Frolov V A, Cho M S, Bronk P, Reese T S, Zimmerberg J
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Traffic. 2000 Aug;1(8):622-30. doi: 10.1034/j.1600-0854.2000.010806.x.
Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (approximately 10-130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but 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 pull 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.
通过快速冷冻、冷冻置换、超薄切片电子显微镜以及全细胞膜导纳和荧光同步记录，研究了流感血凝素糖基磷脂酰肌醇连接的胞外域（GPI-HA）诱导的膜融合中间体。触发后，当通过电子显微镜观察时，先前分离的膜形成了许多沙漏形的膜接触点（腰部约10 - 130纳米）。立体对显示在来自每个膜的互补突起的峰值处有紧密的膜接触。对于HA，接触较少，但融合孔较宽。生理学测量表明，酸化后细胞间脂质染料快速混合，以及融合孔形成或未形成（真正的半融合）。对于最早的孔，HA和GPI-HA检测到相似的电导分布和闪烁孔频率。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，仅在孔开放后观察到脂质混合。我们的发现与一种途径一致，即HA胞外域的构象变化将膜相互拉近以形成接触位点，然后在这些接触位点的一小部分中启动半融合和孔形成。最后，需要HA的跨膜域来完成膜融合以实现大分子内容物混合。
