Karabinos Anton, Schulze Ekkehard, Schünemann Jürgen, Parry David A D, Weber Klaus
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am Fassberg 11, 37077 Goettingen, Germany.
J Mol Biol. 2003 Oct 17;333(2):307-19. doi: 10.1016/j.jmb.2003.08.041.
The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.
分析了四种必需的中间丝（IF）蛋白（A1、A2、A3、B1）和非必需的IF蛋白A4的体外聚合及组织特异性表达模式。重组B1在对11种秀丽隐杆线虫IF蛋白进行印迹覆盖分析时用作探针，与蛋白A1至A4强烈反应，表明存在异型相互作用。电子显微镜证实了体外专性异源多聚体丝的组装。当与等摩尔量的A1、A2或A3混合时，蛋白B1形成长的中间丝。利用绿色荧光蛋白（GFP）启动子报告基因发现了B1与A家族一个或多个成员在发育过程中的共表达。这种共表达模式表明体内存在异源多聚体系统。B1基因的一个或两个剪接变体总是以组织特异性方式与A家族的至少一个成员在皮下组织、咽部、咽肠瓣、排泄细胞、子宫、阴门和直肠中共表达。有趣的是，虽然正常情况下肠道缺乏B1/A对，但滞育幼虫的肠道中显示有B1和A4。这些结果与通过基因B1、A2和A3的RNA干扰（RNAi）诱导的皮下组织类似胚胎后表型一致。同样，在胚胎后B1 RNAi表型中观察到的咽部及其含A1-GFP张力丝的缺陷与B1和A1在边缘细胞中的共表达一致。因此，RNAi分析为体内B1/A专性异源多聚体系统的存在提供了独立证据。在咽部张力丝的光漂白实验中，蛋白A1和B1具有相似且相当缓慢的周转率。
