MEMBRANE TRANSPORT AND FUSION

膜拓扑学定义了生命：细胞内部是高度组织化的 在分子水平上，在几乎固态的密度下， 细胞环境通常比较混乱。 了解 细胞控制其膜拓扑结构的机制， 不断开发新方法，运用新老技术研究 细胞膜重塑的两个方面。 一种新的单元格算法 开发了电容测量。该算法利用 由补偿电路中的已知变化引起的校准跳变 膜片钳放大器的参数，以确定相移， 人为衰减，允许自动相位角调整， 重构所形成的电路的未失真导纳值 通过融合细胞和计算单个细胞的电参数， cell. 我们用这个软件测量了 由两种相似但不同的蛋白诱导的融合，Op的GP 64和 杆状病毒Ac株。 OP介导的初始电导 融合孔在0.3 - 1.9 nS范围内变化，平均值为0.7 + 负0.4 nS。 这与AC诱导的孔显著不同。 电导：1.0 ± 0.3 nS。 这奠定了基础， 融合孔表型结构/功能研究。 在第二 膜事件，内吞作用，我们正在探索电 寄生虫入侵的表现，最终在夹断 寄生空泡（PVM） 同步光学的应用 寄生虫入侵期间的电容测量使我们能够 首次显示宿主细胞无显著变化 表面积在弓形虫入侵。 因此， 不会对PVM的形成产生大量的脂质。 此外，可见PVM从宿主细胞膜上夹断 在每种情况下都是Cm突然下降0.2-0.3 pF。 裂变孔形成 在PVM的夹断过程中。 寄生分裂孔 相当于融合孔的内吞作用。 寄生裂变 孔隙呈现闪烁和半稳定阶段，平均电导 2 nS。孔达到小于1 nS的电导之前， 关门了相关的光学和电子记录超过100 入侵表明，膜电流的瞬时变化（尖峰） 发生在宿主细胞第一次接触的时刻， 寄生虫的顶端

Membrane topology defines life: the inside of a cell is highly organized at the molecular level, at almost solid-state densities; the outside environment of cells is usually more chaotic. To understand the mechanisms by which cells control their membranes' topologies, we have continued to develop new methods and use new and old techniques to study two aspects of cell membrane remodeling. A new algorithm for cell capacitance measurement has been developed. This algorithm uses calibration jumps caused by a known change in the compensation circuit parameters of the patch clamp amplifier to determine the phase shift and artifactual attenuation, allowing automatic phase angle adjustments and reconstruction of the undistorted admittance values of circuits formed by fusing cells and calculation of the electrical parameters of a single cell. We used this software to measure the electrical properties of fusion induced by two similar but distinct proteins, GP64 for the Op and Ac strains of baculovirus. The conductance of the Op-mediated initial fusion pore varies within a range 0.3 - 1.9 nS with a mean of 0.7 plus minus 0.4 nS. This differs significantly from the Ac-induced pore conductance: 1.0 plus minus 0.3 nS. This lays the foundation for structure/function studies of fusion pore phenotype. In the second membrane event, endocytosis, we are exploring the electrical manifestations of parasite invasion, which culminates in the pinching-off of a parasitic vacuole (PVM). The application of simultaneous optical and capacitance measurements during parasite invasion enabled us for the first time to show that there is no significant change in host cell surface area during invasion of Toxoplasma gondii. Thus the parasite does not contribute a significant amount of lipid to the PVM formation. In addition, pinching off of the PVM from the host cell membrane is seen in each case as a sudden 0.2-0.3 pF drop in Cm. A fission pore forms during the pinching off process of the PVM. The parasitic fission pore is the endocytotic equivalent to the fusion pore. The parasitic fission pore exhibits flicker and semistable stages with an average conductance of 2 nS. The pore reaches a conductance of less than 1 nS just before closing. Correlated optical and electrical records of more than 100 invasions have shown that transient changes in membrane current (spike) occur at precisely the moment that the host cell is first contacted by the apical end of the parasite.