Integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1

Journal ArticleCell adhesion and migration are dynamic pro- cesses requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture

Integrin Adhesions Suppress Syncytium Formation in the Drosophila Larval Epidermis

Funding Information: We thank members of M.J.G.’s lab for comments; Jodie Polan for confocal assistance; Guy Tanentzapf, Andreas Wodarz, and Talila Volk, for fly stocks/antibodies; the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the Kyoto stock center for fly strains; and the Developmental Studies Hybridoma Bank for antibodies. This work was supported by a March of Dimes Basil O’Connor Award (5-FY06-588) and NIH R01 GM083031 to M.J.G., NIH R01 GM084103 to J.L.K., and European Research Council Starting Grant (2007-StG-208631) to A.J. Publisher Copyright: © 2015 Elsevier Ltd. All rights reserved.Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components β-integrin, PINCH, and integrin-linked kinase (ILK) caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation, including that induced by wounding, genetic loss of FA proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss-of-function phenotype of integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper integrin-based adhesion in suppressing larval epidermal cell-cell fusion - a role that may be conserved in other epithelia.publishersversionpublishe

The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1

Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. Here, we show that PINCH, a protein required for integrin-dependent cell adhesion and actin–membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, we identify RSU-1, a regulator of Ras signaling in mammalian cells, as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. Our results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development

A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation

<p>Abstract</p> <p>Background</p> <p>Many temperate insects survive the harsh conditions of winter by undergoing photoperiodic diapause, a pre-programmed developmental arrest initiated by short day lengths. Despite the well-established ecological significance of photoperiodic diapause, the molecular basis of this crucial adaptation remains largely unresolved. The Asian tiger mosquito, <it>Aedes albopictus </it>(Skuse), represents an outstanding emerging model to investigate the molecular basis of photoperiodic diapause in a well-defined ecological and evolutionary context. <it>Ae. albopictus </it>is a medically significant vector and is currently considered the most invasive mosquito in the world. Traits related to diapause appear to be important factors contributing to the rapid spread of this mosquito. To generate novel sequence information for this species, as well as to discover transcripts involved in diapause preparation, we sequenced the transcriptome of <it>Ae. albopictus </it>oocytes destined to become diapausing or non-diapausing pharate larvae.</p> <p>Results</p> <p>454 GS-FLX transcriptome sequencing yielded >1.1 million quality-filtered reads, which we assembled into 69,474 contigs (N50 = 1,009 bp). Our contig filtering approach, where we took advantage of strong sequence similarity to the fully sequenced genome of <it>Aedes aegypti</it>, as well as other reference organisms, resulted in 11,561 high-quality, conservative ESTs. Differential expression estimates based on normalized read counts revealed 57 genes with higher expression, and 257 with lower expression under diapause-inducing conditions. Analysis of expression by qPCR for 47 of these genes indicated a high correlation of expression levels between 454 sequence data and qPCR, but congruence of statistically significant differential expression was low. Seven genes identified as differentially expressed based on qPCR have putative functions that are consistent with the insect diapause syndrome; three genes have unknown function and represent novel candidates for the transcriptional basis of diapause.</p> <p>Conclusions</p> <p>Our transcriptome database provides a rich resource for the comparative genomics and functional genetics of <it>Ae. albopictus</it>, an invasive and medically important mosquito. Additionally, the identification of differentially expressed transcripts related to diapause enriches the limited knowledge base for the molecular basis of insect diapause, in particular for the preparatory stage. Finally, our analysis illustrates a useful approach that draws from a closely related reference genome to generate high-confidence ESTs in a non-model organism.</p

Elevated Expression of the Integrin-Associated Protein PINCH Suppresses the Defects of <i>Drosophila melanogaster</i> Muscle Hypercontraction Mutants

<div><p>A variety of human diseases arise from mutations that alter muscle contraction. Evolutionary conservation allows genetic studies in <i>Drosophila melanogaster</i> to be used to better understand these myopathies and suggest novel therapeutic strategies. Integrin-mediated adhesion is required to support muscle structure and function, and expression of Integrin adhesive complex (IAC) proteins is modulated to adapt to varying levels of mechanical stress within muscle. Mutations in <i>flapwing</i> (<i>flw</i>), a catalytic subunit of myosin phosphatase, result in non-muscle myosin hyperphosphorylation, as well as muscle hypercontraction, defects in size, motility, muscle attachment, and subsequent larval and pupal lethality. We find that moderately elevated expression of the IAC protein PINCH significantly rescues <i>flw</i> phenotypes. Rescue requires PINCH be bound to its partners, Integrin-linked kinase and Ras suppressor 1. Rescue is not achieved through dephosphorylation of non-muscle myosin, suggesting a mechanism in which elevated PINCH expression strengthens integrin adhesion. In support of this, elevated expression of PINCH rescues an independent muscle hypercontraction mutant in muscle myosin heavy chain, <i>Mhc^Samba1</i>. By testing a panel of IAC proteins, we show specificity for PINCH expression in the rescue of hypercontraction mutants. These data are consistent with a model in which PINCH is present in limiting quantities within IACs, with increasing PINCH expression reinforcing existing adhesions or allowing for the <i>de novo</i> assembly of new adhesion complexes. Moreover, in myopathies that exhibit hypercontraction, strategic PINCH expression may have therapeutic potential in preserving muscle structure and function.</p> </div

Increased expression of a panel of IAC proteins has little effect on hypercontraction mutants.

<p>A) For each genotype, a normalized geotaxis score from >5 independent assays is plotted as the mean±SEM. * indicates p<0.05, and ns indicates p>0.05 when compared to the <i>Samba¹/+</i> data. B) Survival data for <i>flw⁷; 24B>Zyxin-GFP/+</i> is not significantly different than <i>flw⁷</i> alone (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003406#pgen-1003406-g002" target="_blank">Figure 2B</a>). Pupae present at day 12 do not progress to adulthood. C) Survival data for <i>flw⁷; Ubi-Talin/+</i> shows that larval lethality is increased compared to <i>flw⁷</i> alone (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003406#pgen-1003406-g002" target="_blank">Figure 2B</a>).</p

Increased expression of PINCH-Flag rescues geotaxis defects and thoracic indentation, but does not reverse hypercontraction of the IFM in the <i>Samba¹</i> mutant.

<p>A) For each genotype, a normalized geotaxis score from >5 independent assays is plotted as the mean±SEM. *** indicates p<0.001 and ns indicates p>0.05 when compared to the corresponding control data. B) Representative polarized light images of thoraces show that the IFM in the <i>Samba¹</i> mutant exhibits hypercontraction that is not rescued upon expression of PINCH-Flag. Asterisks indicate areas of hypercontraction within the structure of the IFM. C) A dorsal view shows that the thoracic indentations prevalent in <i>Samba1/+</i> adults are rescued upon expression of transgenic PINCH-Flag. White arrowheads indicate the areas of indentation.</p

PINCH^D303V mutation disrupts binding to RSU1.

<p>A) Schematic of PINCH showing its 5 LIM domain structure. The N-terminal ankyrin repeat of ILK binds to LIM1 of PINCH via PINCH^Q38. Parvin associates with PINCH indirectly via binding to the ILK pseudokinase domain. RSU1 binds to LIM5 of PINCH through PINCH^D303. B) Sequence alignment of the C-terminus of fly, mouse and human PINCH sequences demonstrates the conservation of D303 (boxed with arrowhead). Asterisks denote invariant residues, and colons denote conserved residues. C) Sequence alignment using the 5 individual LIM domains of <i>Drosophila</i> PINCH shows that D303 (boxed with arrowhead) is a variable residue within LIM sequences. Dots denote the zinc binding residues. D) Ni-NTA pull-downs of wild type PINCH-His and PINCH^D303VHis expressed in S2 cells show a disruption of RSU1 binding only in the D303V mutant while ILK binding is preserved. E) Flag immunoprecipitations of adult fly lysates from PINCH null mutants rescued with either wild type PINCH-Flag or PINCH^D303VFlag transgenes. <i>w¹¹¹⁸</i> is used as a negative control that does not express Flag. ILK, RSU1 and a PINCH transgene are all expressed in the starting material. PINCH^D303VFlag pulls down ILK but fails to co-precipitate RSU1. In both D and E, densitometric analyses were conducted to compare levels of protein between samples and quantification is provided below each blot. α-tubulin was used as a loading control and values for PINCH, RSU1 and ILK were adjusted to account for variation in loading.</p

PINCH associates with ILK and RSU1 in order to suppress the larval lethality of <i>flw⁷</i> mutants.

<p>Survival curves show normal developmental progression upon expression of transgenic PINCH^Q38AFlag (A) or PINCH^D303VFlag (C) alone. Pupae present at day 12 subsequently progress to adulthood. Upon introduction of the mutant PINCH transgenes into a <i>flw⁷</i> background (B,D), arrest during the larval stages is observed. E) Western blot plus densitometric analysis for PINCH shows the relative level of expression of the mutant Q38A and D303V transgenes as a percentage of endogenous PINCH. The ribosomal protein RACK1 was used as a loading control.</p

Moderately elevated expression of PINCH rescues muscle detachment defects of the <i>flw⁷</i> mutant.

<p>A–E) Representative images of four-day-old larvae of the indicated genotypes are shown. Ventral view allows the VIS muscles to be rapidly scored on a dissecting microscope. One of the medial pair of VIS muscles is boxed in A. White arrowheads show the relevant segment boundaries (T1,T2,T3). Areas of hypercontraction are indicated with an asterisk. Detachments are characterized by a gap in the GFP signal (arrow) Scale bar = 1 mm. F) The graph shows the percent of total animals in which the VIS muscles exhibit detachment. Number of animals examined of each genotype, pooled from triplicate experiments, is shown above the bars. ** indicates p<0.005, * indicates p<0.05, and ns indicates p>0.05.</p