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

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 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

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

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

ABCB11 canalicular trafficking model based on experimental observations in sandwich-cultured mouse hepatocytes.

<p>Taurocholate, cAMP and AICAR enhanced ABCB11 trafficking to the canalicular membrane of hepatocytes. Canalicular delivery of ABCB11 was greatly reduced in the LKB1-decficient cell. The accelerating effects of taurocholate and AICAR were prevented by the disruption of LKB1. In contrast, addition of cAMP augmented ABCB11 trafficking even in the LKB1-deficient cells. Activation of Epac by CTP-cAMP also led to enhanced canalicular trafficking, however, its effect was LKB1-dependent. Specific activation of PKA by 6-Bnz-cAMP results in increased canalicular trafficking of ABCB11 independently of LKB1. The accelerating effect of cAMP was blocked by specific inhibition of PKA in LKB1-deficient cells, suggesting a PKA-dependent regulatory pathway in control of ABCB11 trafficking. PP2C – Protein phosphatase 2C, the major phosphatase dephosphorylating phospho-AMPK.</p

ABCB11 trafficking in control and LKB1 −/− hepatocytes.

<p>(<b>A</b>) FRAP studies with control and LKB1 −/− mouse hepatocytes transduced with YFP-tagged ABCB11 were performed as described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091921#pone-0091921-g002" target="_blank">Figs. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091921#pone-0091921-g003" target="_blank">3</a>. The second phase of fluorescence recovery was markedly slower in the LKB1 −/− cells (○) as compared to control cells (•). Taurocholate (TC) accelerated the fluorescence recovery only in control hepatocytes (▪), and had no effect in LKB1 −/− cells (□). (<b>B–C</b>) The slopes of the second recovery phase (parameter <b><i>B</i></b> in the equation), which reflect initial rate of canalicular trafficking, were averaged and normalized to the untreated wild type cells. Pretreatments: TC – 100 µM taurocholate, AICAR - 500 µM AICAR, cAMP - 200 µM 8-Br-cAMP, PKAact - 50 µM 6-Bnz-cAMP, Epac - 3 µM 8-CTP-cAMP, PKAi - 500 nM PKA inhibitor. Means ± S.E.M. of at least three independent experiments are shown. Asterisks denote significant differences as compared to untreated control hepatocytes (*), to untreated LKB1 −/− cells (**), or to 8-Br-cAMP-treated cells (***), p<0.05. n.d. – not determined. Taurocholate, AICAR, and cAMP accelerated canalicular trafficking of ABCB11 in control hepatocytes. The effects were not additive. Basal level of ABCB11 trafficking to the canaliculi was reduced in the LKB1 −/− cells as compared to control cells. Taurocholate and AICAR were ineffective in these cells, however, the effect of cAMP persisted. Activation of PKA resulted in accelerated canalicular trafficking in both cell types, whereas inhibition of PKA abolished the effect of cAMP in LKB1-deficient hepatocytes.</p

Kinetic analysis of intracellular particle movements.

<p>Several time lapse image series of ABCB11-YFP-transduced hepatocytes similar to that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091921#pone.0091921.s009" target="_blank">Videos S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091921#pone.0091921.s010" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091921#pone.0091921.s011" target="_blank">S5</a> were acquired. A large number of ABCB11-YFP-containing intracellular vesicles were tracked by custom-written algorithm (see detail in Methods section), and their movements were analyzed (n>36). Distribution of average velocity (<b>A–C</b>), instantaneous velocity (<b>D–F</b>), and mean square displacement (<b>G–I</b>) are indicated for untreated control (<b>A, D, G</b>), untreated LKB1 −/− (<b>B, E, H</b>), and LKB1 −/− cells pretreated with 200 µM 8-Br-cAMP (<b>C, F, I</b>). Particle movements, especially directed motions are disrupted in LKB1 −/− hepatocytes, which can be restored by addition of cAMP.</p