Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation
Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation
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DOI：
10.1034/j.1600-0854.2000.010806.x
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发表时间：
2000-08-01
期刊：
影响因子：
4.5
通讯作者：
Zimmerberg, J
中科院分区：
文献类型：
--
作者：
Frolov, VA;Cho, MS;Zimmerberg, J
用快速冷冻、冷冻替代、薄片电子显微镜、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-磷脂酰肌醇连接的胞外区(GPI-HA)诱导的膜融合中间产物。在触发时，先前分离的膜在电子显微镜下观察时形成了许多沙漏状的膜接触点(类似于10-130 nm的腰围)。立体对在互补突起的峰处显示紧密的膜接触，这些突起来自每个膜。HA组接触较少，但融合孔较宽。生理测量显示酸化后细胞间脂类染料快速混合，形成融合孔或缺乏融合孔(真正的半融合)。对于最早的孔，HA和GPI-HA的电导分布和闪烁孔的频率相似。对于GPI-HA，在开孔前、开孔期间或开孔后检测到脂质混合，而对于HA，只有在开孔后才能看到脂质混合。我们的发现与这样一种途径是一致的，即HA胞外结构域的构象变化将膜拉向彼此形成接触部位，然后在这些接触部位中的一小部分开始半融合和孔形成。最后，需要HA的跨膜区来完成膜融合，以进行大分子含量的混合。
Membrane fusion intermediates induced by the glycosyl-phosphatidylinositol-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 (similar to 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.