Regulation of clathrin-mediated endocytosis by membrane curvature.

通过膜曲率调节网格蛋白介导的内吞作用。

• 批准号: 8984163
• 负责人: Jessica Marks
• 金额: $ 5.24万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8984163
• 关键词: Actins; Active Sites; Adaptor Signaling Protein; Address; Area; Cell membrane; Cells; Clathrin; Data; Detection; Development; Disease; Dynamin; Early Diagnosis; Endocytosis; Event; Guanosine Triphosphate Phosphohydrolases; Life; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Microscopy; Modeling; Nanostructures; Neck; Nutrient; Play; Positioning Attribute; Process; Protein Binding; Proteins; Recruitment Activity; Recycling; Regulation; Research Design; Resolution; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Site; Staging; Structure; System; Techniques; Testing; Vesicle; Work; base; cellular imaging; coated pit; design; genome editing; membrane model; nanofabrication; nanoscale; novel; public health relevance; receptor; reconstitution; scaffold; sensor; spatiotemporal

 DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis (CME) is essential for regulating signaling pathways and internalizing nutrients. During CME initiation, scaffolding proteins that bind to the plasma membrane and adaptor proteins that bind cargo receptors nucleate assembly of clathrin-coated pits (CCP). As the CCP mature, membrane curvature progresses until scission by the GTPase dynamin separates the newly formed vesicle from the plasma membrane. Many curvature effectors have been associated with CME. These proteins are thought to help stabilize membrane curvature as it forms, and promote the membrane deformations necessary for the formation of a vesicle, but their exact role in CME regulation remains elusive. Our preliminary data show that curvature effectors are recruited to areas of artificially induced curvature. Further, live-cell imaging of CME markers revealed that these were active sites of CME. While these findings establish that the number of active CME sites is increased at areas of curvature, the mechanism of this effect is unclear. The objective of the work proposed here is to define the role of membrane curvature in initiation and progression of endocytosis. Specific aims: Regions of mechanically induced curvature are active sites for CME. Whether this increase in CME is due to the ability of the curved membrane to act as a signal of CME site initiation or due to a change sequential recruitment of CME curvature sensors is unknown. Therefore, we propose the following specific aim: to determine the role of membrane curvature in CME initiation and progression. Additionally, aim 2 addresses the role of curvature effector proteins in dynamin fission. Study design. We will analyze the dynamics of CME in cells expressing endogenously-tagged fluorescent CME curvature effector proteins and CME marker proteins on novel nano-fabricated substrates, recently designed by our collaborators. These substrates have nano scale structures that induces curvature of the plasma membrane. To address what role membrane curvature plays in CME initiation and regulation, we will quantify by confocal and super-resolution microscopy the number of initiation events at positions of induced curvature in relation to the planar areas of plasma membrane. In addition, we will define the order of recruitment of key CME proteins to determine if curvature acts as checkpoint to regulate late-stage CCP formation and vesicle scission. We will also determine the role curvature effector domains in dynamin- mediated fission through live cell studies using the nanostructure arrays and apply our findings to reconstitute vesicle fission in a model membrane system composed of unroofed mammalian cells and cytosolic extract derived from genome edited cells.

 描述（由适用提供）：网格蛋白介导的内吞作用（CME）对于调节信号通路和内在营养物质至关重要。在CME启动过程中，结合质膜和衔接蛋白结合的脚手架蛋白结合了货物蛋白涂层坑（CCP）的货物组装。随着CCP成熟的成熟，膜曲率会发展，直到通过GTPase Dynamin进行分离，将新形成的囊泡与质膜分开。许多曲率效应器与CME相关。这些蛋白质被认为有助于稳定膜曲率，并促进形成囊泡所需的膜声明，但它们在CME调节中的确切作用仍然难以捉摸。我们的初步数据表明，将曲率效应募集到人为诱导的曲率领域。此外，CME标记的活细胞成像表明，这些是CME的活跃位点。尽管这些发现表明，在曲率区域，活动性CME位点的数量增加，但这种效果的机制尚不清楚。这里提出的工作的目的是定义膜曲率在内吞作用的启动和进展中的作用。具体目的：机械诱导的曲率区域是CME的活性位点。 CME的增加是由于弯曲膜充当CME位点启动信号的能力，还是由于CME曲率传感器的顺序募集的变化而造成的。因此，我们提出以下特定目的：确定膜曲率在CME计划和进展中的作用。此外，AIM 2解决了曲率效应蛋白在动力蛋白裂变中的作用。研究设计。我们将分析表达内源性荧光CME CME曲率效应蛋白和CME标记蛋白在新型纳米型底物上的CME的动力学，最近由我们的合作者设计。这些底物具有诱导质膜曲率的纳米尺度结构。为了解决膜曲线在CME倡议和调节中的作用，我们将通过共焦和超分辨率显微镜量化诱导曲率位置相对于质膜平面区域的启动事件的数量。此外，我们将定义关键CME蛋白的募集顺序，以确定曲率是否起到调节后期CCP形成和蔬菜科学的检查点。我们还将通过使用纳米结构阵列的活细胞研究来确定在动力学介导的裂变中的作用曲率效应域，并应用我们的发现在模型膜系统中重新构建了囊泡裂变，该模型膜系统由未盖的哺乳动物细胞和由基因组编辑的细胞得出的细胞质提取物组成。