Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II–containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.

The assembly of a three-dimensional actin cytoskeleton is largely mediated by local Rho GTPases. Restriction of microvilli to the epithelial apical surface requires local regulation of actin structure. On epithelial cells, the presence of microvilli depends on RhoA activation of its effector kinases LOK and SLK that phosphorylate ezrin to function as a membrane-actin linker. Further, previous work has revealed that active ezrin negatively regulates RhoA, but the mechanism was unknown. Here, we show this regulation is mediated by recruitment and activation of ARHGAP18 by active ezrin, which stimulates its GTPase activity towards RhoA. Loss of ARHGAP18 enhances ezrin phosphorylation and results in abundant stubby microvilli that turn over rapidly. This phenotype can be rescued by re-expression of ARHAGP18, and by a construct that targets just the GTPase domain to active ezrin, but not a GAP-dead version. Loss of ARHGAP18 also results in enhanced phosphorylation of myosin light chain, the classic response to elevated RhoA levels. This leads to inappropriate assembly of non-muscle myosin-II into microvilli and the disruption of the normal apical actomyosin organization. Thus, the localization and activation of ARGHAP18 by active ezrin constitutes a local auto-regulatory module to fine tune RhoA to ensure appropriate microvilli organization.

AbstractActivated Ezrin-Radixin-Moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate domain specific structures, such as microvilli. Among many kinases implicated in ERM activation are the homologs LOK and SLK. CRISPR/Cas9 was used to knockout all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM activating phosphorylation. The apical domain of cells lacking LOK/SLK or ERMs is strikingly similar, with loss of microvilli, alterations in junctions, induction of myosin-II containing apical stress-fiber-like structures with enhanced myosin-light-chain (MLC) phosphorylation, and enhanced apical tension. Both knockout lines have elevated active RhoA, revealing that active ERMs are negative regulators of RhoA. Constitutively active ezrin can reverse the phenotypes of either ERMs or LOK/SLK knockouts, showing that the major function of LOK/SLK is to activate ERMs. Thus, LOK/SLK and ERM proteins work as a module to regulate RhoA and contribute to the proper apical morphology of epithelial cells.

Coke Zero 375ml cans as sold in vending machines were launched in the US market in 2005, as a sugar-free version of Coca-Cola that maintains the same taste. The product is controlled by the Coca-Cola Company, a multinational firm that is most famous for its namesake product CocaCola, but also produces a range of sodas, spring waters, bottled teas, and other beverages.1 Cans of Coke Zero sold through vending machines are marketed as a convenient product for 18-22-year-old health-conscious male cola-drinkers who identify with masculinity. As the product is currently within the growth stage of the product life cycle and market penetration is the most appropriate strategy, its marketing mix strategy has evolved accordingly. Product features, convenience pricing strategy, customer relationship management (CRM) promotional strategy, and place utility with vending machine distribution have all been effectively utilised in order to maximise market penetration among the targeted customer segment.