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 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

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

Acute loss of muscle attachment causes disorganisation and collapse of arrayed sarcomeres.

<p>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. The 20 most Unc 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). A) Percentage of animals where only normal arrays of sarcomeres were observed is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying normal arrays of sarcomeres (as indicated by straight parallel lines of GFP), these are enlarged 300% to the right of the micrograph. B) Percentage of animals where disorganisation of sarcomere arrays were observed is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying disorganised arrays of sarcomeres (as indicated by lack of straight parallel lines of GFP), these are enlarged 300% to the right of the micrograph. C) Percentage of animals where sarcomere arrays have collapsed into ball like structures is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying a collapsed array of sarcomeres (as indicated by parallel lines of GFP that are not straight, long, lines), these are enlarged 300% to the right of the micrograph. A sample image of an RNAi treatment against each gene can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s001" target="_blank">Figure S1</a>, not displayed here are animals with tears in the arrayed sarcomeres (see <i>unc-52</i> in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s001" target="_blank">Figure S1</a>). **Significant difference from control t = 72 h, P<0.001 (two way repeated measures ANOVA). Scale bars represent 15 µm.</p

Loss of muscle attachment results in mitochondrial fragmentation.

<p>Animals expressing GFP-tagged mitochondria were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). 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 an additional 72 h (mid-adulthood) at 20°C. The 20 most Unc animals were picked and scored for identical defects in mitochondrial 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). A) Percentage of animals where only networked mitochondria were observed is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying networked mitochondria (as indicated by the largely continuous parallel lines of GFP), these are enlarged 300% to the right of the micrograph and look arrayed like the sarcomeres (compare to enlargement in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen-1002471-g003" target="_blank">Figure 3A</a>). B) Percentage of animals where disorganisation of the mitochondrial network was observed is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying disorganisation of the mitochondrial network (as indicated by the lack of parallel lines of GFP), these are enlarged 300% to the right of the micrograph. C) Percentage of animals where moderate fragmentation (>30%) of the mitochondrial network is observed is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying moderate fragmentation of the mitochondrial network (as indicated by the largely non-continuous parallel lines of GFP), these are enlarged 300% to the right of the micrograph. D) Percentage of animals where severe fragmentation (>90%) of the mitochondrial network is observed is displayed as average ± SEM. Below the graph is an example of an RNAi treated animal displaying severe fragmentation of the mitochondrial network (as indicated by sparse GFP puncta), these are enlarged 300% to the right of the micrograph. A sample image of an RNAi treatment against each gene can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471.s002" target="_blank">Figure S2</a>. *, **Significant difference from control t = 72 h, (P<0.01, P<0.001 two way repeated measures ANOVA). Scale bars represent 15 µm.</p

Acute loss of muscle attachment results in a movement defect that does not occur in <i>dim-1(ra102)</i> mutants.

<p>A) Age synchronised wild-type (+/+), <i>unc-112^ts</i> heterozygotes (+/−) and <i>unc-112^ts</i> homozygotes (−/−) were grown to young adulthood at 16°C (t = 0 h) and cultured after temperature-shift to 25°C (mutation active temperature) for an additional 72 h. At t = 0 h, 24 h, 48 h and 72 h animals were analysed for movement rate by placing animals in BU buffer and counting the number of sinusoidal movements completed in one minute. Values shown are the average ± SEM of 5 animals measured 10 times to give a total of 50 independent measurements. **Significant movement decrease versus +/+ and +/− at respective time point (P<0.001). B) Wild-type and C) <i>dim-1(ra102)</i> animals were grown to young adulthood and cultured on RNAi plates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002471#pgen.1002471-Fraser1" target="_blank">[87]</a> against genes indicated on x-axis for an additional 72 h. Movement rate was measured as in A at t = 0 h and 72 h. Values shown are the average ± SEM of 10 animals measured 10 times to give a total of 100 independent measurements. **Significant movement decrease on respective RNAi treatment at 72 h versus wild-type control (P<0.001). ††Significant movement decrease in <i>dim-1(ra102)</i> mutants versus wild-type controls at 72 h (P<0.001). All experiments were repeated at least twice. All significance values are from two way repeated measures ANOVA.</p

Calpain inhibition blocks protein degradation induced by acute loss of attachment.

<p>A) Age synchronised L1 stage <i>unc-112^ts</i> mutants were gown to young adulthood at 16°C (t = 0 h) before being temperature shifted to 25°C and cultured for a further 72 h under normal conditions (top row), or on plates seeded with bacteria plus proteasome pathway inhibitors (+Lev, second row; +ZLLL, third row) or a lysosome pathway inhibitor (+SB202190, fourth row). B) <i>ced-3</i> (caspase 3) mutants were age synchronised at the L1 stage and grown to adulthood at 20°C (t = 0 h) before being cultured on RNAi plates against <i>unc-97</i> or <i>unc-112</i> for a further 72 h. In both A and B, at t = 0 h and 72 h approximately 20–30 animals were stained for β-galactosidase activity (blue). C) <i>unc-52^ts</i> and D) <i>unc-112^ts</i> mutants were cultured for two generations at 16°C (permissive, mutation inactive) under either normal, control conditions or on RNAi against <i>clp-1</i>, <i>clp-4</i>, <i>tra-3</i>, <i>clp-6</i> or <i>clp-7</i>. Second generation animals were then age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h) before being temperature shifted to 25°C (non-permissive, mutation active) and cultured on the respective condition for a further 72 h. Some temperature shifted animals were also placed on calpain inhibitor II or calpain inhibitor III drug plates (5 µg/ml) at t = 0 h and cultured on drug plates for a further 72 h. Approximately 20–30 animals were stained for β-galactosidase (blue) at t = 0 h, and at 24 h, 48 h (not shown) and 72 h. All experiments were performed at least three times. 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>unc-112^ts</i> in A and D). Scale bars represent 100 µm.</p

RNAi against <i>gfp</i> reduces chromosomal GFP expression in spaceflight and ground control (GC).

<p>Animals fed RNAi vector control for 4 d from L1 larvae developed into normal adults in GC and spaceflight conditions. These animals also displayed GFP expression in oocytes and embryos in GC and spaceflight. Animals fed <i>gfp</i> RNAi for 4 d also developed normally to adulthood in GC and spaceflight, and demonstrated a loss of GFP expression in both GC and spaceflight. Scale bars represent 50 µm.</p

Gene expression of the RNAi apparatus is unaltered by spaceflight.

<p>Adult hermaphrodites (2^nd generation) collected at 8 d during spaceflight showed no change in gene expression for components of the RNAi machinery, which were not different ground controls (<i>P</i>>0.05). mRNA expression values are the average of 18 separate probes over six microarrays, and are relative to an internal control (1G controls).</p