Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans

Effects of a balanced translocation between chromosomes 1 and 11 disrupting the DISC1 locus on white matter integrity

Objective Individuals carrying rare, but biologically informative genetic variants provide a unique opportunity to model major mental illness and inform understanding of disease mechanisms. The rarity of such variations means that their study involves small group numbers, however they are amongst the strongest known genetic risk factors for major mental illness and are likely to have large neural effects. DISC1 (Disrupted in Schizophrenia 1) is a gene containing one such risk variant, identified in a single Scottish family through its disruption by a balanced translocation of chromosomes 1 and 11; t(1;11) (q42.1;q14.3). Method Within the original pedigree, we examined the effects of the t(1;11) translocation on white matter integrity, measured by fractional anisotropy (FA). This included family members with (n = 7) and without (n = 13) the translocation, along with a clinical control sample of patients with psychosis (n = 34), and a group of healthy controls (n = 33). Results We report decreased white matter integrity in five clusters in the genu of the corpus callosum, the right inferior fronto-occipital fasciculus, acoustic radiation and fornix. Analysis of the mixed psychosis group also demonstrated decreased white matter integrity in the above regions. FA values within the corpus callosum correlated significantly with positive psychotic symptom severity. Conclusions We demonstrate that the t(1;11) translocation is associated with reduced white matter integrity in frontal commissural and association fibre tracts. These findings overlap with those shown in affected patients with psychosis and in DISC1 animal models and highlight the value of rare but biologically informative mutations in modeling psychosis

Cancer cachexia impairs neural respiratory drive in hypoxia but not hypercapnia

Abstract Background Cancer cachexia is an insidious process characterized by muscle atrophy with associated motor deficits, including diaphragm weakness and respiratory insufficiency. Although neuropathology contributes to muscle wasting and motor deficits in many clinical disorders, neural involvement in cachexia‐linked respiratory insufficiency has not been explored. Methods We first used whole‐body plethysmography to assess ventilatory responses to hypoxic and hypercapnic chemoreflex activation in mice inoculated with the C26 colon adenocarcinoma cell line. Mice were exposed to a sequence of inspired gas mixtures consisting of (i) air, (ii) hypoxia (11% O2) with normocapnia, (iii) hypercapnia (7% CO2) with normoxia, and (iv) combined hypercapnia with hypoxia (i.e. maximal chemoreflex response). We also tested the respiratory neural network directly by recording inspiratory burst output from ligated phrenic nerves, thereby bypassing influences from changes in diaphragm muscle strength, respiratory mechanics, or compensation through recruitment of accessory motor pools. Results Cachectic mice demonstrated a significant attenuation of the hypoxic tidal volume (0.26mL±0.01mL vs 0.30mL±0.01mL; p0.05), breathing frequency (392±5bpm vs 408±5bpm; p>0.05) and phrenic nerve (93.1±8.8% vs 111.1±13.2%; p>0.05) responses were not affected. Further, the concurrent hypercapnia/hypoxia tidal volume (0.45±0.01mL vs 0.45±0.01mL; p>0.05), breathing frequency (395±7bpm vs 400±3bpm; p>0.05), and phrenic nerve (106.8±7.1% vs 147.5±38.8%; p>0.05) responses were not different between C26 cachectic and control mice. Conclusions Breathing deficits associated with cancer cachexia are specific to the hypoxic ventilatory response and, thus, reflect disruptions in the hypoxic chemoafferent neural network. Diagnostic techniques that detect decompensation and therapeutic approaches that support the failing hypoxic respiratory response may benefit patients at risk for cancer cachectic‐associated respiratory failure

Synthetic microbe-to-plant communication channels

Abstract Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a “sender device” in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a “receiver device” in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia

Acute loss of integrin-based attachment induces general cytosolic protein degradation via a common mechanism.

<p>A) Age synchronised wild-type L1 larvae were grown to young adulthood at 16°C (t = 0 h) before transferring to NGM RNAi plates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471-Fraser1" target="_blank">[87]</a> seeded with bacteria expressing dsRNA against genes indicated for an additional 72 h (mid-adulthood) at 20°C. The blue stain that appears as circles in the centre of t = 72 h animals is stain in muscles of developing embryos (for example+<i>pat-2</i> and <i>deb-1</i> RNAi). The blue stain that appears as lines in the t = 72 h animals is indicative of <i>lacZ</i> expressing bacteria in the gut (typically near the head, for example+<i>unc-112</i> RNAi). B) <i>dim-1(ra102)</i> mutants were cultured identically to A. C) Wild-type, <i>unc-52^ts</i>, <i>unc-112^ts</i>, <i>unc-112^ts</i>; <i>dim-1(gk54)</i>, and <i>unc-112^ts</i>; <i>dim-1(ra102)</i> animals were age synchronised at L1 stage and grown to young adulthood (t = 0 h) at 16°C, and cultured for an additional 72 h at either 16°C (permissive temperature for the mutation) or 25°C (non-permissive temperature). <i>unc-52^ts</i> and <i>unc-112^ts</i> animals were also cultured under the same conditions in the presence of cycloheximide (+CHx) at 400 µg/ml. In A, B and C approximately 20–30 animals were stained for β-galactosidase activity (blue) at t = 0 h and after 24 h, 48 h (not shown) and 72 h. D) Representative immunoblot analysis of 146-kDa β-galactosidase fusion protein in 30-worm lysates, cultured under the same conditions as in C after temperature-shift to 25°C only. All experiments in A, B, C and D were repeated a minimum of three times. E) Kinetics of loss of β-galactosidase protein from 16°C (t = 0 h) after temperature-shift to 25°C in wild-type (solid line), <i>unc-52^ts</i> (large dashed line) or <i>unc-112^ts</i> (small dashed line) animals. *,**Significant difference between <i>unc-112^ts</i> versus wild-type (P<0.01, P<0.001). †Significant difference between <i>unc-52^ts</i> versus wild-type (P<0.01). F) Kinetics of loss of β-galactosidase protein from 16°C (t = 0 h) after temperature-shift to 25°C in <i>unc-112^ts</i> (small dashed line), <i>unc-112^ts</i>; <i>dim-1(gk54)</i> (large dashed line) or <i>unc-112</i>^ts; <i>dim-1(ra102)</i> (solid line) animals. **Significant difference between <i>unc-112^ts</i> versus <i>unc-112^ts</i>; <i>dim-1(gk54)</i> and <i>unc-112^ts</i>; <i>dim-1(ra102)</i> (P<0.001). Values in E and F are the average of three immunoblots ± SEM. Level of significance in all indicated cases from two way repeated measures ANOVA. Scale bars represent 100 µm.</p

Calpains are important for maintenance of adult <i>C. elegans</i> muscle.

<p>A) Animals expressing a full length translational fusion of <i>gfp</i> to <i>myo-3</i> (myosin heavy chain A) were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). Adult animals were then transferred to NGM RNAi plates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471-Fraser1" target="_blank">[87]</a> seeded with bacteria expressing dsRNA against genes indicated for a further 72 h to mid-adulthood. 20 random animals were picked and scored for identical defects in sarcomere structure in at least two muscles within the animal and this was repeated for 5 independent RNAi treatments (n = 100 animals per condition/time point). Displayed is the percentage of animals where torn or collapsed arrays of sarcomeres were observed (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s005" target="_blank">Figure S5</a>. **Significant difference from control (t = 72 h, (P<0.001)). B) Animals expressing GFP labelled mitochondria and nuclei were grown, treated and analysed as in A with the exception that mitochondrial structure was scored. Displayed is the percentage of animals where moderate fragmentation of the mitochondrial network was observed (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s005" target="_blank">Figure S5</a>. *, **Significant difference from control (t = 72 h, (P<0.01, P<0.001)). C) Animals expressing GFP labelled attachment complexes (UNC-95::GFP) were grown, treated and analysed as in A with the exception that attachment complex structure was scored. Displayed is the percentage of animals where torn or collapsed arrays of sarcomeres were observed were observed (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s005" target="_blank">Figure S5</a>. **Significant difference from control (t = 72 h, (P<0.001)). D) Wild-type, <i>unc-112^ts</i>, and <i>unc-112^ts</i>; <i>dim-1</i>(<i>gk54</i>) were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). Adult animals were then transferred to 25°C and grown for a further 72 h to mid-adulthood. Some <i>unc-112^ts</i> animals were also placed on calpain inhibitor II drug plates (5 µg/ml) at t = 0 h and cultured on drug plates for a further 72 h. 30 animals were picked for western blot analysis of DEB-1 levels at t = 0 h, and at 72 h. All experiments were performed at least three times. Displayed are representative western blots for each condition and a graph of the initial DEB-1 remaining at 72 h (average ± SEM for three independent experiments). **Significant difference from all other conditions (P<0.001). All significance values are from two way repeated measures ANOVA.</p

Calpains are important for development of <i>C. elegans</i> muscle.

<p>Wild-type animals expressing a full length translational fusion of <i>gfp</i> to <i>myo-3</i> (myosin heavy chain A), GFP labelled mitochondria and nuclei, or GFP labelled attachment complexes (UNC-95::GFP) were cultured from L4 stage to young adulthood under normal conditions at 20°C and on RNAi targeting <i>clp-1</i>, <i>clp-4</i>, <i>tra-3</i>, <i>clp-6</i> or <i>clp-7</i>. 20 random animals were scored for identical sub-cellular defects in at least two muscles at adulthood and 24 and 48 h post-adulthood in both the F1 and F2 generations (e.g. n = 120 per condition). A) Percentage of animals with normally arrayed sarcomeres (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s006" target="_blank">Figure S6</a>. *, **Significant difference from control (P<0.01, P<0.001). B) Percentage of animals with networked mitochondria (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s006" target="_blank">Figure S6</a>. *Significant difference from control (P<0.01). C) Percentage of animals with arrayed attachment complexes (average ± SEM). Example images for each treatment can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s006" target="_blank">Figure S6</a>. *Significant difference from control (P<0.01). All significance values are from one way ANOVA.</p

Acute loss of core integrin attachment complex members leads to collapse of attachments and multiple pathologies.

<p>Collapse of attachment complexes into ball like structures was assessed (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s003" target="_blank">Figure S3</a>) and compared with the severity of other pathologies associated with attachment complex disruption (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen-1002471-g001" target="_blank">Figure 1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen-1002471-g002" target="_blank">Figure 2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen-1002471-g003" target="_blank">Figure 3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen-1002471-g004" target="_blank">Figure 4</a>, and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s001" target="_blank">Figures S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s002" target="_blank">S2</a>). Pathologies identified as significantly different from matched controls (t = 72 h, P<0.001 two way repeated measures ANOVA) were placed into identical groupings based upon severity of defect. In the case of the movement defect the extent of severity was established as follows. First, the core complex members were considered as a group and examined against the remaining components for lack of significant difference from any member of the group (t = 72 h, P>0.05 one way ANOVA). Next, the remaining components were examined for groups of components where a significant difference between individual components within a group of components did not exist (t = 72 h, P>0.05 one way ANOVA) but a significant difference between every member of the group and all other components did exist (P<0.01, one way ANOVA). Thus, the colour coding for the extent of pathology as displayed in the inset legend reflects statistically significant differences in severity of defects.</p