Components And Kinetics In Exocytosis

胞吐作用的组成和动力学

• 批准号: 7208909
• 负责人: JOSHUA ZIMMERBERG
• 金额: --
• 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/7208909
• 关键词: adipocytes; bioenergetics; cholesterol; exocytosis; fluorescence microscopy; glucose transporter; insulin; intermolecular interaction; intracellular transport; laboratory rat; membrane fusion; membrane proteins; membrane structure; protein structure function; sphingolipids; tissue /cell culture; vesicle /vacuole

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.

该项目集中在胞吐作用的机理上，胞吐作用是无处不在的真核过程，囊泡通过该过程将其融合到质膜并释放其含量。我们今年报告了两个子项目 1。胰岛素刺激大鼠脂肪细胞中移动glut4囊泡的停止，绑扎和融合： 胰岛素通过细胞内部和葡萄糖转运蛋白4（GLUT4）的细胞内部和质膜（PM）之间的细胞内重新分布来调节肌肉和脂肪细胞中的葡萄糖转运。脂肪细胞的质膜（PM）中的GLUT4含量取决于其胞吐作用和内在化之间的动态平衡。在基底脂肪细胞中，含量 PM中的GLUT4保持较低（5％），因为GLUT4的内在速度比传递到PM的速度快10倍。胰岛素大大刺激了GLUT4胞吐作用的速率，对内在化速率的影响相对较小。因此，在胰岛素激活后，将50％的细胞内GLUT4转移到PM，从而在细胞表面的转运蛋白量增加了10倍。 GLUT4通过专门的微管隔室（此处称为Glut4囊泡）将其携带到PM上。 因此，我们应用了延时的总内反射荧光显微镜，以在原代培养物中大鼠脂肪细胞中这种GLUT4易位的中间体解剖。没有胰岛素，Glut4囊泡沿着覆盖整个PM的微管网络迅速移动，并通过松散地绑在PM上，定期停止（通常简短地停止）。胰岛素通过将囊泡紧紧地绑在PM形成簇并慢慢融合到PM的情况下，从而停止了这种交通。 GLUT4的缓慢释放决定了PM GLUT4的总体增加。因此，胰岛素最初募集了在PM附近的移动囊泡中隔离的GLUT4。 GLUT4易位中胰岛素作用的主要机制可能是刺激运输囊泡的束缚和融合到PM中的特定融合位点。 总而言之，我们建议Glut4囊泡遵循组成型胞吐作用的常见途径，在通往PM的途中利用微管轨道并揭示了膜货物的约束释放。但是，这些囊泡与PM的束缚和融合的可能性对胰岛素特别敏感。已知胰岛素通常会刺激构成胞吐作用，尽管比GLUT4胞吐作用的程度较小。我们目前正在研究提供胰岛素作用对GLUT4囊泡的特异性的分子机制。 2。膜筏的线张力和相互作用能量是根据脂质散布和倾斜计算的： 有人建议在某些膜微区域中胞吐作用发生。称为筏子的膜结构域富含胆固醇和鞘脂，被认为比周围的膜厚。如果是这样，单层应弹性变形，以免疏水表面暴露于筏边界处的水。我们计算了避免这种疏水性暴露所需的张力和倾斜变形的能量。每单位长度的能量值（线张力G）的派生值取决于木筏和周围膜的弹性模量；随着筏和周围的厚度的初始差异，它倍增。并且在两者之间的自发曲率中，它通过正或负差异降低。对于零自发曲率，与实验测量一致的单层高度不匹配的G为1 pn。我们的模型揭示了可以防止木筏形成的条件，以及一种可能导致筏子小的机制。预防筏的形成是基于我们的发现，即如果筏和周围的自发曲率的差异差异足够大，则计算出的线张力为负：如果G，除非分子相互作用（模型中忽略）足以使总线张力呈阳性，否则木筏将无法形成。对大小的控制是基于我们的发现，即从木筏到包围的高度轮廓并不能单调减小，而是表现出阻尼的振荡行为。重要的结果是，计算出的筏之间相互作用的能量也随着分离的距离而减小，从而在紧密齐全的筏之间产生能屏障。主要屏障的高度是筏和周围的自发曲线的复杂功能。这种障碍可以运动稳定与合并的木筏。因此，我们的物理理论量化了使筏形成并进一步定义控制筏合并的参数的条件。