Request for administrative supplement to Parent R01 GM073165 entitled "Mechanisms regulating clathrin-mediated endocytosis"

请求对母体 R01 GM073165 进行行政补充，标题为“调节网格蛋白介导的内吞作用的机制”

• 批准号: 9304692
• 负责人: Gaudenz Danuser
• 金额: $ 21.29万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304692
• 关键词: Achievement; Adhesions; Administrative Supplement; Affect; Algorithmic Software; Algorithms; Behavior; Biological Assay; Cardiovascular Diseases; Cell Surface Receptors; Cell membrane; Cell physiology; Cells; Clathrin; Clathrin-Coated Vesicles; Complex; Computer Simulation; Consensus; Data; Defect; Detection; Development; Diabetes Mellitus; Disease; Dynamin; Endocytosis; Engineering; Environment; Equilibrium; Event; Fluorescence Microscopy; Funding; Generations; Goals; Grant; Growth; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Image; Image Analysis; Individual; Kinetics; Label; Life; Link; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Measures; Mediating; Membrane; Methods; Microscopy; Modeling; Molecular; Neurologic; Nutrient; Parents; Pathology; Pathway interactions; Pattern; Physiology; Play; Population; Process; Proteins; Regulation; Role; Signal Transduction; Slave; Small Interfering RNA; Staging; Surface; Testing; Transcription Factor AP-2 Alpha; Variant; Work; base; cell motility; coated pit; feeding; genome editing; human disease; hypercholesterolemia; imaging modality; insight; interdisciplinary collaboration; interest; knock-down; live cell imaging; mathematical model; model building; molecular dynamics; nervous system disorder; novel therapeutic intervention; particle; programs; protein function; quantitative imaging; receptor; sensor; uptake

DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis (CME) is the major pathway for internalization from, and remodeling of the plasma membrane (PM) in mammalian cells. Thus, CME plays a fundamental role in all aspects of cell physiology, including nutrient uptake, signal transduction, cell motility, adhesion, polarity and differentiation. Consequently, defects in CME impinge on many human diseases, including cancer, cardiovascular disease, diabetes, neurological defects and others. CME is a multistep process initiated by the assembly of clathrin and the AP2 adaptor complex to form nascent CCPs. Subsequent steps including CCP stabilization, maturation and their scission from the PM to release clathrin coated vesicles are orchestrated by a myriad of endocytic accessory proteins (EAPs). Under the auspices of this grant, we have built an interdisciplinary team of biochemists, cell and molecular biologists, microscopists, engineers and computational biologists whose long- term goal is to define the mechanisms that regulate CME. Over the past 8 years, we have developed accurate and highly sensitive particle detection and tracking software and algorithms to quantitatively measure multiple orthogonal parameters relating to the dynamics of clathrin-eGFP labeled CCPs imaged by live cell total internal reflection fluorescence microscopy. We identified 3 kinetically-distinc subpopulations of CCPs, two short-lived abortive populations and longer-lived productive CCPs, as well as factors that differentially affect CCP initiation, stabilization and maturation. These data led us to propose that CCP maturation is gated during the first ~30s after initiation by an "endocytic check-point" that is, in part, regulated by the GTPase dynamin. Early recruitment of AP2-interacting EAPs to CCPs is also required for efficient curvature generation and to regulate multiple stages of the maturation process. The overarching goal of this proposal is to test this checkpoint hypothesis by identifying the determinants (i.e. individual or sets of EAPs) that function as potential sensors or effectors of the checkpoint, as well as those required for efficient CCP stabilization and maturation. To accomplish these goals, we propose three Specific Aims: 1) To measure the effect of individual and pairwise knockdown of EAPs on stages of CME through quantitative multi-parametric analyses of CCP dynamics; 2) To develop a molecularly explicit, mathematical model of the multi-step CCP maturation program and calibrate model parameters against measured CCP lifetime distributions in siRNA-treated cells, and 3) To, directly test, under minimally perturbing conditions, the functional assignments of EAPs predicted from the studies in Aim 1 and 2, and define the functional hierarchy of EAPs in regulating CCP maturation and progression beyond the endocytic checkpoint. These studies will provide unprecedented insights into the function of individual EAPs and their role in regulating key early stages of CCP stabilization of maturation. While most studies have focused on readily detectable early (CCP initiation) or late (CCV budding) events, we focus here on the less well understood stages of CCP stabilization and maturation that are central to the regulation of CME.

描述（由申请人提供）：网格蛋白介导的内吞作用（CME）是哺乳动物细胞中质膜（PM）的内在化和重塑的主要途径。因此，CME在细胞生理的各个方面都起着基本作用，包括养分吸收，信号转导，细胞运动，粘附，极性和分化。因此，CME缺陷会影响许多人类疾病，包括癌症，心血管疾病，糖尿病，神经系统缺陷等。 CME是由Clathrin和AP2适配器复合物组装起来以形成新生CCP的多步过程。随后的步骤，包括CCP稳定，成熟及其从PM释放蛋白网蛋白涂层的囊泡的分裂，由无数的内吞辅助蛋白（EAP）精心策划。在这笔赠款的主持下，我们建立了一个由生物化学家，细胞和分子生物学家，显微镜，工程师和计算生物学家组成的跨学科团队，其长期目标是定义调节CME的机制。在过去的8年中，我们开发了准确且高度敏感的粒子检测和跟踪软件和算法，以定量测量与通过活细胞总内反射荧光显微镜成像的网格蛋白EGFP动力学相关的多个正交参数。我们确定了CCP的3个动力学亚群，两个短寿命的流产人群和寿命较长的生产性CCP，以及差异影响CCP启动，稳定和成熟的因素。这些数据导致我们提出，在启动后的第一个〜30s中，CCP成熟是由“内吞检查点”开始的，而GTPase Dynamin则部分调节。对于有效的曲率产生并调节成熟过程的多个阶段，还需要早期募集AP2相互作用与CCPS的EAP。该提案的总体目标是通过识别确定功能作为检查点的潜在传感器或效应子以及有效的CCP稳定和成熟所需的决定因素（即，EAP的个体或EAP集合）来检验此检查点假设。为了实现这些目标，我们提出了三个具体目标：1）通过CCP动力学的定量多参数分析来衡量EAP对CME阶段的个人和成对敲低的影响； 2）开发多步CCP成熟程序的分子明确的数学模型，并校准模型参数，以针对siRNA处理的细胞中测得的CCP寿命分布进行校准，3）直接测试，在最小扰动条件下，在AIP的功能分配中，在AIM和DEFINE的功能分配中，在AIM和2和2的功能分配中，CCP和3）内吞检查点以外的成熟和进展。这些研究将提供对单个EAP功能的前所未有的见解及其在调节成熟的CCP稳定的关键早期阶段中的作用。尽管大多数研究都集中在易于检测到的早期（CCP启动）或晚期（CCV萌芽）事件上，但我们在这里集中在不太了解的CCP稳定和成熟阶段，这对于CME调节至关重要。