Most polarized cells form specialized cortical domains that are essential for their functions, such as microvilli in epithelia and axons in neurons. Proteins of the conserved Ezrin-Radixin-Moesin (ERM) family play critical roles in the formation of apical membrane domains by directly linking the plasma membrane to the actin cytoskeleton, and by controlling the spatial distribution of proteins. The activity of ERM proteins depends on a conformational change that turns an inactive cytoplasmic form into an active membrane- and actin-bound form. In vitro data suggests an activation model dependent on PIP2-binding followed by phosphorylation of a conserved C-terminal residue. However, in vivo data supporting this model is scarce and contradictory. We investigate the regulation of the single C. elegans ERM ortholog, ERM-1, and its contribution to the establishment of the intestinal apical membrane. Using CRISPR/Cas9 gene editing and live-cell imaging, we show that an ERM-1 PIP2-binding mutant mimics the
erm-1 null phenotype, consistent with binding to PIP2 being critical for ERM-1 function. However, phosphorylation mimicking and blocking mutants are viable and show defects whose severity varies between tissues. We show that apical enrichment and stability of ERM-1 are decreased in intestinal cells of phosphorylation mutants. Furthermore, loss of phosphorylation affects actin organization and trafficking at the apical membrane. As phosphorylation was not essential for ERM-1 functioning, we investigated various conserved ERM-1 regulators, including NRFL-1, the sole C. elegans ortholog of the NHERF family, and a candidate kinase. Surprisingly, depletion of NRFL-1 synergizes with loss of ERM-1 phosphorylation, resulting in intestinal defects resembling complete loss of
erm-1. We also identified a candidate ERM-1 kinase, whose loss caused major intestinal development defects similar to combined loss of
nrfl-1 and ERM-1 phosphorylation. Our data indicates that the kinase phosphorylates ERM-1 but also controls the localization of NRFL-1. Currently, we are investigating these genetic interactions on a mechanistic and molecular level.