integrative Cell Biophysics

综合细胞生物物理学

• 批准号: 9150141
• 负责人: Ralph Nossal
• 金额: $ 23.58万
• 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/9150141
• 关键词: Action Potentials; Affect; Animals; Area; Axon; Behavior; Binding; Biochemistry; Biogenesis; Biological; Biophysics; Blood Cells; Capsid Proteins; Cell membrane; Cell physiology; Cell surface; Cells; Characteristics; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Coated vesicle; Complex; Data; Defect; Dependence; Dependency; Development; Diffusion; Endocytosis; Equation; Eukaryota; Eukaryotic Cell; Exhibits; Film; Growth; Hodgkin Disease; Intracellular Transport; Investigation; Ion Channel; Kinetics; Lead; Ligands; Link; Lipid Bilayers; Lipids; Loligo; Malaria; Malignant Neoplasms; Mediating; Membrane; Membrane Potentials; Metabolic Diseases; Methods; Modeling; Molecular; Monte Carlo Method; Nutrient; Organism; Parasites; Pathway interactions; Phase Transition; Play; Pregnancy; Probability; Process; Production; Property; Protein Binding; Proteins; Publishing; Rana; Rattus; Regulation; Research; Role; Side; Signal Transduction; Signaling Molecule; Solvents; Squid; Staging; Structure; Temperature; Theoretical model; Time; Transport Process; Vesicle; Virus; Work; base; biological systems; cell type; coated pit; design; mathematical methods; mathematical model; microorganism; nanoparticle; nanosized; nervous system disorder; neurophysiology; particle; physical model; physical property; protein complex; receptor internalization; receptor mediated endocytosis; research study; response; simulation; therapy outcome; uptake; voltage

In general, we employ physical and mathematical methods to understand the biophysics of cellular processes. For the past several years, 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. Defects in CME can lead to metabolic disorders, aberrant signaling related to various cancers, and neurological disorders. A second major research area involves using published neurophysiological data to develop deeper basic understanding of the biophysical behavior of cell membranes at the normal (growth or gestation) temperatures of organisms (see below). Additionally, in the recent past we have collaborated on the development of a mathematical model to explore how difference in the average number of parasites (merozoites) released from malaria-infected blood cells can affect outcomes of therapy. The early stage of receptor mediated endocytosis involves the formation of transient structures known as clathrin coated pits (CCPs). This process depends on the detailed energetics of protein binding and associated membrane transformations. The CCPs either mature into clathrin coated vesicles (CCVs) or regress and vanish from the cell surface. During CCP formation, clathrin and several other proteins assemble to form a coat on the cytoplasmic side of the outer cell membrane. We previously developed a simple physical model for CCP dynamics and have carried out Monte Carlo simulations to investigate the time development of CCP size. By fitting the results of the simulations to experimental data, we have been able to estimate values of the kinetic parameters related to the formation of clathrin-associated protein complexes that comprise the coat. Recently, we have extended that model to investigate the role that CME plays in the uptake of viruses and nanoparticles (NPs). Understanding how nano-sized particles interact with the clathrin coat is important when designing NPs for biomedical purposes, as well as for developing stategies to inhibit cellular entry of viruses. Heretofore, theoretical models on this subject did not take clathrin coat assembly into explicit consideration. Thus we have developed a framework to study the endocytosis of single NPs, which focuses on protein coat assembly, and have derived a simple analytical formula for the mean internalization time of NPs, defined as the average time between the binding of a NP to the plasma membrane and its entry into the cell. We have studied the dependence of this quantity on coat parameters as well as NP size. Experiments indicate, for example, that there is a maximum size beyond which uptake via clathrin-mediated endocytosis does not occur. Moreover, there seems to be an optimal size at which cellular uptake is highest. Various published results indicate that these sizes seem to be independent of the type of cells, nanoparticles, and ligands. We have been able to show that such observations are consequences of the energetics and kinetics of protein coat formation during CCP production. As indicated above, we also have investigated putative temperature-dependent lipid phase transitions occurring in higher eukaryotes. It is well established that microorganisms adjust the lipid composition of their membranes in response to changes in the temperature at which they are grown. Moreover, investigations carried out over many years have demonstrated that whole lipid extracts from various higher organisms, as well as microorganisms, exhibit singular properties at their growth temperatures. We have investigated previously-published data concerning the temperature dependence of the electrophysiological responses of cells obtained from representative animals (e.g., frog, squid; rat), searching for unusual features occurring at the gestation/growth temperature, Tg, of these animals. Special emphasis has been given to the giant axons of the temperate squids Loligo forbesi and Loligo pealei, where the temperature dependencies of the resting potentials and action potentials exhibit behaviors that strongly suggest the onset of a membrane state change at Tg, mirroring, in the case of squids, anomalies seen in the physical properties of films formed of whole lipid extracts obtained from those organisms. Based on approximations to the classic Hodgkin-Huxley equations, analysis of axonal responses indicates that observed changes in these electrophysiological characteristics most likely reflect reversible molecular couplings between voltage-switchable ion channels and surrounding lipids in the plasma membrane, which can affect the probability of channel opening. The change in electrophysiological properties with increasing temperature for these animals yields activation energies close to values noted for other putative lipid-bilayer-linked kinetic processes which we have examined, as well as being seen in the diffusion of exogenous probes in lipid extracts (e.g., A.J. Jin, M. Edidin, R. Nossal, and N.L. Gershfeld, Biochemistry 38:13275-8, 1999). These, and other observations, substantiate the view that to first order the lipid bilayer acts as a universal solvent for the embedded, integral proteins found in the plasma membrane.

一般来说，我们采用物理和数学方法来理解细胞过程的生物物理学。在过去的几年里，重点一直是在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。CME缺陷可导致代谢紊乱、与各种癌症相关的异常信号传导和神经系统疾病。第二个主要研究领域涉及使用已发表的神经生理学数据，以加深对生物体正常（生长或妊娠）温度下细胞膜生物物理行为的基本理解（见下文）。此外，在最近的过去，我们合作开发了一个数学模型，以探索从疟疾感染的血细胞释放的寄生虫（裂殖子）的平均数量的差异如何影响治疗结果。 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。这个过程取决于蛋白质结合和相关膜转化的详细能量学。CCP要么成熟为网格蛋白包被的囊泡（CCV），要么从细胞表面退化并消失。在CCP形成过程中，网格蛋白和其他几种蛋白质组装在细胞外膜的细胞质侧形成外壳。我们以前开发了一个简单的物理模型CCP动力学，并进行了Monte Carlo模拟研究CCP大小的时间发展。通过拟合的模拟结果的实验数据，我们已经能够估计的网格蛋白相关的蛋白质复合物，包括外套的形成相关的动力学参数的值。最近，我们扩展了该模型，以研究CME在病毒和纳米颗粒（NP）摄取中的作用。当设计用于生物医学目的的纳米颗粒以及开发抑制病毒进入细胞的策略时，了解纳米尺寸的颗粒如何与网格蛋白涂层相互作用是重要的。因此，关于该主题的理论模型没有明确考虑网格蛋白被膜组装。因此，我们已经开发了一个框架来研究单个NP的内吞作用，其重点是蛋白质外壳组装，并推导出NP的平均内化时间的简单分析公式，其定义为NP与质膜结合和进入细胞之间的平均时间。我们已经研究了这个量对涂层参数以及NP尺寸的依赖性。 实验表明，例如，有一个最大的尺寸，超过该尺寸，通过网格蛋白介导的内吞作用的摄取不会发生。此外，似乎存在细胞摄取最高的最佳尺寸。 各种已发表的结果表明，这些大小似乎与细胞，纳米颗粒和配体的类型无关。我们已经能够表明，这样的观察结果的能量和动力学的蛋白质外壳形成过程中CCP生产的后果。 如上所述，我们还研究了高等真核生物中发生的假定的温度依赖性脂质相变。众所周知，微生物响应于它们生长的温度的变化来调节它们的膜的脂质组成。此外，多年来进行的研究表明，来自各种高等生物以及微生物的全脂质提取物在其生长温度下表现出独特的性质。我们已经研究了先前发表的关于从代表性动物（例如，青蛙，鱿鱼;大鼠），寻找这些动物在妊娠/生长温度Tg下发生的不寻常特征。特别强调的是温带鱿鱼Loligo forbesi和Loligo pealei的巨大轴突，其中的静息电位和动作电位的温度依赖性表现出的行为，强烈表明在Tg膜状态变化的开始，镜像，在鱿鱼的情况下，从这些生物体中获得的整个脂质提取物形成的膜的物理性质中看到的异常。基于对经典的Hodgkin-Huxley方程的近似，轴突反应的分析表明，观察到的这些电生理特征的变化最有可能反映电压可切换离子通道与质膜中周围脂质之间的可逆分子耦合，这可能影响通道开放的概率。这些动物的电生理特性随温度升高而变化，产生的活化能接近我们已经检查过的其他假定的脂质双层连接的动力学过程的值，以及在脂质提取物中的外源性探针的扩散中看到的值（例如，A.J. Jin，M.埃迪丁河Nossal和N.L. Gershfeld，Biochemistry 38：13275-8，1999）。这些和其他观察结果证实了这样的观点，即首先，脂质双层作为质膜中发现的嵌入的、整合的蛋白质的通用溶剂。