MEMBRANE REMODELING DURING VIRAL INDUCED SYNCYTIA FORMATION & PARASITE INVASION

我们正在继续我们关于融合毛孔的工作，这是第一个可以检测到的 生物融合的中间体。我们之前描述了诱导的毛孔 通过流感包膜蛋白血凝素(HA)。我们现在报道 另一种病毒蛋白杆状病毒诱导的融合毛孔特征 GP64。与透明质酸不同，GP64诱导的Sf9细胞融合孔具有 初始电导大，不闪烁，形成速度快得多 最适合多个独立的并行模型的动力学 元素。调查融合孔的特征是否仅被确定 通过融合蛋白或它们可能受到其他病毒被膜的影响 蛋白质或靶膜的特性，我们在一个 稳定表达Op-1D细胞株及其与Op-1D细胞融合的研究 红血球。红血球以前被用作靶细胞 羟基磷灰石诱导融合的研究。我们的初步分析显示 其他杆状病毒包膜蛋白或靶膜特性 不太可能干扰初始孔电导，但更有可能 对熔融孔隙动力学的影响。 另一个在膜重塑过程中形成的孔洞是裂变孔。多数 细胞内病原体通过以下方式避免在入侵期间裂解宿主细胞 将自己包裹在液泡膜中。这个寄生的液泡 膜(PVM)通常被保留，作为一种关键的运输 寄生虫和宿主细胞细胞质之间的界面。为了测试 弓形虫寄生虫形成的PVM是否来源于 寄主细胞膜或来自寄生虫分泌的脂类，我们使用 时间分辨电容测量和视频显微镜检测宿主 侵袭过程中的细胞表面积。我们没有观察到显著的变化 PVM形成过程中的宿主细胞表面积，表明PVM 主要由内陷的宿主细胞膜组成。掐掉…… 来自宿主细胞膜的PVM发生在意外的延迟之后 (S 34-305)，被认为电容下降了0.219+/-0.006 pF， 这与整个PVM的预测表面积很好地对应 (30-33平方米)。裂变孔的形成和闭合 细胞外培养液和空泡间隙检测为PVM 被掐掉了。寄生虫进入的最后阶段是在没有 细胞膜完整性的任何破坏。

We are continuing our work on fusion pores, the first detectable intermediate in biological fusion. We previously described pores induced by the influenza envelope protein hemagglutinin (HA). We now report features of fusion pores induced by another viral protein, baculovirus GP64. Unlike for HA, the GP64-induced fusion pore on Sf9 cells has a large initial conductance, does not flicker, and forms much faster with kinetics which fits best to a parallel model with many independent elements. To investigate if features of fusion pores are determined only by fusion proteins or they could be influenced by other viral envelope proteins or characteristics of target membranes, we express GP64 in a stably transfected cell line (Op-1D) and study fusion of Op-1D cells with erythrocytes. Red blood cells were previously used as target cells for research on HA-induced fusion. Our preliminary analysis showed that other baculovirus envelope proteins or features of target membrane unlikely interfere with initial pore conductance but rather have an impact on fusion pore kinetics. Another pore figures in membrane remodelling, the fission pore. Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 s) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 m2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.