MEMBRANE REMODELING DURING VIRAL INFECTION, PARASITE INVASION, AND APOPTOSIS

我们继续研究致病过程的膜生物学。 用快速冷冻、冷冻置换、薄层切片电镜、全细胞导纳和荧光同步记录等方法研究了流感血凝素糖基磷脂酰肌醇连接胞外域（GPI-HA）诱导的膜融合中间产物。在触发时，当通过电子显微镜观察时，先前分离的膜形成膜接触位点的许多沙漏形点（约10-130 nm腰部）。立体对表现出密切的膜接触的互补突起，从每个膜的峰。 使用HA时，接触较少，融合孔较宽。 生理测量显示酸化后细胞之间的快速脂质染料混合，以及融合孔形成或缺乏融合孔（真半融合）。 对于最早的孔，HA和GPI-HA检测到类似的电导分布和闪烁孔的频率。 对于GPI-HA，在孔开放之前、期间或之后检测到脂质混合，而对于HA，仅在孔开放之后观察到脂质混合。 我们的研究结果是一致的途径，其中在HA的胞外域的构象变化拉膜朝向对方，形成一个接触点，然后半融合和孔形成开始在这些接触点的一小部分。 最后，需要HA的跨膜结构域来完成膜融合以用于大分子内容物混合。疟疾寄生虫在人类红细胞（RBC）中的生长伴随着许多溶质的增加，包括阴离子，糖，嘌呤，氨基酸和有机阳离子。虽然这种摄取的药理学性质和选择性表明涉及氯离子通道，但尚未确定确切的机制。此外，这种摄取在感染的红细胞中的位置是未知的，因为示踪剂研究由于可能通过液相胞饮或膜导管摄取而变得复杂。在这个项目中，我们使用全细胞电压钳方法研究了受感染的红细胞的渗透性，以前从未对受感染的细胞进行过。使用这种方法，未感染的RBC具有小于100 pS的欧姆全细胞电导，这与它们的低示踪剂渗透性一致。相比之下，滋养体感染的红细胞表现出电压依赖性，非饱和电流的150倍大，主要是由阴离子和突然取消通道阻断剂。膜片钳测量和光谱分析证实，感染红细胞表面上的一个小（< 10 pS）离子通道（每个细胞约有1,000个拷贝）负责这些电流。由于其药理学性质和底物选择性与示踪剂研究中观察到的那些相匹配，因此该通道解释了感染RBC中小溶质摄取增加的原因。这个新的通道的表面位置和它的渗透性寄生虫生长所需的有机溶质表明，它可能有一个主要的作用，在一个连续的扩散途径寄生虫营养物质的收购。 这个通道可能被阻断，因此它是药物开发的一个有吸引力的目标。

We continued our work on the membrane biology of pathogenic processes. Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapidly freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed, when viewed by electron microscopy, numerous hourglass shaped points of membrane contact sites (~10-130 nm waist). Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, with wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pulls membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing. Growth of the malaria parasite in human red blood cells (RBCs) is accompanied by an increased uptake of many solutes including anions, sugars, purines, amino acids and organic cations. Although the pharmacological properties and selectivity of this uptake suggest that a chloride channel is involved, the precise mechanism has not been identified. Moreover, the location of this uptake in the infected RBC is unknown because tracer studies are complicated by possible uptake through fluid-phase pinocytosis or membranous ducts. In this project, we have studied the permeability of infected RBCs using the whole-cell voltage-clamp method, never before performed on infected cells. With this method, uninfected RBCs had ohmic whole-cell conductances of less than 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents that were 150-fold larger, predominantly carried by anions and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirmed that a small (< 10 pS) ion channel on the infected RBC surface, present at about 1,000 copies per cell, is responsible for these currents. Because its pharmacological properties and substrate selectivities match those seen with tracer studies, this channel accounts for the increased uptake of small solutes in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth indicate that it may have a primary role in a sequential diffusive pathway for parasite nutrient acquisition. This channel may be blocked, and so it is an attractive target for drug development.