Abstract
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.
Highlights
The establishment of molecularly and functionally distinct apical, basal, and lateral domains is a key feature of polarized epithelial cells
ezrin/ radixin/moesin (ERM) proteins consist of an N-terminal band Four-pointone/ezrin/radixin/moesin (FERM) domain that mediates binding to the plasma membrane and Redundant Control of Lumen Morphogenesis in C. elegans membrane-associated proteins, a C-terminal tail that mediates actin binding, and a central α-helical linker region (Fehon et al, 2010; McClatchey, 2014)
The observed distribution of NRFL-1::mCherry is consistent with previous observations in C. elegans (Hagiwara et al, 2012), as well as with localization of ERM-binding phosphoprotein 50 (EBP50) in mammalian epithelial tissues (Ingraffea, 2002; Morales et al, 2004; Kreimann et al, 2007)
Summary
The establishment of molecularly and functionally distinct apical, basal, and lateral domains is a key feature of polarized epithelial cells. The specialization of the apical domain and microvilli formation requires the activities of the ezrin/ radixin/moesin (ERM) family of proteins. ERM proteins consist of an N-terminal band Four-pointone/ezrin/radixin/moesin (FERM) domain that mediates binding to the plasma membrane and Redundant Control of Lumen Morphogenesis in C. elegans membrane-associated proteins, a C-terminal tail that mediates actin binding, and a central α-helical linker region (Fehon et al, 2010; McClatchey, 2014). Binding to the plasma membrane lipid phosphatidylinositol-(4,5) bisphosphate (PIP2) as well as phosphorylation of a conserved C-terminal threonine residue (T567 in ezrin) promote the transition to an open and active conformation that can link the plasma membrane to the underlying actin cytoskeleton and control the spatial distribution of protein complexes at the membrane (Simons et al, 1998; Nakamura et al, 1999; Barret et al, 2000; Coscoy et al, 2002; Yonemura et al, 2002; Fievet et al, 2004; Hao et al, 2009; Roch et al, 2010)
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