[PMC free article] [PubMed] [Google Scholar]Paul NR, Jacquemet G, Caswell PT

[PMC free article] [PubMed] [Google Scholar]Paul NR, Jacquemet G, Caswell PT. substratum, typically the ECM. Small GTPases of the Rho family mediate front/rear polarization (Ridley is usually a valuable model for the genetic analysis of wound healing, as it has been studied at several developmental stages (Kiehart mutants (Campos as a model to study integrin function in wound RE and statement here our systematic analysis of the epithelial requirements for specific and integrins and core adhesion components in wound closure, the pathways regulating their accumulation, and their contributions to cell polarization. RESULTS AND Conversation PS2/PS and PS3/PS integrin dimers both contribute to epithelial wound closure To examine integrin involvement in wound healing RE, we knocked down and integrins by expressing transgenes in the larval epidermis using (Galko and Krasnow, 2004 ). For each gene, we tested nonoverlapping RNA interference (RNAi) lines to exclude off-target effects (Mohr and Perrimon, 2012 ) and coexpressed when necessary to enhance knockdown efficiency (Dietzl (knockdown in the larval epidermis of third instar larvae, denoted only control. (F) (J), (L), and (N) larvae. Yellow arrowheads show trachea. Scale bar: 25 m (ICN). (O) Quantification of the wound closure defects in the larvae of indicated genotypes. At least six animals were analyzed for each genotype. Epidermal knockdown of (knockdown larvae experienced closed, in a delayed manner (Physique 1, D and O, and Supplemental Physique S1B). Wound measurements 20 min after injury in wild-type (WT) vs. knockdown larvae found no significant difference in size (Supplemental Physique S2). In further support for any model whereby slowed wound healing in (Physique 1, I and J, and Supplemental Physique S1C). Homozygotes for null alleles of the other -integrin, (((in the wing disk phenocopied the wing blisters of null mutants (unpublished data; Brower and Jaffe, 1989 ), indicating the RNAi constructs were functional. Anti-PS2 and anti-PS3 antibodies CP-724714 detected the corresponding proteins primarily in the plasma membrane of epidermal cells (Physique CP-724714 1, K and M); in the knockdown larvae, expression levels were severely reduced (Physique 1, L and N, and Supplemental Physique S1, L and M). Altogether, these results indicate that the lack of phenotype was not due to knockdown failure. We then examined double knockdowns for the three PS genes. Of the three different combinations, only the RNAi pair showed defects, with 96%C82% of the larvae displaying open wounds at 14 h after wounding, (Physique 1, ECH and O, and Supplemental Physique S1K). This reveals that PS2 and PS3 integrins do function in larval epidermal wound closure but may compensate for each other. This CP-724714 is reminiscent of the contemporaneous but unique functions that PS2 and PS3 play in CP-724714 glial cell migration and axon guidance (Tavares caused wound closure defects as severe as the defects, with 100% of the wounds still open at 16 h (Physique 2, A and B, and Supplemental Physique S1, NCP). Open in a separate window Physique 2: Talin is required for efficient wound closure, unlike other components and regulators of integrin-containing adhesive structures. Analysis of wound closure in the epidermis of late third instar larvae at 16 h. (A) was added to to enhance knockdown efficiency. For each genotype, at least six wounded animals were analyzed. (C) Analysis of the knockdown efficiency Abcc4 of each transgene using and qRT-PCR. Cross symbols indicate not tested (for to increase efficiency, but in no case observed a.