E-Cadherin/HMR-1 Membrane Enrichment Is Polarized by WAVE-Dependent Branched Actin.

Polarized epithelial cells adhere to each other at apical junctions that connect to the apical F-actin belt. Regulated remodeling of apical junctions supports morphogenesis, while dysregulated remodeling promotes diseases such as cancer. We have documented that branched actin regulator, WAVE, and apical junction protein, Cadherin, assemble together in developing C. elegans embryonic junctions. If WAVE is missing in embryonic epithelia, too much Cadherin assembles at apical membranes, and yet apical F-actin is reduced, suggesting the excess Cadherin is not fully functional. We proposed that WAVE supports apical junctions by regulating the dynamic accumulation of Cadherin at membranes. To test this model, here we examine if WAVE is required for Cadherin membrane enrichment and apical-basal polarity in a maturing epithelium, the post-embryonic C. elegans intestine. We find that larval and adult intestines have distinct apicobasal populations of Cadherin, each with distinct dependence on WAVE branched actin. In vivo imaging shows that loss of WAVE components alters post-embryonic E-cadherin membrane enrichment, especially at apicolateral regions, and alters the lateral membrane. Analysis of a biosensor for PI(4,5)P2 suggests loss of WAVE or Cadherin alters the polarity of the epithelial membrane. EM (electron microscopy) illustrates lateral membrane changes including separations. These findings have implications for understanding how mutations in WAVE and Cadherin may alter cell polarity.

WAVE regulates Cadherin junction assembly and turnover during epithelial polarization.

Actin is an integral component of epithelial apical junctions, yet the interactions of branched actin regulators with apical junction components are still not clear. Biochemical data have shown that α-catenin inhibits Arp2/3-dependent branched actin. These results suggested that branched actin is only needed at earliest stages of apical junction development. We use live imaging in developing C. elegans embryos to test models for how WAVE-induced branched actin collaborates with other apical junction proteins during the essential process of junction formation and maturation. We uncover both early and late essential roles for WAVE in apical junction formation. Early, as the C. elegans intestinal epithelium becomes polarized, we find that WAVE components become enriched concurrently with the Cadherin components and before the DLG-1 apical accumulation. Live imaging of F-actin accumulation in polarizing intestine supports that the Cadherin complex components and branched actin regulators work together for apical actin enrichment. Later in junction development, the apical accumulation of WAVE and Cadherin components is shown to be interdependent: Cadherin complex loss alters WAVE accumulation, and WAVE complex loss increases Cadherin accumulation. To determine why Cadherin levels rise when WVE-1 is depleted, we use FRAP to analyze Cadherin dynamics and find that loss of WAVE as well as of the trafficking protein EHD-1/RME-1 increases Cadherin dynamics. EM studies in adults depleted of branched actin regulators support that WVE-1 maintains established junctions, presumably through its trafficking effect on Cadherin. Thus we propose a developmental model for junction formation where branched actin regulators are tightly interconnected with Cadherin junctions through their previously unappreciated role in Cadherin transport.

Bacterial communities in the small intestine respond differently to those in the caecum and colon in mice fed low- and high-fat diets.

Bacterial communities in the mouse caecum and faeces are known to be altered by changes in dietary fat. The microbiota of the mouse small intestine, by contrast, has not been extensively profiled and it is unclear whether small intestinal bacterial communities shift with dietary fat levels. We compared the microbiota in the small intestine, caecum and colon in mice fed a low-fat (LF) or high-fat (HF) diet using 16S rRNA gene sequencing. The relative abundance of major phyla in the small intestine, Bacteriodetes, Firmicutes and Proteobacteria, was similar to that in the caecum and colon; the relative abundance of Verrucomicrobia was significantly reduced in the small intestine compared to the large intestine. Several genera were uniquely detected in the small intestine and included the aerotolerant anaerobe, Lactobacillus spp. The most abundant genera in the small intestine were accounted for by anaerobic bacteria and were identical to those identified in the large intestine. An HF diet was associated with significant weight gain and adiposity and with changes in the bacterial communities throughout the intestine, with changes in the small intestine differing from those in the caecum and colon. Prominent Gram-negative bacteria including genera of the phylum Bacteroidetes and a genus of Proteobacteria significantly changed in the large intestine. The mechanistic links between these changes and the development of obesity, perhaps involving metabolic endotoxemia, remain to be determined.

WAVE/SCAR promotes endocytosis and early endosome morphology in polarized C. elegans epithelia.

Cells can use the force of actin polymerization to drive intracellular transport, but the role of actin in endocytosis is not clear. Studies in single-celled yeast demonstrate the essential role of the branched actin nucleator, Arp2/3, and its activating nucleation promoting factors (NPFs) in the process of invagination from the cell surface through endocytosis. However, some mammalian studies have disputed the need for F-actin and Arp2/3 in Clathrin-Mediated Endocytosis (CME) in multicellular organisms. We investigate the role of Arp2/3 during endocytosis in Caenorhabditis elegans, a multicellular organism with polarized epithelia. Arp2/3 and its NPF, WAVE/SCAR, are essential for C. elegans embryonic morphogenesis. We show that WAVE/SCAR and Arp2/3 regulate endocytosis and early endosome morphology in diverse tissues of C. elegans. Depletion of WAVE/SCAR or Arp2/3, but not of the NPF Wasp, severely disrupts the distribution of molecules proposed to be internalized via CME, and alters the subcellular enrichment of the early endosome regulator RAB-5. Loss of WAVE/SCAR or of the GEFs that regulate RAB-5 results in similar defects in endocytosis in the intestine and coelomocyte cells. This study in a multicellular organism supports an essential role for branched actin regulators in endocytosis, and identifies WAVE/SCAR as a key NPF that promotes Arp2/3 endocytic function in C. elegans.

Arp2/3 promotes junction formation and maintenance in the Caenorhabditis elegans intestine by regulating membrane association of apical proteins.

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin-radixin-moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes.

Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis.

The TOCA family of F-BAR-containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell-cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP-dependent actin-dynamics.

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis.

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.

The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance.

In the developing nervous system, axons are guided to their targets by the growth cone. Lamellipodial and filopodial protrusions from the growth cone underlie motility and guidance. Many molecules that control lamellipodia and filopodia formation, actin organization, and axon guidance have been identified, but it remains unclear how these molecules act together to control these events. Experiments are described here that indicate that, in Caenorhabditis elegans, two WH2-domain-containing activators of the Arp2/3 complex, WVE-1/WAVE and WSP-1/WASP, act redundantly in axon guidance and that GEX-2/Sra-1 and GEX-3/Kette, molecules that control WAVE activity, might act in both pathways. WAVE activity is controlled by Rac GTPases, and data are presented here that suggest WVE-1/WAVE and CED-10/Rac act in parallel to a pathway containing WSP-1/WASP and MIG-2/RhoG. Furthermore, results here show that the CED-10/WVE-1 and MIG-2/WSP-1 pathways act in parallel to two other molecules known to control lamellipodia and filopodia and actin organization, UNC-115/abLIM and UNC-34/Enabled. These results indicate that at least three actin-modulating pathways act in parallel to control actin dynamics and lamellipodia and filopodia formation during axon guidance (WASP-WAVE, UNC-115/abLIM, and UNC-34/Enabled).