Components And Kinetics In Exocytosis

胞吐作用的组成和动力学

• 批准号: 8736843
• 负责人: JOSHUA ZIMMERBERG
• 金额: $ 116.97万
• 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/8736843
• 关键词: Adipocytes; Adipose tissue; Affect; Beta Cell; Binding; Biological; Biological Assay; Body mass index; Catalysis; Cell membrane; Cell secretion; Cell surface; Cells; Chemicals; Clinical; Confocal Microscopy; DNA Sequence Rearrangement; Defect; Development; Diffuse; Diffusion; Dissociation; Docking; Dynamin; Endocytosis; Ensure; Epitopes; Event; Exhibits; Exocytosis; Extravasation; Fatty acid glycerol esters; Functional disorder; GLUT4 gene; Glucose Transporter; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Image; Insulin; Insulin Resistance; Intracellular translocation; Kinetics; Left; Life; Link; Lipid Bilayers; Lipids; Liver; Maintenance; Measures; Mediating; Membrane; Membrane Proteins; Metabolic; Microtubules; Modeling; Molecular; Monitor; Muscle; Muscle Cells; Nanotubes; Neck; Non-Insulin-Dependent Diabetes Mellitus; Obesity; PH Domain; Pathway interactions; Permeability; Play; Polymers; Proteins; Radial; Regulation; Role; Rupture; Scaffolding Protein; Shapes; Signal Transduction; Staging; Stochastic Processes; Stress; Structure; Subgroup; Surface; System; Testing; Tissues; Total Internal Reflection Fluorescent; Translating; Vesicle; adipokines; blood glucose regulation; constriction; genetic regulatory protein; glucose metabolism; glucose transport; glucose uptake; human subject; insulin sensitivity; insulin signaling; lipid metabolism; membrane model; monomer; nanoscale; novel; polymerization; protein complex; response; scaffold; trafficking

1. Since the hallmark of glucose metabolism is insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) and the hallmark of membrane protein organization is its domain structure, we examined insulins effect on GLUT4 organization in PM of adipose cells. After delivery to PM, all GLUT4 monomers outside domains diffuse freely, but GLUT4 within elongated domains (sized 60-240 nm) diffuse with confinement. Insulin stimulates dissociation of GLUT4 monomers from these domains but does not stimulate monomer-domain association, thereby shifting most PM GLUT4 from clustered to dispersed states. While outside domains, GLUT4 monomers collide frequently but do not form new domains; GLUT4 domain formation is only observed immediately upon exocytosis. Insulin also inhibits exit of GLUT4 from PM, which occurs through endocytosis only at the domains. Thus, insulin not only regulates both exocytosis and endocytosis of GLUT4, it also regulates molecular details of its diffusion, all to control glucose homeostasis. 2. Adipose cells from insulin-resistant human subjects exhibit decreased levels of glucose transporter-4 (GLUT4) and impaired insulin signaling. Here we investigated the dynamics of GLUT4 trafficking and the insulin-stimulated translocation of GLUT4 in adipose cells isolated from human subjects with varying body mass indexes (BMI) and insulin sensitivities (SI). Cells were transfected with HA-GLUT4-GFP/mCherry, and imaged live using total internal reflection fluorescent microscopy to monitor GLUT4 storage vesicle (GSV) trafficking and fusion with the plasma membrane (PM). Confocal microscopy was used to assess the redistribution of HA-GLUT4-GFP to PM using the surface-exposed HA epitope, and to distinguish dispersed from clustered transporters. Without insulin, GSV trafficking on microtubules and fusion with PM, and total cell-surface GLUT4, do not vary with donor subject SI. However, while insulin in cells from insulin-sensitive subjects halts GSV trafficking by stimulating tethering and fusion to PM, and thereby increases cell-surface GLUT4, these effects diminish with decreasing SI, without affecting PM GLUT4 cluster number. In a subgroup of subjects with BMIs of 25 to 35, we found that altered GLUT4 trafficking highly correlated with systemic insulin resistance, independent of BMI. We suggest that development of systemic insulin resistance is associated with maintenance of basal GLUT4 trafficking and PM clusters, but diminished insulin-stimulated GSV tethering and fusion, and cell-surface GLUT4, independent of obesity, and that this altered insulin responsiveness in adipose cells may represent a fundamental mechanistic link between cellular and systemic dysfunction. 3. Biological membrane fission requires protein-driven stress. The GTPase dynamin builds up curvature stress by polymerizing into a helical collar, but the mechanism by which these dynamin collars ensure non-leaky membrane remodeling is actively debated. Using short lipid nanotubes as substrates to directly measure geometric intermediates of the fission pathway, we found that GTP hydrolysis-mediated assembly and disassembly cycles drive dynamin polymerization into short, metastable collars that are optimal for fission. Collars as short as two-rungs can translate radial constriction to reversible hemifission via membrane wedging of the pleckstrin homology domains (PHD) of dynamin. Modeling reveals that tilting of the PHDs to conform with membrane deformations creates the lowest possible energy pathway for hemifission. This local coordination of dynamin and lipids suggests a novel paradigm of membrane remodeling in cells. The theoretical analysis reveals that a stable dynamin polymer constrains highly curved membrane structures thereby effectively inhibiting topological transitions, just as tighter substrate binding inhibits enzymatic catalysis. These constraints are partially relaxed in short and metastable dynamin scaffolds, which not only apply elastic stress (mechano-chemical effects) but also, in coordination with lipids, participate in stochastic searching for the optimal pathway of membrane rearrangements (catalytic effects). The nanoscale coordination between the geometry of the protein scaffold, concerted membrane wedging and the shape of the lipid bilayer results in a distinct geometric catalytic pathway to hemifission. This coordination can be supported by any membrane-inserting protein complex with a ring-like structure, providing a new structural rationale for protein regulatory and catalytic function in membrane remodeling.

1.由于葡萄糖代谢的标志是胰岛素刺激的葡萄糖转运蛋白-4（GLUT 4）向质膜（PM）的递送，而膜蛋白组织的标志是其结构域结构，因此我们研究了胰岛素对脂肪细胞PM中GLUT 4组织的影响。 在递送至PM后，结构域外部的所有GLUT 4单体自由扩散，但细长结构域（尺寸为60-240 nm）内的GLUT 4扩散受限。 胰岛素刺激GLUT 4单体从这些结构域解离，但不刺激单体-结构域缔合，从而使大多数PM GLUT 4从聚集状态转变为分散状态。在结构域之外，GLUT 4单体经常碰撞，但不形成新的结构域; GLUT 4结构域的形成仅在胞吐作用后立即观察到。 胰岛素还抑制GLUT 4从PM的退出，其仅在结构域通过内吞作用发生。因此，胰岛素不仅调节GLUT 4的胞吐和胞吞作用，还调节其扩散的分子细节，所有这些都是为了控制葡萄糖稳态。 2.来自胰岛素抵抗人类受试者的脂肪细胞表现出葡萄糖转运蛋白-4（GLUT 4）水平降低和胰岛素信号传导受损。在这里，我们研究了从不同体重指数（BMI）和胰岛素敏感性（SI）的人受试者中分离的脂肪细胞中GLUT 4运输和胰岛素刺激的GLUT 4易位的动力学。 用HA-GLUT 4-GFP/mCherry转染细胞，并使用全内反射荧光显微镜实时成像以监测GLUT 4储存囊泡（GSV）运输和与质膜（PM）的融合。 使用共聚焦显微镜来评估使用表面暴露的HA表位将HA-GLUT 4-GFP重新分配到PM，并区分分散的转运蛋白和聚集的转运蛋白。 在没有胰岛素的情况下，GSV在微管上的运输和与PM的融合以及总细胞表面GLUT 4不随供体受试者S1而变化。 然而，虽然来自胰岛素敏感受试者的细胞中的胰岛素通过刺激束缚和融合到PM来停止GSV运输，从而增加细胞表面GLUT 4，但这些作用随着SI的降低而减弱，而不影响PM GLUT 4簇数。 在BMI为25至35的受试者亚组中，我们发现改变的GLUT 4运输与全身胰岛素抵抗高度相关，与BMI无关。 我们认为，系统性胰岛素抵抗的发展与维持基础GLUT 4运输和PM集群，但减少胰岛素刺激的GSV拴系和融合，和细胞表面GLUT 4，独立于肥胖症，这种改变的胰岛素在脂肪细胞的反应性可能代表细胞和全身功能障碍之间的基本机制联系。 3.生物膜分裂需要蛋白质驱动的压力。 GT3发动蛋白通过聚合成螺旋环来建立曲率应力，但是这些发动蛋白环确保非渗漏膜重塑的机制正在积极辩论。使用短的脂质纳米管作为基板直接测量裂变途径的几何中间体，我们发现GTP水解介导的组装和拆卸周期驱动发动蛋白聚合成短的，亚稳态的衣领，是最佳的裂变。短至两个梯级的套环可以通过发动蛋白的普列克底物蛋白同源结构域（PHD）的膜楔入将径向收缩转化为可逆的半裂。建模表明，倾斜的PHDs，以符合膜变形创造了最低可能的能量通路半裂变。发动蛋白和脂质的这种局部协调表明细胞中膜重塑的新范例。理论分析表明，一个稳定的发动蛋白聚合物约束高度弯曲的膜结构，从而有效地抑制拓扑转变，就像更紧密的底物结合抑制酶催化。这些限制在短的亚稳态发动蛋白支架中部分放松，其不仅施加弹性应力（机械化学效应），而且与脂质协调，参与随机搜索膜重排的最佳途径（催化效应）。蛋白质支架的几何形状之间的纳米级协调，一致的膜楔入和脂质双层的形状导致在一个独特的几何催化途径半裂变。这种协调可以通过任何具有环状结构的膜插入蛋白质复合物来支持，为膜重塑中的蛋白质调节和催化功能提供了新的结构原理。