MEMBRANE REMODELING DURING VIRAL INFECTION, PARASITE INVASION, AND APOPTOSIS

病毒感染、寄生虫入侵和细胞凋亡期间的膜重塑

• 批准号: 6432565
• 负责人: JOSHUA 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/6432565
• 关键词: hemagglutinin; influenzavirus A; membrane fusion; microorganism hemagglutinin; protein folding; protein structure function; tissue /cell culture; video microscopy; virus cytopathogenic effect; virus protein

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.

我们继续研究致病过程的膜生物学。采用快速冷冻、冷冻取代、薄切片电镜以及同时记录全细胞导纳和荧光的方法，研究了由流感血凝素（GPI-HA）的糖基磷脂酰肌醇连接外结构域诱导的膜融合中间体。触发后，先前分离的膜形成，在电子显微镜下观察，许多沙漏状的膜接触点（~10-130 nm腰）。立体对在互补突起的峰值处显示出紧密的膜接触。羟基磷灰石的接触少，融合孔宽。生理测量显示酸化后细胞间脂质染料快速混合，融合孔形成或缺失（真正的半融合）。对于HA和GPI-HA的最早孔隙，检测到相似的电导分布和闪烁孔隙频率。对于GPI-HA，在开孔之前、开孔期间和开孔之后都可以检测到脂质混合，而对于HA，只有在开孔之后才能看到脂质混合。我们的发现与HA外结构域的构象变化将膜拉向彼此形成接触位点的途径一致，然后在这些接触位点的一小部分开始半融合和孔形成。最后，需要HA的跨膜结构域来完成大分子含量混合的膜融合。疟疾寄生虫在人红细胞中的生长伴随着对许多溶质的增加摄取，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氯离子通道参与其中，但其确切机制尚未确定。此外，这种摄取在感染红细胞中的位置尚不清楚，因为示踪研究可能通过液相胞饮作用或膜管摄取。在这个项目中，我们使用全细胞电压钳法研究了感染红细胞的渗透性，这种方法以前从未在感染细胞上进行过。用这种方法，未感染红细胞的欧姆全细胞电导小于100 pS，这与它们的低示踪剂渗透率一致。相比之下，滋养体感染的红细胞表现出电压依赖性的非饱和电流，其电流大150倍，主要由阴离子携带，并被通道阻滞剂突然消除。膜片钳测量和光谱分析证实，受感染红细胞表面的一个小（< 10ps）离子通道，每个细胞约有1000个拷贝，是产生这些电流的原因。由于其药理学性质和底物选择性与示踪剂研究结果相符，该通道可解释受感染红细胞对小溶质的吸收增加。这个新通道的表面位置及其对寄生虫生长所需的有机溶质的渗透性表明，它可能在寄生虫养分获取的顺序扩散途径中起主要作用。这个通道可能被阻断，因此它是一个有吸引力的药物开发靶点。