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

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

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

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

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

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

Moderately elevated expression of wild-type PINCH rescues the size and motility defects of <i>flw⁷</i> mutants.

<p>A) The lengths of four-day-old larvae were measured, normalizing to the mean of a parallel <i>w¹¹¹⁸</i> control. Mean±SEM for each genotype is shown, with the number of animals measured (n) shown above the bar on the graph. **** indicates p<0.0001 compared to the <i>w¹¹¹⁸</i> control. B) Representative phase contrast images of six L3 larvae of the indicated genotypes. Scale bar = 1 mm. C) The larval motility assay uses a 5 cm grape agar plate divided into zones as shown. D) The average percentage of larvae located in each zone at the end of the motility assay is plotted for the indicated genotypes, arranged in order of decreasing motility.</p

LIM Domains Target Actin Regulators Paxillin and Zyxin to Sites of Stress Fiber Strain

<div><p>Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton. </p> </div

Paxillin aids stabilization of strain sites by altering actin dynamics independently of the zyxin.

<p>Comparison of normalized average paxillin and zyxin fluorescent intensity at strain site over time (A). Line scans through the strain site (B) show the spatial distribution of paxillin and zyxin fluorescence intensity (AIU) at strain sites. (C) Comparison of the catastrophic SF break frequency in paxillin null and paxillin rescued cells. (D) The actin recovery rate, as expressed by the slope of a line fit to actin intensity curve, in strain sites that do not break. (E) Western blot showing paxillin RNAi knocked down in zyxin null cells. (F) The actin recovery rate in cells lacking zyxin and with paxillin knock down. (G) Kymograph analysis of fluorescent labeled SF in zyxin null with paxillin knockdown cells. Note the SFSS slowly pull apart without repairing, but they rarely undergo catastrophic breaks within the imaging period.</p

Paxillin does not stabilize stress fiber strain sites through integrin signaling.

<p>Micrographs of fixed fibroblasts labeled for paxillin interacting protein FAK in conjunction zyxin-GFP, and Lifeact-mApple (A), and linescans through either a focal adhesion (B), or stress fiber strain site (C). FAK is present in focal adhesions, but is not found at stress fiber strain sites while zyxin localizes to both sites. Micrographs of fixed fibroblasts labeled for phospo-paxillin Y118 in conjunction zyxin-GFP, and Lifeact-mApple (D). Phospho-paxillin Y118 labels focal adhesions (E) but not strain sites (F). Similarly, phospho-tyrosine immunolocalization in conjunction zyxin-GFP, and Lifeact-mApple (G) detected phopho-tyrosine activation at focal adhesions (H), but not at strain sites (I). Scale bar=10um.</p