MEMBRANE TRANSPORT AND FUSION

膜运输和融合

• 批准号: 3842367
• 负责人: J 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/3842367
• 关键词: biological transport; cell fusion; cell membrane; chimeric proteins; cytoplasm; electron microscopy; electrophysiology; exocytosis; laboratory rat; mast cell; membrane fusion; membrane permeability; membrane proteins; protein structure; sea urchins

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.

我们继续研究融合孔的特征， 胞吐作用使用米色小鼠肥大细胞，电子显微镜， 通过电生理学，可以确定事件的顺序 在胞吐过程中。 刺激后，质膜上的一个凹痕 与分泌颗粒膜形成小的接触面积。 在这 区，没有有序的蛋白质专业化，一个小的融合 孔隙形成，其迅速加宽至电导为1 nS。 然后 融合孔保持在1和20 nS之间的半稳定电导， 在10 ms和15000 ms之间的宽时间范围内。这些电导 对应于高达25 nm的孔径。 超微结构数据显示 沙漏状小孔构成的生物膜 与质膜和颗粒膜共面。 后来，融合 孔隙电导迅速增加，与观察到的一致 Omega图形的形态学 并没有出现类似漩涡般的波动。 由于小孔的形态显示连续的断裂面， 电数据表示含有脂质的孔，与我们的实验结果一致。 先前提出的脂质/蛋白质复合物介导融合的模型。 为了更好地理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 proteins. 杆状病毒\昆虫细胞系统进行了测试，发现 具有非常活跃的pH依赖性融合活性。 只有10秒 需要酸性介质的脉冲来触发合胞体形成。 染料 转移，电容变化和形态变化很容易 检测到 这一新的体系为研究pH活化提供了方便 细胞-细胞融合 我们已经开始了电生理学研究， 表征该系统中的融合孔发育。 为了充分研究生物膜融合，我们启动了两个 纯化介导GTP触发大鼠肝脏的膜蛋白的项目 微粒体-微粒体融合和海胆皮层，基于 通过共价修饰抑制膜融合。 我们也 研究细胞质膜的大量融合， 分裂。