Components And Kinetics In Exocytosis

这个项目是集中在胞吐作用的机制，普遍存在的真核细胞的过程中，囊泡融合到质膜和释放其内容物。今年我们报告两个子项目 1.胰岛素刺激大鼠脂肪细胞中移动的GLUT 4囊泡的停止、束缚和融合： 胰岛素通过葡萄糖转运蛋白4（GLUT 4）在细胞内部和质膜（PM）之间的细胞内再分布来调节肌肉和脂肪细胞中的葡萄糖转运。脂肪细胞质膜（PM）中的GLUT 4含量由其胞吐和内化之间的动态平衡决定。在基底脂肪细胞中， PM中的GLUT 4仍然很低（5%），因为GLUT 4内化的速度比其递送到PM的速度快10倍。胰岛素显著刺激GLUT 4胞吐速率，而对内化速率的影响相对较小。因此，50%的细胞内GLUT 4在胰岛素活化后易位到PM，使细胞表面上的转运蛋白的量增加10倍。GLUT 4通过与转运蛋白紧密包装的专门的管泡隔室（在此称为GLUT 4囊泡）被运送到PM。 因此，我们应用延时全内反射荧光显微镜来解剖大鼠脂肪细胞原代培养中GLUT 4易位的中间体。在没有胰岛素的情况下，GLUT 4囊泡沿着覆盖整个PM的微管网络快速移动，周期性地停止，大多数情况下只是短暂地，通过松散地拴在PM上。胰岛素通过将囊泡紧紧地拴在PM上来阻止这种交通，在PM上囊泡形成簇并缓慢地融合到PM上。GLUT 4的这种缓慢释放决定了PM GLUT 4的总体增加。因此，胰岛素最初募集隔离在PM附近的移动的囊泡中的GLUT 4。胰岛素在GLUT 4易位中作用的主要机制可能是刺激运输囊泡与PM中特定融合位点的束缚和融合。 总之，我们建议GLUT 4囊泡遵循共同的组成性胞吐途径，利用微管轨道上的方式PM和揭示限制释放的膜货物。然而，这些囊泡与PM的拴系和融合的可能性对胰岛素特别敏感。已知胰岛素通常刺激组成型胞吐，尽管程度低于GLUT 4胞吐。我们目前正在研究提供胰岛素作用于GLUT 4囊泡的特异性的分子机制。 2.根据脂质展开和收缩计算的膜筏的线张力和相互作用能： 有迹象表明，胞吐作用发生在某些膜微区。膜结构域被称为筏富含胆固醇和鞘脂，被认为比周围的膜厚。如果是这样，单层应该弹性变形，以避免在筏边界处疏水表面暴露于水。我们计算了避免这种疏水暴露所需的张开和倾斜变形的能量。每单位长度的能量的导出值，线张力g，取决于筏和周围膜的弹性模量;它与筏和周围膜之间的初始厚度差成二次方地增加;并且它因两者之间的自发曲率的正或负差异而减少。对于零自发曲率，g为1 pN的单层高度失配为0.3 nm，与实验测量一致。我们的模型揭示了可能阻止筏形成的条件，以及可能导致筏保持较小的机制。防止筏的形成是基于我们的发现，如果筏和周围的自发曲率的差异足够大，计算的线张力是负的：如果g小于0，筏不能形成，除非分子相互作用（在模型中忽略）足够强，使总的线张力为正。尺寸控制基于我们的发现，即从木筏到周围的高度轮廓不会单调下降，而是表现出阻尼、振荡行为。作为一个重要的结果，计算的筏之间的相互作用的能量也振荡，因为它随着距离的分离，创造紧密并列筏之间的能量障碍。主要屏障的高度是筏和周围环境的自发曲率的复杂函数。这个屏障可以在动力学上稳定筏，防止合并。因此，我们的物理理论量化的条件，允许筏形成，并进一步定义控制筏合并的参数。

This project is centered on the mechanisms of exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. We report two subprojects this year 1. Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells: Insulin regulates glucose transport in muscle and adipose cells through the intracellular redistribution between the cell interior and the plasma membrane (PM) of the glucose transporter 4 (GLUT4). GLUT4 content in the plasma membrane (PM) of adipose cells is determined by a dynamic equilibrium between its exocytosis and internalization. In basal adipose cells, the content of GLUT4 in the PM remains low (5%) as GLUT4 internalizes 10 times faster than it is delivered to the PM. Insulin considerably stimulates the rate of GLUT4 exocytosis with relatively little effect on the rate of internalization. Consequently, 50% of intracellular GLUT4 is translocated to the PM upon insulin activation, providing a 10-fold increase in the amount of transporter on the cell surface. GLUT4 is carried to the PM by specialized tubulovesicular compartments (referred to here as GLUT4 vesicles) tightly packed with the transporter. Thus we applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM. In summary, we propose that GLUT4 vesicles follow common pathways of constitutive exocytosis, exploiting microtubule tracks on their way to the PM and revealing constrained release of membrane cargo. However, the probability of tethering and fusion of these vesicles to the PM is specifically sensitive to insulin. Insulin is known to stimulate constitutive exocytosis in general, though to a lesser extent than GLUT4 exocytosis. We are currently investigating molecular mechanisms providing the specificity of insulin action on the GLUT4 vesicles. 2. Line Tension and Interaction Energies of Membrane Rafts Calculated from Lipid Splay and Tilt: There are suggestions that exocytosis takes place in certain membrane microdomains. Membrane domains known as rafts are rich in cholesterol and sphingolipids, and are thought to be thicker than the surrounding membrane. If so, monolayers should elastically deform so as to avoid exposure of hydrophobic surfaces to water at the raft boundary. We calculated the energy of splay and tilt deformations necessary to avoid such hydrophobic exposure. The derived value of energy per unit length, the line tension g, depends on the elastic moduli of the raft and the surrounding membrane; it increases quadratically with the initial difference in thickness between the raft and surround; and it is reduced by differences, either positive or negative, in spontaneous curvature between the two. For zero spontaneous curvature, g is 1 pN for a monolayer height mismatch of 0.3 nm, in agreement with experimental measurement. Our model reveals conditions that could prevent rafts from forming, and a mechanism that can cause rafts to remain small. Prevention of raft formation is based on our finding that the calculated line tension is negative if the difference in spontaneous curvature for a raft and the surround is sufficiently large: rafts cannot form if g is less than 0, unless molecular interactions (ignored in the model) are strong enough to make the total line tension positive. Control of size is based on our finding that the height profile from raft to surround does not decrease monotonically, but rather exhibits a damped, oscillatory behavior. As an important consequence, the calculated energy of interaction between rafts also oscillates as it decreases with distance of separation, creating energy barriers between closely apposed rafts. The height of the primary barrier is a complex function of the spontaneous curvatures of the raft and the surround. This barrier can kinetically stabilize the rafts against merger. Our physical theory thus quantifies conditions that allow rafts to form, and further, defines the parameters that control raft merger.