Nolan Scott, Cowan Ann E, Koppel Dennis E, Jin Hui, Grote Eric
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Mol Biol Cell. 2006 May;17(5):2439-50. doi: 10.1091/mbc.e05-11-1015. Epub 2006 Feb 22.
Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.
交配的酵母细胞为细胞融合研究提供了一个基因易获取的系统。通过追踪融合伙伴之间荧光标记物的交换,分析了酵母细胞间融合孔的动态变化。质膜融合时,细胞质中的绿色荧光蛋白(GFP)和红色荧光蛋白(DsRed)以与融合孔大小成比例的速率在细胞间扩散。GFP通透率测量显示,典型的融合孔会突然打开,然后逐渐扩大。在一些交配配对中,发现GFP通透率突然增加,这与第二个孔的打开一致。相反,其他融合孔在允许有限量的细胞质交换后关闭。两个交配伙伴的FUS1基因缺失导致融合孔的初始通透率和扩张速率降低10倍以上。虽然fus1交配配对在质膜融合前降解分隔交配伙伴的细胞壁方面也存在缺陷,但其他具有细胞壁重塑缺陷的细胞融合突变体对融合孔通透率的影响较小。在fus1交配配对中,核融合延迟超过1小时,这可能是融合孔扩张受阻的结果。