Integrative Cell Biophysics

综合细胞生物物理学

• 批准号: 7968773
• 负责人: Ralph Nossal
• 金额: $ 18.47万
• 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/7968773
• 关键词: Accounting; Affect; Antibiotics; Architecture; Area; Atomic Force Microscopy; Bacteria; Behavior; Biogenesis; Biological Process; Biophysics; Buffers; Cell Adhesion; Cell physiology; Cells; Charge; Clathrin; Coated vesicle; Communities; Complex; Coupled; Data; Development; Disease; Distress; Electron Microscopy; Elements; Endocytic Vesicle; Endocytosis; Environment; Eukaryotic Cell; Film; Gel; Goals; Growth; Heterogeneity; Immune; Intracellular Transport; Investigation; Kinetics; Laboratories; Ligand Binding; Measures; Mechanics; Mediating; Membrane; Metabolism; Methods; Microbial Biofilms; Microscopic; Modeling; Molecular; Morphology; Natural regeneration; Nucleic Acids; Nutrient; Optical Instrument; Patients; Persons; Phosphatidylinositols; Polymers; Polysaccharides; Process; Prokaryotic Cells; Property; Proteins; Protons; Research; Resolution; Role; Sampling; Scanning Electron Microscopy; Scanning Probe Microscopes; Scheme; Shapes; Signaling Molecule; Site; Solutions; Stimulus; Streptococcus mutans; Structure; Study models; Surface; Systems Biology; Techniques; Tissues; Transport Process; Vesicle; Work; antimicrobial drug; biological systems; extracellular; human disease; improved; instrument; interest; microorganism; pathogen; physical property; receptor; receptor mediated endocytosis; response; theories

We utilize advanced physical and mathematical methods to understand the biophysics of complex cellular processes. Phenomena under study include the stochastic biogenesis of coated vesicles involved in endocytosis and other intracellular transport processes, and the structural organization of multicellular biofilms arising from the attachment of prokaryotes to surfaces in nutrient-rich environments. These studies are of interest to persons studying basic cell biological processes, but they also are relevant to disease processes and normal and abnormal tissue development. Each requires the integration of several complicated processes, utilizing information obtained through reductionist studies but here focusing on behaviors emerging from both synergistic and competitive interactions. We have constructed a complex, multi-element model of receptor mediated endocytosis that encompasses cargo recognition, phosphoinositide metabolism, and clathrin coat formation and dissolution. The analysis demonstrates how the inter-related kinetic elements of these processes determine whether an endocytic vesicle will form. Not only does the model explain how vesicle biogenesis is triggered by, e.g., the binding of ligands to receptors at specific sites, but it also can rationalize the observed probabilistic quality of cell response in the presence of a stimulus. To gather data needed to refine the model, we have performed Atomic Force Microscopic investigations of clathrin triskelia, devising a scheme for examining fluctuations in triskelial shape while free in solution. We also perfected a technique for viewing dry samples that yields triskelial morphology with resolution at least as good as that obtainable by electron microscopy (EM). The latter was used to examine the formation of assembly intermediates when the triskelia are allowed to polymerize into clathrin cages in low pH buffers. Another area of complex systems biology currently under investigation in our laboratory pertains to bacterial biofilms. The latter are surface-attached communities of microorganisms that express a polymer coating--the extracellular polymeric substance (EPS)--that protects the attached bacterial colonies from antimicrobial agents. Biofilms are ubiquitous in the natural and technologically-modified worlds, yet little is really understood about their formation and viability; in human disease many bacterial pathogens form biofilms which resist destruction, causing great distress for patients who are unfortunate enough to be infected. We have focused on measuring the mechanical and transport properties of the EPS as a function of environmental parameters such as pH and externally-induced shear forces. One goal of this research is to identify factors that affect the flow of antibiotics within a biofilm and to understand how the EPS mediatesthe activity of immune cells. Another is to understand how the transport of nutrients and signaling molecules within a film is coupled to the spatially-heterogenous structures that develop, with a view towards understanding how various agents might mediate the growth of the bacteria. We also are investigating how biofilms, which are amenable to external manipulation, can serve as rudimentary models for studying the growth and regeneration of more complex cell communities. In order to characterize the mechanical properties of the EPS, we have developed methods involving atomic force microscopy that allow us to take into account the spatial heterogeneity of the colonies. We have found that the soft, hydrated EPS gel, which consists mainly of polysaccharides, proteins and nucleic acids that carry labile charges, softens and stiffens according to the proton concentration in the surrounding environment. We also have developed an improved technique for morphological analysis of bacterial biofilms, using scanning electron microscopy, that preserves the structure of these fragile and highly hydrated materials upon drying, hence revealing finer details about biofilm architecture and cellular adhesion. Finally, we fabricatd a multichannel culture chamber from cast PDMS that allows examination of biofilms with optical instruments as well as with direct contact instruments such as an atomic force microscope.

我们利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。 正在研究的现象包括随机生物发生的包被囊泡参与内吞作用和其他细胞内运输过程，和结构组织的多细胞生物膜所产生的附着的原核生物在营养丰富的环境中的表面。 这些研究对于研究基本细胞生物学过程的人来说是有意义的，但它们也与疾病过程以及正常和异常组织发育有关。 每一个都需要整合几个复杂的过程，利用通过还原论研究获得的信息，但这里关注的是从协同和竞争相互作用中出现的行为。 我们已经构建了一个复杂的，多元素的受体介导的内吞作用，包括货物识别，磷酸肌醇代谢，网格蛋白外套的形成和溶解模型。 分析表明，这些过程的相互关联的动力学元素如何决定是否会形成内吞囊泡。 该模型不仅解释了囊泡生物发生是如何被触发的，例如，配体与受体在特定位点的结合，但它也可以合理化在刺激存在下观察到的细胞反应的概率质量。 为了收集改进模型所需的数据，我们对网格蛋白triskelia进行了原子力显微镜研究，设计了一个方案，用于检查自由溶液中triskelia形状的波动。 我们还完善了一种观察干样品的技术，该技术产生的三维形貌的分辨率至少与电子显微镜（EM）获得的分辨率一样好。 后者用于检查当使三孢菌在低pH缓冲液中进入网格蛋白笼时组装中间体的形成。 我们实验室目前正在研究的另一个复杂系统生物学领域涉及细菌生物膜。 后者是表面附着的微生物群落，表达聚合物涂层-胞外聚合物物质（EPS）-保护附着的细菌菌落免受抗菌剂的侵害。 生物膜在自然界和技术改造的世界中无处不在，但人们对它们的形成和生存能力知之甚少;在人类疾病中，许多细菌病原体形成生物膜，抵抗破坏，给不幸被感染的患者带来巨大痛苦。 我们专注于测量EPS的机械和运输性能作为环境参数的函数，如pH值和外部诱导的剪切力。这项研究的一个目标是确定影响生物膜内抗生素流动的因素，并了解EPS如何介导免疫细胞的活性。 另一个是了解膜内营养物质和信号分子的运输如何与发展的空间异质结构相结合，以了解各种试剂如何介导细菌的生长。 我们也正在研究如何生物膜，这是服从外部操纵，可以作为研究更复杂的细胞群落的生长和再生的基本模型。为了表征EPS的机械性能，我们已经开发了涉及原子力显微镜的方法，使我们能够考虑到殖民地的空间异质性。 我们已经发现，主要由携带不稳定电荷的多糖、蛋白质和核酸组成的柔软的水合EPS凝胶根据周围环境中的质子浓度而软化和硬化。 我们还开发了一种使用扫描电子显微镜对细菌生物膜进行形态分析的改进技术，该技术在干燥后保留了这些脆弱且高度水合材料的结构，从而揭示了有关生物膜结构和细胞粘附的更精细细节。 最后，我们制作了一个多通道的培养室，从铸造PDMS，允许检查生物膜的光学仪器，以及与直接接触的仪器，如原子力显微镜。