Image_1_PATs and SNATs: Amino Acid Sensors in Disguise.pdf

<p>Solute Carriers (SLCs) are involved in the transport of substances across lipid bilayers, including nutrients like amino acids. Amino acids increase the activity of the microenvironmental sensor mechanistic Target of Rapamycin Complex 1 (mTORC1) to promote cellular growth and anabolic processes. They can be brought in to cells by a wide range of SLCs including the closely related Proton-assisted Amino acid Transporter (PAT or SLC36) and Sodium-coupled Neutral Amino acid Transporter (SNAT or SLC38) families. More than a decade ago, the first evidence emerged that members of the PAT family can act as amino acid-stimulated receptors, or so-called “transceptors,” connecting amino acids to mTORC1 activation. Since then, further studies in human cell models have suggested that other PAT and SNAT family members, which share significant homology within their transmembrane domains, can act as transceptors. A paradigm shift has also led to the PATs and SNATs at the surface of multiple intracellular compartments being linked to the recruitment and activation of different pools of mTORC1. Much focus has been on late endosomes and lysosomes as mTORC1 regulatory hubs, but more recently a Golgi-localized PAT was shown to be required for mTORC1 activation. PATs and SNATs can also traffic between the cell surface and intracellular compartments, with regulation of this movement providing a means of controlling their mTORC1 regulatory activity. These emerging features of PAT and SNAT amino acid sensors, including the transceptor mechanism, have implications for the pharmacological inhibition of mTORC1 and new therapeutic interventions.</p

<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> 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

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

DCGs are released from SCs during mating, activating BMP signalling.

<p><b>A, B</b>. SCs from 6-day-old esgF/O^ts virgin males (A) and from males immediately after mating (B) were fixed and stained with an anti-pMad antibody (red) and DAPI (blue), revealing that the proportion of SCs with detectable nuclear pMad is higher in mated animals. <b>C, D.</b> Immediately after mating, living SCs (D) have less GFP-GPI-labelled DCGs than virgins (C). Image shows a single z-plane of gland stained with Lysotracker Red; not all compartments are in the focal plane. Note that the largest MVBL in (C; arrowhead) contains GFP, probably because of fusion between a DCG compartment and the MVBL [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006366#pgen.1006366.ref017" target="_blank">17</a>]. <b>E</b>. Graph shows proportion of SCs with nuclear pMad in 6-day-old virgin, and mated males (dissected 8 min into mating [Mid] and immediately after mating), and mated males expressing <i>dpp</i>-RNAi and Dad in SCs from eclosion onwards using the <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4</i> driver. <b>F</b>. Graph shows number of GFP-GPI-positive DCG compartments in 6-day-old virgin and mated males (using the <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> driver line; Double is twice mated in 2 h), and at different times after single mating in control SCs. Compartments were also counted in SCs expressing <i>Snap24</i> RNAi post-eclosion in virgins and immediately after mating. Labelled compartments were counted using a complete z-series for each cell. Genotypes for images are: <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP/+; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4/+</i> (A, B); <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI/CyO</i> (C, D).***P<0.001, Kruskal-Wallis test, n>15. Scale bar for A-B is 20 μm and C-D is 10 μm.</p

Model to explain autocrine regulation of DCG replenishment by BMP signalling in SCs.

<p>Schematics of a single SC immediately before and after mating. (1) In virgin males, Dpp is trafficked to and stored in DCGs. Sporadic release of these DCGs activates BMP signalling, and sustains a basal level of growth, DCG biogenesis and exosome secretion. (2) During mating, about 4 mature DCGs are released (3), resulting in an increase in BMP signalling (4), primarily via an autocrine mechanism and probably in pulses. This stimulates growth, but also increases biosynthesis of new DCG compartments (5; solid arrow), ensuring that the total number of DCGs is fully replenished within 24 h. Dashed arrows highlight other parts of the secretory/endolysosomal system that might be affected by altered BMP signalling. Previous data (Corrigan et al., 2014) and data presented here suggest that long-term elevated BMP signalling enhances endolysosomal trafficking.</p

Autocrine Dpp regulates SC growth, SV number, endolysosomal trafficking and exosome secretion.

<p><b>A</b>. Expression of <i>dpp</i>-RNAi during the first six days of adulthood using the esgF/O^ts driver reduces the size of SCs and their nuclei (green arrows) relative to MCs (red arrows). <b>B</b>. Relative SC:MC nuclear size for SCs expressing RNAis targeting <i>dpp</i> and <i>gbb</i>, or GFP-tagged Dpp and Gbb, revealing specific effects of Dpp on growth. <b>C</b>. <i>dpp</i>^<i>blk</i>-GAL4 drives expression of a UAS-coupled nuclear GFP exclusively in SCs of the AG. <b>D, E.</b> Mosaic expression of <i>dpp</i>-RNAi or Dpp-GFP in a subset of SCs has a stronger effect on nuclear growth in expressing cells (on–green arrows) than in non-expressing (off–red arrows; white dashed circle) SCs, although <i>dpp</i> knockdown also reduces growth in the latter. <b>F-K.</b> Co-expression of <i>dpp</i>-RNAi with CD63-GFP using the <i>dsx</i>-GAL4 driver (G) reduces non-acidic SV number (eg., marked by arrowhead) and increases GFP fluorescence in largest MVBL (arrow; stained with Lysotracker Red) compared to controls (F); the statistical analysis of these changes for two independent RNAis is shown in H and I respectively. Knockdown of <i>dpp</i> either results in a small increase in the size of the largest MVBL or no significant size change (J), and reduces exosome secretion (K). Confocal images are from fixed glands (A, C, D) stained with DAPI (blue) and for Fas3 (yellow) or from living glands (F, G). Genotypes for images are: <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4/P[TRiP</i>.<i>HMS00011]attP2</i> (A and mosaic in D; the esgF/O^ts driver was also used to generate data in E); <i>w; P[w</i>^<i>+</i> <i>UAS-GFP</i>_<i>nls</i><i>]; P[w</i>^<i>+</i> <i>dpp</i>^<i>blk</i><i>-GAL4]</i> (C); <i>w; UAS-CD63-GFP tub-GAL80</i>^<i>ts</i><i>; dsx-GAL4</i> combined with no other transgene (F) or <i>P[TRiP</i>.<i>HMS00011]attP2</i> (III) (G). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for A, D is 20 μm, F, G, 10 μm, and for C, 50 μm.</p

Rapid replenishment of DCGs after mating is BMP-dependent.

<p><b>A</b>. Schematic representation of pulse-chase experiments shown in B-F, indicating the duration of GFP-GPI and <i>Dad</i> overexpression and timings of mating events. <b>B-D</b>. 6-day-old flies were shifted to 28.5°C for 24 h to allow expression of GFP-GPI in virgins (B) or in males mated 8 h after the start of the pulse (C). The number of GFP-GPI-labelled DCGs in SCs was reduced in virgin males co-expressing Dad (D). <b>E.</b> Graph shows a significant increase in the number of labelled DCGs if males are mated at 8h during a 24 h GFP-GPI pulse. The number of DCGs labelled in virgin and mated males is reduced if <i>Dad</i> is co-expressed. <b>F</b>. The increase in labelled compartments after mating is also reduced by Dad co-expression. The <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> line was used to generate data in E and F. *P<0.05, ***P<0.001, Kruskal-Wallis test, n = 15. Scale bar in B-D, 10 μm.</p

Multiple BMP signalling components regulate nuclear growth in SCs.

<p><b>A.</b> Schematic of the paired male accessory glands (arrows), which pump their contents into the ejaculatory duct (arrowhead) during mating. Left inset shows the epithelial secretory monolayer containing secondary cells (SCs; green) and main cells (MCs), all of which are binucleate. Right inset is a section through the gland revealing the large lumen (asterisk). <b>B.</b> SC (circled) expressing a gene trap for the BMP type I receptor <i>tkv</i>, and stained with an antibody against the BMP type II receptor Wit. These receptors are present both on the plasma membrane (arrowhead) and co-localise in intracellular compartments (arrow). DAPI marks nuclei blue in SCs (asterisks) and surrounding non-expressing MCs. <b>C-F.</b> Accessory glands (AGs) from 6-day-old controls (C) and males expressing RNAis targeting <i>tkv</i> (D) or <i>Mad</i> (F) or expressing the BMP signalling antagonist <i>Dad</i> (E) in adult SCs under the control of esgF/O^ts after temperature shift at eclosion. AGs were dissected, fixed and imaged by confocal microscopy. Glands were stained with DAPI (blue) to mark nuclei and an anti-Fas3 antibody (yellow) to mark cell boundaries. SCs express nuclear GFP, which is also present in the cytosol. Pairs of nuclei from binucleate SCs and MCs are indicated by green and red arrows respectively. <b>G.</b> Bar chart showing SC nuclear size relative to that of MC neighbours for glands expressing different transgenes in SCs under esgF/O^ts control, normalized to the ratio for controls. Note that all manipulations that decrease BMP signalling significantly reduce SC nuclear size. Genotypes for images are: <i>w; PBac[544</i>.<i>SVS-1]tkv</i>^{<i>CPTI002487</i>} (B); <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4</i> combined with no other transgene (C); <i>P[TRiP</i>.<i>JF01485]attP2</i> (III) (D); <i>P[w</i>^<i>+</i> <i>UAS-Dad]</i> (II) (E); <i>P[TRiP</i>.<i>JF01263]attP2</i> (III) (F). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for (B) is 10 μm, for all other images it is 20 μm.</p