<i>Pten</i>^<i>5</i> transheterozygous mutants exhibit <i>Pten</i>-associated locomotive phenotypes.

<p><b>(A,B)</b> Flight tests of adult females of different genotypes over a 9 day <b>(A)</b> and 25 day <b>(B)</b> period. <b>(A)</b><i>Pten</i>^<i>5</i> transheterozygous mutant female flightlessness rises between 3 and 9 days (***<i>P</i><0.001) and is significantly higher than <i>w</i>^<i>1118</i> and heterozygous <i>Pten</i>^<i>5</i><i>/CyORoi</i> controls over a 9 day period; ***<i>P</i> < 0.001 relative to both controls. There was no statistically significant difference between wild type <i>w</i>^<i>1118</i> and heterozygous control <i>Pten</i>^<i>5</i><i>/CyORoi</i> at any time point; graphs represent pooled data from six experiments, n ≥ 120. <b>(B)</b> Frequency of flightless phenotype for <i>Pten</i>^<i>5</i> transheterozygous mutant female flies continues to increase (<i>P</i> < 0.001 from days 2–9, 9–16 and 16–25 days) and be significantly greater than control <i>w</i>^<i>1118</i> and <i>Pten</i>^<i>5</i><i>/CyORoi</i> females over a 25 day period. Data from at least six independent experiments. **<i>P</i> < 0.01, ***<i>P</i> < 0.001, n ≥ 100, determined by two-way ANOVA with Bonferroni post-hoc correction for <b>A</b> and <b>B</b>. <b>(C)</b> Flightless phenotype in 9-day-old <i>Pten</i>⁵ transheterozygous female flies is strongly rescued by a <i>Pten</i> genomic construct; pooled data from six experiments; *** <i>P</i> < 0.001, n ≥ 100. <b>(D)</b> 9-day-old <i>Pten</i>^<i>5</i> transheterozygous mutant males display a defective geotaxic phenotype compared to <i>w</i>^<i>1118</i> controls, <i>Pten</i>^<i>5</i><i>/CyORoi</i> heterozygotes or genomic rescue flies, assessed by scoring flies that failed to climb 6 cm in 30 sec; n ≥ 50, *** <i>P</i> < 0.001. Significance determined by one-way ANOVA with Bonferroni post-hoc correction for <b>C</b> and <b>D</b>. Graphs present as mean ± SEM.</p

Transheterozygous <i>Pten</i>^<i>5</i> mutants exhibit defects in mitochondrial structure in IFM and upregulation of the oxidative stress response gene, <i>GstD1</i>.

<p>(<b>A</b>) qRT-PCR of <i>Pink1</i> mRNA expression levels in third instar larvae carrying different mutations affecting IIS/mTORC1 signalling normalised to wild type <i>w</i>^<i>1118</i> control animals. (<b>B</b>) Levels of the anti-oxidative enzyme-encoding <i>GstD1</i> transcript are elevated significantly in <i>Pten</i> mutant backgrounds compared to <i>w</i>^<i>1118</i> controls. However, there is no significant modulation in the transcript expression levels of <i>GstD1</i> in either <i>foxo</i> or <i>4E-BP</i> mutants or <i>Pten</i> heterozygous animals. Data are presented as mean ± SEM. * <i>P</i> <0.05; ** <i>P</i> < 0.001; ***<i>P</i> < 0.0001, and are from three independent experiments. Significance was determined by one-way ANOVA with Bonferroni post-hoc correction test. (<b>C</b>-<b>H</b>) Longitudinal sections of thoraces of 26-day-old female flies either stained with toluidine blue and visualized by light microscopy (scale bar: 100μm; <b>C</b>,<b>D</b>) or imaged by transmission electron microscopy (TEM; scale bar: 1μm) to visualize ultrastructure of IFMs (<b>E</b>-<b>H</b>). The sarcomeric structure of mutant muscle appears relatively normal (black arrows in <b>F</b>,<b>H</b> compared to controls in <b>E</b>,<b>G</b>), but mitochondrial morphology in the mutant is severely disrupted (white arrows in <b>F</b>,<b>H</b> compared to controls in <b>E</b>,<b>G</b>).</p

Transheterozygous <i>Pten</i>^<i>5</i> mutant flies have a highly penetrant eye phenotype.

<p><b>A-J</b>. Low (<b>A-E</b>) and high (<b>F-J</b>) magnification views of eyes from females of different genotypes. A mild disorganisation of the ommatidia in the posterior region of the eye is observed in <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>1</i> (<b>C</b>,<b>H</b>) and <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i> (<b>D</b>, <b>I</b>) flies, as shown by black arrows in <b>H</b> and <b>I</b>, but not in wild type <i>CantonS</i> (<b>A</b>,<b>F</b>) and <i>Pten</i>^<i>5</i> heterozygous control females (<i>Pten</i>^<i>5</i><i>/CyO</i>) (<b>B</b>,<b>G</b>). Almost all female mutant animals carrying a <i>Pten</i> genomic rescue construct (<i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i><i>;[R]</i>) (<b>E</b>,<b>J</b>) do not display the eye phenotype. (<b>K</b>) Histogram presented as mean percentage of flies exhibiting disorganised eye phenotype. Error bars indicate standard error of mean (SEM). *** <i>P</i> < 0.001, from two separate experiments n ≥ 100. (<b>L</b>) The mean body mass of different <i>Pten</i> mutant females, <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>1</i> and <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i>, is not significantly heavier than wild type <i>w</i>^<i>1118</i>. Surprisingly, <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i> rescue females have significantly higher body mass than all other genotypes. Data are presented as mean body mass per fly ± SEM. Pooled from two independent experiments, n ≥60. Statistical significance was determined by two-tailed unpaired Student’s <i>t</i>-test. Scale bar: 100μm.</p

<i>Pten</i>^<i>5</i> transheterozygous mutant flies are sensitive to a wide range of stresses.

<p>Survival of <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i> transheterozygous mutants (orange), mutants carrying a <i>Pten</i> genomic rescue construct (<i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i><i>;[R]</i>) (green), and wild type control <i>w</i>^<i>1118</i> (pink) males after they were exposed to (<b>A</b>) 5mM rotenone, (<b>B)</b> 2mM paraquat, (<b>C)</b> water-only diet and <b>(D)</b> 500mM NaCl. The mean survival times (in hours) for <i>Pten</i>^<i>5</i> transheterozygous mutants, rescue flies, and wild type control <i>w</i>^<i>1118</i> flies respectively are: rotenone = 40.4, 89.2 and 107.1; paraquat = 8.6, 24.2, and 50.3; water starvation = 9.2, 19.1 and 24.2; NaCl = 15.4, 19.4 and 30.9. In all four stress assays, <i>Pten</i>^<i>5</i> transheterozygous mutants were short lived compared with wild type <i>w</i>^<i>1118</i> (<i>P</i> < 0.001), and for all but NaCl stress had a significantly shorter mean survival time compared to rescue flies (<i>P</i> < 0.01). For each experiment, flies were grouped into at least 6–8 batches of 20, these experiments were then repeated four times and data pooled together, n ≥ 480). Statistical significance was determined by Mantel-Cox Log rank test and Wilcoxon test using GraphPad5. Graphs presented as pooled data of percentage mean of survival for each genotype. Graphs presented as mean ± SEM.</p