MEMBRANE TRANSPORT AND FUSION

我们一直在描述熔化气孔的特征， 胞吐。使用米色小鼠肥大细胞、电子显微镜和 电生理学，有可能确定事件的顺序 在胞吐过程中。刺激后，质膜上出现一个凹陷 与分泌颗粒膜形成小的接触区。在这个范围内 没有有序的蛋白质特化的区域，一个小的融合 气孔形成，电导率迅速扩大到1纳秒。此后， 熔融孔保持在1到20 ns之间的半稳定电导 时间范围很广，在10到15000毫秒之间。这些电导 对应的孔径最大可达25纳米。超微结构数据显示 由生物膜组成的沙漏状小孔 与质膜和颗粒膜共面。后来，聚变 气孔的电导率迅速增加，这与观察到的一致 欧米伽图形的形态。没有看到通道状的波动。 由于小孔的形态表现为连续的断裂面，因此 电学数据代表含有脂质的毛孔，与我们的 先前提出的脂质/蛋白质复合体介导融合的模型。 为了更好地了解融合蛋白结构在融合孔中的作用 结构，我们一直在开发融合表达系统 蛋白质。对杆状病毒-昆虫细胞系统进行了测试，发现 具有非常活跃的、依赖于pH的融合活性。只有10秒 触发合胞体的形成需要酸性介质的脉冲。染料 转移、电容变化和形态变化很容易 检测到。该新体系为研究pH活化提供了很大方便 细胞-细胞融合。我们已经开始了电生理学研究 描述了该体系中熔融孔隙的发育特征。 为了充分研究生物膜融合，我们启动了两个 介导GTP触发的大鼠肝脏膜蛋白的纯化方案 微体-微体融合和海胆皮质，基于 通过共价修饰抑制膜融合。我们也是 细胞质膜末端大量融合的研究 有丝分裂。

We have continued to characterize the fusion pore which initiates exocytosis. Using beige mouse mast cells, electron microscopy, and electrophysiology, it is possible to determine the sequence of events during exocytosis. After stimulation, a dimple in the plasma membrane forms a small contact area with secretory granule membrane. Within this zone, which has no ordered proteinaceous specializations, a small fusion pore forms which widens rapidly to 1 nS in conductance. Thereafter, the fusion pore remains at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15000 ms. These conductances correspond to pore diameters of up to 25 nm. Ultrastructural data show small pores of hourglass morphology composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increases in conductance, consistent with the observed morphology of omega-figures. Channel-like fluctuations were not seen. Since the morphology of small pores show contiguous fracture planes, the electrical data represent pores which contain lipid, consistent with our previously proposed model of lipid/protein complexes mediating fusion. To better understand the role of fusion protein structure in fusion pore structure, we have been developing expression systems for fusion proteins. The baculovirus\insect cell system was tested, and discovered to have a very active, pH-dependent fusion activity. Only a 10 second pulse of acidic medium is needed to trigger syncytia formation. Dye transfer, capacitance changes, and morphological changes are readily detected. This new system is very convenient for studying pH activated cell-cell fusion. We have begun electrophysiological studies to characterize fusion pore development in this system. To fully investigate biological membrane fusion, we have initiated two projects to purify membrane proteins mediating GTP-triggered rat liver microsome-microsome fusion and the sea urchin cortex, based upon inhibition of membrane fusion by covalent modification. We are also studying the massive fusion of cytoplasmic membranes at the end of mitosis.