Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components
Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components
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DOI：
10.1016/s0006-3495(01)76185-1
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发表时间：
2001-05-01
影响因子：
3.4
通讯作者：
Baird, B
中科院分区：
文献类型：
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作者：
Pyenta, PS;Holowka, D;Baird, B
为了研究林恩酪氨酸激酶和IgE-Fc ε RI或脂筏的其他组分之间发生的膜相互作用的结构基础，我们制备了林恩的绿色荧光蛋白类似物（PM-EGFP），并使用交叉相关分析来量化IgE-Fc ε RI在细胞表面上交联后发生的聚集体的共再分布，PM-EGFP，该探针在林恩上含有最低限度的棕榈酰化和肉豆蔻酰化位点，与另一种内叶探针EGFP-GG进行了比较，EGFP-GG含有异戊烯化位点和类似于K-ras的多碱基序列。共聚焦荧光显微镜用于检查这些内叶组分与IgE-Fc β RI和外叶筏组分、神经节苷脂GD和糖基磷脂酰肌醇连接的Thy-1的共再分布，条件是后者在外部交联以在细胞表面形成大斑块。互相关分析的开发和表征与模拟代表细胞表面分布，和参数的互相关曲线，rho（o）（峰高）和A（峰面积），被证明是可靠的措施的程度co-redistributed聚集体和它们的大小。然后应用交叉相关分析来量化RBL-2 H3细胞上荧光标记的内瓣叶和外瓣叶组分的共再分布。如以这种方式目视观察和参数化的，发现PM-EGFP与脂筏的共重分布显著多于EGFP-GG或内源性异戊二烯化蛋白Cdc 42。这些定量结果与先前对林恩共再分布的分析一致，并支持以下假设：林恩与交联的IgE- Fc结合蛋白RI的功能重要的相互作用是由于它们与脂筏的相互共缔合。
To investigate the structural basis for membrane interactions that occur between Lyn tyrosine kinase and IgE-Fc epsilon RI or other components of lipid rafts, we prepared a green fluorescent protein analog of Lyn (PM-EGFP) and used cross-correlation analysis to quantify co-redistributions of aggregates that occur after IgE-Fc epsilon RI is cross-linked on the cell surface, PM-EGFP, which contains minimally the palmitoylation and myristoylation sites on Lyn, was compared with another inner leaflet probe, EGFP-GG, which contains a prenylation site and a polybasic sequence similar to K-ras. Confocal fluorescence microscopy was used to examine co-redistributions of these inner leaflet components with IgE-Fc epsilon RI and outer leaflet raft components, ganglioside GD,, and glycosylphosphotidylinositol-linked Thy-1, under conditions where the latter were cross-linked externally to form large patches at the cell surface. The cross-correlation analysis was developed and characterized with simulations representing cell surface distributions, and parameters from the cross-correlation curves, rho (o) (peak height) and A (peak area), were shown to be reliable measures of the extent of co-redistributed aggregates and their size. Cross-correlation analysis was then applied to quantify co-redistributions of the fluorescently labeled inner and outer leaflet components on RBL-2H3 cells. As visually observed and parameterized in this manner, PM-EGFP was found to co-redistribute with lipid rafts significantly more than EGFP-GG or an endogenous prenylated protein, Cdc42. These quantitative results are consistent with previous analyses of Lyn co-redistributions and support the hypothesis that the functionally important interaction of Lyn with cross-linked IgE- Fc epsilon RI is due to their mutual co-association with lipid rafts.