Fat Body Cells Are Motile and Actively Migrate to Wounds to Drive Repair and Prevent Infection

Adipocytes have many functions in various tissues beyond energy storage, including regulating metabolism, growth, and immunity. However, little is known about their role in wound healing. Here we use live imaging of fat body cells, the equivalent of vertebrate adipocytes in Drosophila, to investigate their potential behaviors and functions following skin wounding. We find that pupal fat body cells are not immotile, as previously presumed, but actively migrate to wounds using an unusual adhesion-independent, actomyosin-driven, peristaltic mode of motility. Once at the wound, fat body cells collaborate with hemocytes, Drosophila macrophages, to clear the wound of cell debris; they also tightly seal the epithelial wound gap and locally release antimicrobial peptides to fight wound infection. Thus, fat body cells are motile cells, enabling them to migrate to wounds to undertake several local functions needed to drive wound repair and prevent infections

Integration of contractile forces during tissue invagination

Contractile forces generated by the actomyosin cytoskeleton within individual cells collectively generate tissue-level force during epithelial morphogenesis. During Drosophila mesoderm invagination, pulsed actomyosin meshwork contractions and a ratchet-like stabilization of cell shape drive apical constriction. Here, we investigate how contractile forces are integrated across the tissue. Reducing adherens junction (AJ) levels or ablating actomyosin meshworks causes tissue-wide epithelial tears, which release tension that is predominantly oriented along the anterior–posterior (a-p) embryonic axis. Epithelial tears allow cells normally elongated along the a-p axis to constrict isotropically, which suggests that apical constriction generates anisotropic epithelial tension that feeds back to control cell shape. Epithelial tension requires the transcription factor Twist, which stabilizes apical myosin II, promoting the formation of a supracellular actomyosin meshwork in which radial actomyosin fibers are joined end-to-end at spot AJs. Thus, pulsed actomyosin contractions require a supracellular, tensile meshwork to transmit cellular forces to the tissue level during morphogenesis.American Cancer Society (grant PF-06-143-01-DDC)National Institutes of Health (U.S.) (NIH/NIGMS, P50 grant GM071508)National Institutes of Health (U.S.) (NIH/NIGMS, R01 grant GM079340)Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (grant 5R37HD15587)Howard Hughes Medical Institute (Investigator

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4

Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis

Apical constriction is a cell shape change that promotes epithelial bending. Activation of nonmuscle myosin II (Myo-II) by kinases such as Rho-associated kinase (Rok) is important to generate contractile force during apical constriction. Cycles of Myo-II assembly and disassembly, or pulses, are associated with apical constriction during Drosophila melanogaster gastrulation. It is not understood whether Myo-II phosphoregulation organizes contractile pulses or whether pulses are important for tissue morphogenesis. Here, we show that Myo-II pulses are associated with pulses of apical Rok. Mutants that mimic Myo-II light chain phosphorylation or depletion of myosin phosphatase inhibit Myo-II contractile pulses, disrupting both actomyosin coalescence into apical foci and cycles of Myo-II assembly/disassembly. Thus, coupling dynamic Myo-II phosphorylation to upstream signals organizes contractile Myo-II pulses in both space and time. Mutants that mimic Myo-II phosphorylation undergo continuous, rather than incremental, apical constriction. These mutants fail to maintain intercellular actomyosin network connections during tissue invagination, suggesting that Myo-II pulses are required for tissue integrity during morphogenesis.National Institute of General Medical Sciences (U.S.) (Transgenic RNAi Project at Harvard Medical School, (R01-GM084947)

Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin

Listeria monocytogenes targets accessible E-cadherin expressed on mucus-producing goblet cells to invade the intestinal tissue

RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding.American Cancer Society (125792-RSG-14-039-01-CSM

The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction

Cadherin-based adherens junctions (AJs) mediate cell adhesion and regulate cell shape change. The nectin–afadin complex also localizes to AJs and links to the cytoskeleton. Mammalian afadin has been suggested to be essential for adhesion and polarity establishment, but its mechanism of action is unclear. In contrast, Drosophila melanogaster’s afadin homologue Canoe (Cno) has suggested roles in signal transduction during morphogenesis. We completely removed Cno from embryos, testing these hypotheses. Surprisingly, Cno is not essential for AJ assembly or for AJ maintenance in many tissues. However, morphogenesis is impaired from the start. Apical constriction of mesodermal cells initiates but is not completed. The actomyosin cytoskeleton disconnects from AJs, uncoupling actomyosin constriction and cell shape change. Cno has multiple direct interactions with AJ proteins, but is not a core part of the cadherin–catenin complex. Instead, Cno localizes to AJs by a Rap1- and actin-dependent mechanism. These data suggest that Cno regulates linkage between AJs and the actin cytoskeleton during morphogenesis

Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

The epithelial cadherin (E-cadherin)–catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell’s apical–basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling

Wnt, Hedgehog and Junctional Armadillo/β-Catenin Establish Planar Polarity in the Drosophila Embryo

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. Here we report that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless (Wg or Wnt1), provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. We further find that proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. Our results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm

Genome-Wide Association Analysis of Oxidative Stress Resistance in Drosophila melanogaster

Background: Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress. Methods and Findings: We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genomewide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79 % and 56–66 % of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis. Conclusions: We identified novel candidate genes associated with variation in resistance to oxidative stress that hav