Bossinger Olaf, Fukushige Tetsunari, Claeys Myriam, Borgonie Gaetan, McGhee James D
Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, D-40225 Duesseldorf, Germany.
Dev Biol. 2004 Apr 15;268(2):448-56. doi: 10.1016/j.ydbio.2004.01.003.
We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.
我们希望了解器官特异性结构在胚胎发育过程中是如何组装的。在本文中，我们探讨了决定线虫秀丽隐杆线虫肠道细胞中终末网亚顶端位置的因素。终末网是指极化上皮细胞顶端微绒毛下方缺乏细胞器、富含中间丝的细胞质层。它通常被认为是从微绒毛核心伸出的肌动蛋白小根的锚定物。我们证明：（i）广泛使用的单克隆抗体MH33（仅）与由ifb-2基因编码的肠道特异性中间丝蛋白发生反应；（ii）IFB-2蛋白在胚胎发育的利马豆阶段开始在肠腔附近积累，并在成年期一直与肠腔相关联；（iii）免疫电子显微镜显示，IFB-2蛋白局限于肠道终末网内一个离散的周向亚顶端层（线虫中称为“内管”）；该层直接连接到连接相邻肠道细胞的顶端连接复合体。为了研究在发育过程中终末网组装时是什么决定了含IFB-2结构的布局，我们使用RNA干扰来去除先前已证明参与发育中肠道细胞整体顶-基极性的基因产物的功能。去除dlg-1、ajm-1或hmp-1的功能对终末网含IFB-2层的整体位置或连续性影响很小。相反，去除let-413基因的功能会导致终末网向基底外侧扩展，以至于现在它可以围绕肠道细胞的整个周长延伸。同样的处理也会导致肠道细胞皮质肌动蛋白和肌动蛋白相关蛋白ERM-1向基底外侧一致扩展。LET-413先前已被证明位于基底外侧，并能阻止几种单个顶端蛋白向基底外侧扩展。在本文的背景下，我们得出结论，LET-413对于维持秀丽隐杆线虫肠道细胞顶端表面的整个终末网或刷状缘组装也是必需的，这是上皮细胞连接所谓“栅栏”功能的一个显著例子。另一方面，LET-413在早期发育过程中对于建立这种顶端位置并非必需。最后，终末网中间丝层位于肠道细胞表面（顶端和基底外侧）下方的距离必须独立于顶端连接位置来确定。
