DataSheet1_ERM-1 Phosphorylation and NRFL-1 Redundantly Control Lumen Formation in the C. elegans Intestine.zip

Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na+/H+ exchanger 3 regulating factor (NHERF) family are conserved regulators of cortical specialization. ERM proteins function as membrane-actin linkers and as molecular scaffolds that organize the distribution of proteins at the membrane. NHERF proteins are PDZ-domain containing adapters that can bind to ERM proteins and extend their scaffolding capability. Here, we investigate how ERM and NHERF proteins function in regulating intestinal lumen formation in the nematode Caenorhabditis elegans. C. elegans has single ERM and NHERF family proteins, termed ERM-1 and NRFL-1, and ERM-1 was previously shown to be critical for intestinal lumen formation. Using CRISPR/Cas9-generated nrfl-1 alleles we demonstrate that NRFL-1 localizes at the intestinal microvilli, and that this localization is depended on an interaction with ERM-1. However, nrfl-1 loss of function mutants are viable and do not show defects in intestinal development. Interestingly, combining nrfl-1 loss with erm-1 mutants that either block or mimic phosphorylation of a regulatory C-terminal threonine causes severe defects in intestinal lumen formation. These defects are not observed in the phosphorylation mutants alone, and resemble the effects of strong erm-1 loss of function. The loss of NRFL-1 did not affect the localization or activity of ERM-1. Together, these data indicate that ERM-1 and NRFL-1 function together in intestinal lumen formation in C. elegans. We postulate that the functioning of ERM-1 in this tissue involves actin-binding activities that are regulated by the C-terminal threonine residue and the organization of apical domain composition through NRFL-1.

动物中大多数极化细胞的正常功能依赖于质膜及其下肌动蛋白细胞骨架的重排和组织为特定区域。ezrin-radixin-moesin（ERM）家族和Na+/H+交换因子3调节因子（NHERF）家族的蛋白质是皮质特化保守的调节因子。ERM蛋白作为膜-肌动蛋白连接蛋白，同时也是组织膜上蛋白质分布的分子支架。NHERF蛋白是包含PDZ结构域的适配器，能够与ERM蛋白结合并扩展其支架功能。在本研究中，我们探讨了ERM和NHERF蛋白在秀丽隐杆线虫Caenorhabditis elegans调节肠道腔形成中的作用。C. elegans具有单个ERM和NHERF家族蛋白，分别命名为ERM-1和NRFL-1，且ERM-1已被证实对于肠道腔的形成至关重要。通过CRISPR/Cas9生成的nrfl-1等位基因，我们证实NRFL-1定位于肠道微绒毛，并且这种定位依赖于与ERM-1的相互作用。然而，nrfl-1功能缺失突变体存活且不表现出肠道发育缺陷。有趣的是，将nrfl-1功能缺失与erm-1突变体（无论是阻断还是模拟调节C端苏氨酸残基的磷酸化）相结合，会导致肠道腔形成出现严重缺陷。这些缺陷并非仅存在于磷酸化突变体中，且与强erm-1功能缺失的影响相似。NRFL-1的缺失并未影响ERM-1的定位或活性。综上所述，这些数据表明ERM-1和NRFL-1在C. elegans的肠道腔形成中协同作用。我们推测ERM-1在此组织中的功能涉及受C端苏氨酸残基调节的肌动蛋白结合活性，以及通过NRFL-1组织顶域成分的排列。