Integrin-independent repression of cadherin transcription by talin during axis formation in Drosophila.

The Drosophila melanogaster anterior–posterior axis becomes polarized early during oogenesis by the posterior localization of the oocyte within the egg chamber. The invariant position of the oocyte is thought to be driven by an upregulation of the adhesion molecule DE-cadherin in the oocyte and the posterior somatic follicle cells, providing the first in vivo example of cell sorting that is specified by quantitative differences in cell–cell adhesion. However, it has remained unclear how DE-cadherin levels are regulated. Here, we show that talin, known for its role in linking integrins to the actin cytoskeleton, has the unexpected function of specifically inhibiting Decadherin transcription. Follicle cells that are mutant for talin show a strikingly high level of DE-cadherin, due to elevated transcription of DE-cadherin. We demonstrate that this deregulation of DE-cadherin is sufficient to attract the oocyte to lateral and anterior positions. Surprisingly, this function of talin is independent of integrins. These results uncover a new role for talin in regulating cadherin-mediated cell adhesion

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

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion

Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization

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

Characterization of adult muscle.

<p>The morphology, function and transcriptional activity of wild-type indirect flight muscles (IFM) was assayed at sequential time points during the first 30 days of fly life. (A) Simplified diagram showing the relaxed and contracted state of sarcomeres in a longitudinal section. The structural elements relevant to the work presented in this study have been included. Actin, or thin, filaments are shown in blue; Myosin, or thick, filaments are shown in red; Projectin is shown in green; the M-line is shown in yellow; and the Z-disc is shown in black. Note the slight shortening of the sarcomere in the contracted state, specifically in the H-zone and I-band as is characteristic of the IFM. (B) Simplified diagram of a transverse section of a sarcomere. As in (A), actin filaments are shown in blue and myosin filaments are shown in red. (C) Longitudinal Sections (LS) and (D) Transverse Sections of wild-type IFM sarcomeres show that the Z-disc, M-lines, actin and myosin filaments, and the myosin and actin lattice all exhibit consistent appearance over the first 30 days of fly life. (C: z, Z-disc. m, M-line; D: Larger circles are myosin, smaller dots are actin; Scale bars are in white and are 500 nm; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099362#pone-0099362-g001" target="_blank">Figure 1A</a> for schematic representations). (E) The climbing ability of flies was assayed using a negative geotaxis assay. Over the 30 day time course, the climbing ability of adult flies decreased in a linear fashion by 82%. N is 10 independent replicates each using 10 flies to assay climbing ability. (F) Key sarcomeric genes are continually transcribed in adult flies. RNA was extracted from whole flies and qPCR was used to assay for expression levels of <i>bent</i>, <i>Mhc</i>, and <i>Act88F</i>. GAPDH was used as the internal control for expression. Expression levels were normalized to the Day 0 timepoint. For all three genes, mRNA levels remained close, with slight variations, to the initial timepoint over the first 30 days of fly life. N≥3 for all qPCR time points, where each replicate was an independent RNA extraction using 10 flies.</p

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

Loss of <i>bent</i>, <i>Mhc</i> or <i>Act88F</i> does not disrupt sarcomeric architecture.

<p>Using phalloidin and antibody labelling, IFM integrity was examined before and after <i>bent</i>, <i>Mhc</i> or <i>Act88F</i> RNAi expression. (A-C) qPCR analysis confirmed that expression of RNAi constructs targeting <i>bent</i>, <i>Mhc</i> or <i>Act88F</i> leads to substantial reduction in transcript levels for all three genes compared to control flies of equivalent age. For (A-C), 3 independent RNA extractions using 10 flies for each time-point were performed. GAPDH was used as an internal expression control. (D-G) Phalloidin stainings of sarcomeric F-actin before and after RNAi expression showed no defects in thin filament organization after 6 days of RNAi construct expression for all three genes. Z-discs are bright lines and M-lines are dark lines as indicated by the labels ‘z’ and ‘m’ in the TARGET>OR Day 0 image. (H-K) To examine the integrity of key sarcomeric structures, we used antibodies to label the Z-disc and the M-line. Z-discs were labelled with an α-actinin antibody (green) and M-lines were labelled with an Obscurin antibody (red). No disruption to the integrity of either structure or to sarcomeric actin was observed for any of the three genes. For all panels, scale bars are given below all images in black and are 5 µm; error bars indicate standard error; n.s. indicates not significant; * indicates a p-value<0.05, ** indicates a p-value<0.005, *** indicates a p-value<0.0005.</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