Components And Kinetics In Exocytosis

1.由于葡萄糖代谢的标志是胰岛素刺激下葡萄糖转运蛋白-4(GLUT4)转运到质膜(PM)，而膜蛋白结构的标志是其结构域结构，因此我们检测了胰岛素对脂肪细胞质膜GLUT4组织的影响。当GLUT4被传递到PM后，结构域外的所有GLUT4单体都可以自由扩散，但长链结构域(尺寸为60-240 nm)内的GLUT4在限制条件下扩散。胰岛素刺激GLUT4单体从这些结构域解离，但不刺激单体-结构域的结合，从而将大多数PM GLUT4从簇状状态转变为分散状态。而在结构域外，GLUT4单体经常碰撞，但不形成新的结构域；只有在胞吐时才能立即观察到GLUT4结构域的形成。胰岛素还可以抑制GLUT4从PM中排出，这种情况只发生在这些区域的内吞作用中。因此，胰岛素不仅调节GLUT4的胞吐和内吞，还调节其扩散的分子细节，所有这些都是为了控制葡萄糖的动态平衡。 2.胰岛素抵抗受试者的脂肪细胞表现出葡萄糖转运蛋白4(GLUT4)水平降低和胰岛素信号转导受损。在这里，我们研究了不同体重指数(BMI)和胰岛素敏感性(SI)的受试者分离的脂肪细胞中GLUT4转运和胰岛素刺激的GLUT4转位的动力学。将HA-GLUT4-GFP/mCherry导入细胞，在全内反射荧光显微镜下观察GLUT4储存囊(GSV)的转运和与质膜(PM)的融合。利用表面暴露的HA表位，用共聚焦显微镜评价HA-GLUT4-GFP在PM中的重新分布，并区分分散的和聚集的转运子。在没有胰岛素的情况下，GSV在微管上的转运和与PM的融合，以及细胞表面的总GLUT4，不会随着供体受试者的SI而改变。然而，虽然胰岛素敏感受试者细胞中的胰岛素通过刺激拴系和融合到PM来阻止GSV的运输，从而增加细胞表面GLUT4，但这些影响随着SI的降低而减弱，而不影响PM GLUT4簇数。在BMI在25到35之间的一组受试者中，我们发现GLUT4转运的改变与系统性胰岛素抵抗高度相关，与BMI无关。我们认为，全身性胰岛素抵抗的发生与基础GLUT4转运和PM簇的维持有关，但胰岛素刺激的GSV连接和融合以及细胞表面GLUT4的减少与肥胖无关，并且这种脂肪细胞中胰岛素反应性的改变可能代表了细胞和系统功能障碍之间的基本机制联系。 3.生物膜的分裂需要蛋白质驱动的胁迫。GTPase Dynamin通过聚合形成螺旋环来建立曲率应力，但这些Dynamin环确保无渗漏膜重塑的机制仍有活跃的争论。使用短脂纳米管作为底物直接测量裂变途径的几何中间体，我们发现GTP水解酶介导的组装和拆卸循环推动动力蛋白聚合形成短的、亚稳定的环，这是分裂的最佳条件。短到两级的颈圈可以通过动力素的Pleckstrin同源结构域(PHD)的膜楔入将径向收缩转化为可逆的半分裂。模型显示，PHD的倾斜与膜的变形相一致，为半分裂创造了可能的最低能量途径。这种动力蛋白和脂类的局部协调暗示了细胞膜重塑的新范例。理论分析表明，稳定的动态素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一样。在短的和亚稳态的动力支架中，这些限制被部分地放松，这些支架不仅施加弹性应力(机械力-化学效应)，而且与脂类协调，参与随机寻找膜重排的最佳途径(催化效应)。蛋白质支架的几何形状、协调的膜楔形和脂质双层的形状之间的纳米级协调导致了一条独特的几何催化途径来进行半分裂。这种配位作用可以被任何具有环状结构的膜插入蛋白复合体所支持，为蛋白质在膜重塑中的调节和催化功能提供了新的结构基础。

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.