The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development

Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease

An ongoing role for structural sarcomeric components in maintaining Drosophila melanogaster muscle function and structure.

Animal muscles must maintain their function while bearing substantial mechanical loads. How muscles withstand persistent mechanical strain is presently not well understood. The basic unit of muscle is the sarcomere, which is primarily composed of cytoskeletal proteins. We hypothesized that cytoskeletal protein turnover is required to maintain muscle function. Using the flight muscles of Drosophila melanogaster, we confirmed that the sarcomeric cytoskeleton undergoes turnover throughout adult life. To uncover which cytoskeletal components are required to maintain adult muscle function, we performed an RNAi-mediated knockdown screen targeting the entire fly cytoskeleton and associated proteins. Gene knockdown was restricted to adult flies and muscle function was analyzed with behavioural assays. Here we analyze the results of that screen and characterize the specific muscle maintenance role for several hits. The screen identified 46 genes required for muscle maintenance: 40 of which had no previously known role in this process. Bioinformatic analysis highlighted the structural sarcomeric proteins as a candidate group for further analysis. Detailed confocal and electron microscopic analysis showed that while muscle architecture was maintained after candidate gene knockdown, sarcomere length was disrupted. Specifically, we found that ongoing synthesis and turnover of the key sarcomere structural components Projectin, Myosin and Actin are required to maintain correct sarcomere length and thin filament length. Our results provide in vivo evidence of adult muscle protein turnover and uncover specific functional defects associated with reduced expression of a subset of cytoskeletal proteins in the adult animal

Sarcomeric actin undergoes turnover in adult muscles.

<p>(A) Graphic representation of the experimental design for the pulse-chase experiments. Act88F::GFP or eGFP was expressed under the control of the TARGET system for 28 hours directly after eclosion (the pulse) and then shut off (the chase). (B, B′) Localization of eGFP (B) or Act88F::GFP (B′) when expressed throughout development and adulthood with the Mef2:GAL4 system (F-actin is counterstained with rhodamine-phalloidin). eGFP non-specifically labels the whole sarcomere while Act88F::GFP is specifically localized to the sarcomeric core. (C,D) A time series of representative images of IFMs from flies expressing eGFP (C) or Act88F::GFP (D) under the control of the TARGET system (F-actin counterstained with rhodamine-phalloidin). The locations of the Z-discs and M-lines are indicated by a ‘z’ or ‘m’ in both panels. (E) Quantification of fluorescent intensity at the Z-disc for eGFP during TARGET pulse-chase experiment. Intensity at the Z-disc increased during the 28 hr pulse phase but did not decline afterwards. Instead, intensity remained elevated. (F) Quantification of fluorescent intensity at the Z-disc for Act88F::GFP during TARGET pulse-chase experiment. Intensity at the Z-disc increased during the 28 hour pulse phase and then gradually declined. (G) The ratio of Z-disc to sarcomere body fluorescent intensity during the pulse-chase experiment shows that the increase in staining intensity is specific to the Z-disc for Act88F::GFP. Comparison of Z-disc to sarcomere body intensity ratios for eGFP (grey bars) and Act88F::GFP (black bars) expression shows that while eGFP localization is non-specific, Act88F::GFP is significantly enriched at the Z-disc. (H) Examples showing how Z-disc and sarcomere intensity was measured for both TARGET>eGFP and TARGET>Act88F::GFP myofibrils. Phalloidin counterstains were used to identify relevant areas. Red rectangles indicate the area quantified for Z-disc intensities. Irregular blue tetragons indicate the area quantified for sarcomere body intensities. For (E,F,G) N≥40 individual sarcomeres from ≥5 animals. For all panels, error bars indicate standard error; ns indicates not significant; * indicates a p-value<0.05, ** indicates a p-value<0.005, *** indicates a p-value<0.0005. TARGET>eGFP and TARGET>Act88F::GFP are abbreviated as T>eGFP and T>Act88F::GFP throughout.</p

Results of the Mef2:GAL4 screen of the role cytoskeleton muscle function.

<p>(A) Phenotypic breakdown of results from the Mef2:GAL4 screen. The Mef2:GAL4 screen expressed the RNAi constructs throughout both development and adulthood. By assaying or developmental lethality or climbing ability, we observed that 106 genes had no role in muscle development or maintenance (None, brown), 20 were Embryo Lethal, 68 were Pupal Lethal, and 44 caused a Climbing Defect. (B) Enrichment analysis of Gene Ontology (GO) terms by phenotype. Enrichment ratios were calculated by comparing frequency of a term in a specific phenotypic class to the frequency of the same term in the entire screened set. GO terms are listed on the left. An enrichment ratio of 0 indicates that a given term did not appear in the phenotypic group. An enrichment ratio of <1 indicates that the frequency for the term was reduced in the phenotypic class compared to the whole screened set. An enrichment ratio of 1 indicates that the frequency for the term was the same in the phenotypic class compared to the whole screened set. An enrichment ratio of >1 indicates that the frequency for the term was the enriched in the phenotypic class compared to the whole screened set.</p

Sarcomere length defects are due to disruption of thin filament length.

<p>Distribution of actin across the sarcomere was used to determine sarcomere length, thin filament length and H-zone width. (A) Sample image of a single sarcomere labeled with phalloidin to show actin filaments. Black overlay lines indicate quantified parameters. (B) Sample profile of a sarcomere showing the normalized fluorescent intensities of pixels found along the overlay line labeled ‘Sarcomere’ in A. Measured parameters, “Sarcomere Length”, “Thin Filament Length” and “H-zone Width”, are indicated. (C-E) Quantification of Sarcomere Length, Thin Filament Length, and H-zone width respectively in control and <i>bent</i> RNAi construct-expressing flies. Sarcomere and thin filament length does not significantly change in the control flies, but significantly shortens in the <i>bent</i> RNAi construct-expressing flies over six days. (F-H) Quantification of Sarcomere Length, Thin Filament Length, and H-zone width respectively in control and <i>Mhc</i> RNAi construct-expressing flies. Sarcomere and thin filament does not significantly change in the control flies, but significantly increases in the <i>Mhc</i> RNAi construct-expressing flies over six days. (I-K) Quantification of Sarcomere Length, Thin Filament Length, and H-zone width respectively in control and <i>Mhc</i> RNAi construct-expressing flies. Sarcomere and thin filament length does not significantly change in the control flies, but significantly increases in the <i>Mhc</i> RNAi construct-expressing flies over six days. Representative images of phalloidin stainings can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099362#pone-0099362-g007" target="_blank">Figure 7D-G</a> For all panels, error bars indicate standard error; n.s. indicates a p-value>0.05, *** indicates a p-value<0.0005. For (C-K) N≥30 sarcomeres from 5 animals at each time-point.</p

Sample assay graphs from TARGET screen.

<p>Severity of the climbing phenotypes identified in the TARGET screen were classified based on when flies lost the ability to climb. Loss of climbing ability between Day 0 and Day 9 were classified as ‘Severe’; between Day 12 and Day 21 were classified as ‘Intermediate’; between Day 24 and Day 30 were classified as ‘Weak’; and ‘None’ if climbing ability was not altered. Representative samples for each of the four classes were selected. (A-C) Examples of ‘Severe’ phenotypes. <i>up</i> RNAi (A), <i>Mhc</i> RNAi (B), and <i>Act88F</i> RNAi (C). In total, 5 genes had caused ‘Severe’ climbing defects. (D-F) Examples of ‘Intermediate’ phenotypes. <i>bent</i> RNAi (D), <i>act79B</i> RNAi (E) and <i>rab5</i> RNAi (F). In total, 13 genes caused ‘Intermediate’ phenotypes. (G-I) Examples of ‘Weak’ phenotypes. <i>EB1</i> RNAi (G), <i>myo28B</i> RNAi (H) and actr13E RNAi (I). In total, 28 genes caused Weak' phenotypes. J-L. Examples of ‘None’ phenotypes. <i>shot</i> RNAi (J), <i>msps</i> RNAi (K) and <i>βTub60D</i> RNAi (L). In total, 86 genes had no effect on adult climbing ability. For all graphs, blue lines are the control and red lines are the RNAi-mediated gene knockdown line. Error bars are standard error. For all lines, climbing ability was normalized to the first timepoint, Day 0, before RNAi construct expression was started.</p