Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics

The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3′-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly

The GATOR2 Component Wdr24 Regulates TORC1 Activity and Lysosome Function.

TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes

Mio and Seh1 influence lysosome dynamics independent of TORC1 activity.

<p>(A-H) Depleting <i>nprl2</i> and <i>nprl3</i> by germline specific driver Nanos-Gal4 fails to rescue the LysoTracker accumulation phenotype in <i>mio</i>^<i>2</i> and <i>seh1</i>^<i>Δ15</i> ovaries. Ovarioles from WT (A) <i>mio</i>^<i>2</i><i>; mCherry</i>^<i>RNAi</i> (B), <i>mio</i>^<i>2</i><i>; nprl2</i>^<i>RNAi</i> (C) and <i>mio</i>^<i>2</i><i>;nprl3</i>^<i>RNAi</i> (D) or from <i>WT</i> (E), <i>seh1</i>^<i>Δ15</i><i>; mCherry</i>^<i>RNAi</i> (F), <i>seh1</i>^<i>Δ15</i><i>; nprl2</i>^<i>RNAi</i> (G) and <i>seh1</i>^<i>Δ15</i><i>;nprl3</i>^<i>RNAi</i> (H) females were stained with LysoTracker and Hoechst. Size bar is 10 μm. (I and J) Proteins isolated from WT, <i>mio</i>^<i>2</i><i>; mCherry</i>^<i>RNAi</i>, <i>mio</i>^<i>2</i><i>; nprl2</i>^<i>RNAi</i>, <i>mio</i>^<i>2</i><i>;nprl3</i>^<i>RNAi</i> ovaries, or protein isolated from <i>WT</i>, <i>seh1</i>^<i>Δ15</i><i>; mCherry</i>^<i>RNAi</i>, <i>seh1</i>^<i>Δ15</i><i>; nprl2</i>^<i>RNAi</i> and <i>seh1</i>^<i>Δ15</i><i>;nprl3</i>^<i>RNAi</i> ovaries were analyzed by Western blot probed with pS6K and S6K antibodies. (K and L) Quantification of phospho-S6K levels relative to the total S6K. Error bars represent the standard deviation for three independent experiments. ** p value < 0.01.</p

Germline depletions of <i>nprl2</i> and <i>nprl3</i> in <i>wdr24</i>^<i>1</i> mutant ovaries.

<p>(A-D) Dissected ovaries from wild type (WT) (A), <i>GFP</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> (B), <i>nprl2</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> (C) and <i>nprl3</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> (D) females. <i>GFP</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> was used as a negative control. Size bar is 100 μm. (E) Bar graph shows the number of eggs laid by WT, <i>GFP</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i>, <i>nprl2</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> <i>and nprl3</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> females. Error bars represent the standard deviation for three independent experiments. ** p value < 0.01 (F-I’) Depleting <i>nprl2</i> and <i>nprl3</i> fails to rescue the LysoTracker accumulation phenotype in <i>wdr24</i>^<i>1</i> ovaries. Ovarioles from WT (F and F’) <i>GFP</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> (G and G’), <i>nprl2</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> (H and H’) and <i>nprl3</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> (I and I’) females were stained with LysoTracker and Hoechst. Size bar is 10 μm. (J) Proteins isolated from WT, <i>GFP</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i>, <i>nprl2</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> <i>and nprl3</i>^<i>RNAi</i><i>; wdr24</i>^<i>1</i> ovaries were analyzed by Western blot probed with pS6K and S6K antibodies. (K) Quantification of phospho-S6K levels relative to the total S6K. Error bars represent the standard deviation for four independent experiments. ** p value < 0.01.</p

Wdr24 influences lysosome dynamics independent of TORC1 activity.

<p>(A) Proteins isolated from wild type (WT), <i>wdr24</i>^<i>1</i>, <i>nprl3</i>^<i>1</i>, <i>wdr24</i>^<i>1</i> and <i>nprl3</i>^<i>1</i> third instar larvae were analyzed by Western blot probed with pS6K and S6K antibodies. (B) Quantification of phospho-S6K levels relative to the total S6K. Error bars represent the standard deviation for three independent experiments. * p value < 0.05. (C-F’) Fat bodies from GFP-Lamp1/ CyO (C-C”), GFP-Lamp1/ CyO<i>; wdr24</i>^<i>1</i> (D-D”), GFP-Lamp1/ CyO<i>; wdr24</i>^<i>1</i>, <i>nprl3</i>^<i>1</i> (E-E”) and GFP-Lamp1/ CyO<i>; nprl3</i>^<i>1</i> (F-F”) third instar larvae stained with GFP, Atg8a antibodies and Hoechst. Size bar is 10 μm.</p

Wdr24 promotes TORC1 activity and cell growth in both germline and somatic cells.

<p>(A) Dissected ovaries from control <i>wdr24</i>^<i>1</i>/<i>TM6</i> (control), <i>wdr24</i>^<i>1</i> and <i>wdr24</i>^<i>1</i><i>/Df</i> females. Size bar is 100 μm. (B) Bar graph shows the number of eggs laid by control <i>wdr24</i>^<i>1</i>/<i>TM6</i>, <i>wdr24</i>^<i>1</i> and <i>wdr24</i>^<i>1</i><i>/Df</i> females. Error bars represent the standard deviation for three independent experiments. ** p value < 0.01 (C) Ovariole containing a <i>wdr24</i>^<i>1</i> mutant germline clone stained with anti-GFP and DAPI. Egg chambers containing <i>wdr24</i>^<i>1</i> germline clones are marked by the absence of GFP. Note that the <i>wdr24</i>^<i>1</i> mutant egg chamber (yellow arrow) is smaller than a younger WT egg chamber (white arrowhead). Size bar is 10 μm (D) Representative images of control <i>wdr24</i>^<i>1</i>/<i>TM6</i>, <i>wdr24</i>^<i>1</i> and <i>wdr24</i>^<i>1</i><i>/Df</i> adult males. Size bar is 100 μm. (E) Quantification of body weights of <i>wdr24</i>^<i>1</i>/<i>TM6</i>, <i>wdr24</i>^<i>1</i> and <i>wdr24</i>^<i>1</i><i>/Df</i> adult males. Error bars represent the standard deviation for three independent experiments (8 males per group). **p value < 0.01 (F and G) Somatically derived cells from an adult fat body (F) and follicle cells (G) from a stage 10B egg chamber stained with anti-GFP and DAPI. The <i>wdr24</i>^<i>1</i> mutant cells are marked by the absence of GFP and are outlined by a red line. Note that <i>wdr24</i>^<i>1</i> mutant cells have a smaller nuclear size suggesting decreased ploidy. Size bar is 10 μm (H and I) Quantification of nuclear size fold change of <i>wdr24</i>^<i>1</i> mutant cells compared to wild type cells from adult fat bodies (H) and follicle cells (I). Error bars represent the standard deviation from 4 individual fat body clones and 9 individual follicle cell clones. **p value < 0.01, *** p value < 0.001 (J) Proteins isolated from WT, <i>wdr24</i>^<i>1</i> and starved WT (positive control) females and males were analyzed by Western blot probed with pS6K and S6K antibodies (K) Quantification of phospho-S6K levels relative to the total S6K. Error bars represent the standard deviation for three independent experiments. **p value < 0.01.</p

Wdr24 associates with GATOR complex components and localizes to lysosomes and autolysosomes.

<p>(A-C) S2 cells were co-transfected with HA-tagged Seh1, HA-tagged Mio, V5-tagged Nprl3 and GFP-tagged Wdr24 or GFP (control) plasmids. Cells were lysed and immunoprecipitated with GFP antibody. Cell lysates (input) and immunoprecipitates (IP) were detected by Western blot using HA, V5 and GFP antibodies. (D-K) Live cell imaging of <i>Drosophila</i> egg chambers from females cultured on standard fly medium (fed) or on 20% sucrose (starved). (D-E”) Wdr24-mCherry co-localizes with GFP-Lamp1. (F-G”) GFP-Wdr24 co-localizes with LysoTracker. (H-I”) GFP-Wdr24 co-localizes with mCherry-Atg8 under starvation conditions. (J-K”) Wdr24-mCherry co-localizes with GFP-Nprl2. Size bar is 10 μm.</p

WDR24 regulates lysosomal acidification and autophagic flux in HeLa cells.

<p>(A) Proteins isolated from WT and <i>wdr24</i>^<i>-/-</i> HeLa cells were analyzed by Western blot probed with pS6K, S6K, LC3, p62 and actin antibodies. (B-E) Wild type (WT) (B and D) and <i>wdr24</i>^<i>-/-</i> (C and E) HeLa cells were stained with LC3 antibody, LysoTracker and DAPI. (F) Lysates from WT and <i>wdr24</i>^<i>-/-</i> HeLa cells treated or untreated with chloroquine were analyzed by Western blot probed with LC3 and GAPDH antibodies. (G) Quantification of relatively fold changes of LC3II level after chloroquine treatment. Error bars represent the standard deviation for three independent experiments. ** p < 0.01. (H and I) Confocal images show the fluorescent degradation products of the DQ-BSA in lysosomes in WT (H) and <i>wdr24</i>^<i>-/-</i> (I) HeLa cells. (H’ and I’) Bright field images of WT and <i>wdr24</i>^<i>-/-</i> HeLa cells in H and I respectively. (J) Western blot probed with antibodies against Cathepsin D. Actin was used as an internal control. (K) Quantification of cleaved Cathepsin D levels relative to actin is shown. Error bars represent the standard deviation for five independent experiments. ** p < 0.01. (L) Quantification of relatively fluorescent intensity from microplate reader measurement of Lysosensor DND-189 stained cells. Error bars represent the standard deviation for three independent experiments. * p< 0.05. (M) Quantification of lysosomal pH in lysosomes in WT and <i>wdr24</i>^<i>-/-</i> HeLa cells as determined by lysosensor Yellow/Blue DND-160 stained cells. Error bars represent the standard deviation for three independent experiments. * p< 0.05. (N-O’) Wild type (WT) (N, N’) and <i>wdr24</i>^<i>-/-</i> (O, O’) HeLa cells were stained with TFEB antibody and DAPI.</p

A dual role for the GATOR2 component Wdr24 in the regulation of TORC1 activity and lysosome function.

<p>Wdr24 promotes TORC1 activation by opposing the activity of the GATOR1 complex. Additionally, independent of TORC1 status, Wdr24 promotes lysosome acidification, which is required for autophagic flux.</p