Silencing class II PI3Ks decreases autophagy in both control and <i>Vps34</i> null MEFs.

<p>(<b>A</b>) Control MEFs were transfected with mock or PI3K-C2α/β siRNA as well as GFP-WIPI-1 for 48 hrs, cultured in normal media or HBSS for 90 min and fixed. Right: Confocal microscopy images of GFP-WIPI-1 fluorescence in mock or PI3K-C2α/β siRNA-treated control cells after HBSS starvation for 90 min. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of GFP-WIPI-1 puncta observed after 90 min HBSS starvation (n=11-15 cells). (<b>B</b>) Cells prepared as in (A) were fixed and immunostained. Right: Confocal microscopy images showing endogenous LC3 (green) in cells cultured in HBSS in the presence of 50 nM Bafilomycin (St+B) for 30 min. DAPI is shown in blue. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of LC3 puncta observed under normal media (Nm), HBSS (St) and HBSS in the presence of Bafilomycin (St+B) conditions (n=12-19, 23-40 and 26-53 cells for Nm, St and St+B conditions, respectively). Scale bars: 10 µm.(<b>C</b>) Control and <i>Vps34</i> KO MEFs were transfected for 48 hrs with mock or PI3K-C2α/β siRNA, cultured in normal medium (N), HBSS (St) or HBSS with 50 nM Bafilomycin (St+B) for 30 min, lysed and analyzed by immunoblotting using the indicated antibodies (n=3).</p

A higher affinity PI3P-binding probe, 4x-FYVE^Hrs, reveals a larger pool of intracellular PI3P in the absence of Vps34 compared to the conventional 2x-FYVE^Hrs probe.

<p>Control and <i>Vps34</i> KO MEFs were transiently transfected with both RFP-2x-FYVE^Hrs and GFP-4x-FYVE^Hrs PI3P-binding constructs for 24hr, grown in normal media and fixed. Confocal microscopy analysis of RFP-2x-FYVE^Hrs (red) and GFP-4x-FYVE^Hrs (green) is shown. Scale bar: 10 µm.</p

Macroautophagy-mediated protein degradation is partially impaired in <i>Vps34</i> null MEFs.

<p>(<b>A</b>) Quantification of total [¹⁴C]-valine long-lived protein degradation induced by nutrient deprivation (HBSS) in control and <i>Vps34</i> KO MEFs. (<b>B</b>) Assessment of autophagy efficiency in control and <i>Vps34</i> KO MEFs by ¹⁴C-valine long-lived protein degradation under starvation (HBSS), starvation with 3MA or starvation with NH₄Cl conditions (see Methods) (n=8 for both A and B).</p

<i>Vps34</i> KO MEFs show a decrease in LC3 conjugation and LC3 puncta formation upon starvation.

<p>(<b>A</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal medium (N) or HBSS (St) in the presence or absence of 50nM Bafilomycin (Nm+B or St+B, respectively) for 90 min. Right: Lysates were analyzed by immunoblotting using the indicated antibodies. Left: Relative LC3-II levels normalized to actin (n=4). (<b>B</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal media (Nm) or HBSS (St) for 30 and 90 min, fixed and immunostained. Confocal analysis of LC3, p62 and GFP-Cre fluorescence, which is artificially shown in green, red and blue colors, respectively. Arrowheads indicate LC3 and p62 colocalization (yellow). Scale bar: 10 µm. (<b>C</b>) Quantification of the number of LC3 (left panel) and p62 puncta (middle panel) per cell. Colocalization of p62 with LC3 puncta is also shown (right panel) (colocalization was defined as the number of pixels overlapping in the p62 and LC3 channels normalized per cell) (n=35-45 cells).</p

Recruitment of WIPI-1, a PI3P-binding protein, to sites of AP biogenesis occurs in <i>Vps34</i> KO MEFs but at diminished levels.

<p>Control and <i>Vps34</i> KO MEFs were transiently transfected with GFP-WIPI-1 for 24 hrs, cultured in HBSS for 90 min (90 min St) and fixed. Left: Confocal microscopy analysis of GFP-WIPI-1 fluorescence (green). Nuclear inactive and active Tomato-Cre is shown in red. The contrast was enhanced to reveal the WIPI puncta over the cytosolic background fluorescence. Right: Quantification of the number of GFP-WIPI-1 puncta (n=13-15 cells). Scale bar: 10 µm.</p

Ablation of Vps34 alters levels of Vps34 complex proteins.

<p>(<b>A</b>) Western blot analysis showing a time-course of Vps34 protein levels in <i>Vps34</i>^Flox/Flox MEFs infected with lentiviruses expressing either an inactive Cre (Δ) or active (CRE) full-length Cre recombinase for 7-13 days. (<b>B</b>) Western blot using an antibody directed to the NH₂-terminus of Vps34 in control and <i>Vps34</i> KO cell extracts. (<b>C</b>) Western blot analysis of Vps34 complex components and endosomal protein levels in control and <i>Vps34</i> KO MEF lysates. Quantification of protein levels after normalization to tubulin (n=3).</p

PLD deletion results in abnormal phagosomal cup formation.

<p>Confocal microscopy of primary BMDM transfected via nucleofection with an EGFP-fused Spo20-PA sensor to image PA (green) in unstimulated WT BMDM (A) or in WT (B), <i>Pld1</i>^−/− (C), <i>Pld2</i>^−/− (D), or FIPI-treated WT (E) BMDM that were cultured with opsonized beads for 5 min. F-actin was visualized using rhodamine phalloidin (red). Scale bar, 5 µm. Best representative images from at least 3 sets of independently-isolated BMDMs. F) Circles shown indicating thickness of cup in WT BMDM superimposed on <i>Pld1</i>^−/− and <i>Pld2</i>^−/− cups, with quantitation of the thickness of the PA-visualized cups. At least 10 cups were measured for each experimental setting, and the experiments were performed 3 or more times. ***  =  p<0.0001.</p

DOCK2 mislocalizes in the absence of PLD1 or PLD2.

<p>A) Confocal microscopy of WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min followed by immunostaining for DOCK2 (green) and rhodamine phalloidin staining of F-actin (red); *, region magnified in inset. B) Graph of distinct and indistinct DOCK2 cups as well as actin cups, quantitated as in Fig. 2. C) Western blot of DOCK2 in WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM, best representative images from at least 3 blots. Bar, 7.5 µm. ***  =  p<0.0001.</p

Functional interactions between ER-PM tethers and PI4P regulators.

<p><b>A.</b><i>OSH4</i> deletion in Δ-s-tether cells results in synthetic lethality. WT (SEY6210), Δtether (ANDY198), Δ-s-tether (CBY5838), <i>osh4</i>Δ Δtether (CBY5940), and <i>osh4</i>Δ Δ-s-tether cells (CBY5988) were transformed with an episomal copy of the <i>SCS2</i> tether gene (+ [<i>SCS2</i>]; pCB1183) and streaked onto selective solid media with and without choline supplementation. The presence of the <i>SCS2</i> gene provides an ER-PM tether that confers robust growth, even in the absence of all other tether genes. On growth medium selecting against the <i>SCS2</i> plasmid (− [<i>SCS2</i>]), <i>osh4</i>Δ Δ-s-tether cells were inviable with or without choline. <b>B.</b> <i>OSH6</i> expression suppresses the synthetic lethality of <i>osh4</i>Δ in Δ-s-tether cells. WT and <i>osh4</i>Δ Δ-s-tether cells containing an episomal copy of <i>SCS2</i> were transformed with either the high-copy vector control (YEplac181), <i>OSH4</i> (pCB598), or <i>OSH6</i> (pCB1266) and streaked onto solid growth media. On a medium selecting against the <i>SCS2</i> plasmid, <i>OSH4</i> or <i>OSH6</i> expression suppressed <i>osh4</i>Δ Δ-s-tether synthetic lethality, whereas vector control did not. <b>C.</b> Representative images of WT, Δtether, and Δ-s-tether cells by DIC with corresponding fluorescence microscopy showing the localization of the PI4P sensor GFP-2xPH^<i>OSH2</i> (pTL511). Scale bar = 2 μm. <b>D.</b> Bar graphs quantifying the number of GFP-2xPH^<i>OSH2</i> fluorescent Golgi spots (lower and upper boundaries of boxes correspond to data quartiles; the white bar indicates the median; lines represent the range of spots/cell) and the percentage of GFP-2xPH^<i>OSH2</i> fluorescent mothers detected in WT, Δtether, and Δ-s-tether cells. <b>E.</b> <i>SAC1</i> deletion in Δ-s-tether cells results in a synthetic lethal interaction. WT, Δtether, <i>sac1</i>Δ Δtether (CBY6142), Δ-s-tether, and <i>sac1</i>Δ Δ-s-tether cells (CBY6146) were transformed with an episomal copy of <i>SCS2</i> and streaked onto selective solid media with and without choline supplementation. On a medium that selects against the <i>SCS2</i> plasmid, <i>sac1</i>Δ Δ-s-tether cells were inviable whether or not choline was added. Numerical data presented in this figure may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.s003" target="_blank">S1 Data</a>. Δ-s-tether, Δ-super-tether; DIC, differential interference contrast; ER, endoplasmic reticulum; PI4P, phosphatidylinositol-4-phosphate; PM, plasma membrane; WT, wild type.</p

Bidirectional transport of ergosterol from the ER to the PM is unaffected in Δ-s-tether cells.

<p><b>A.</b> Outline of the transport assay. <b>B.</b> Characterization of subcellular fractions. Top, immunoblots using antibodies against Pma1 (PM), Dpm1 (ER), and Vph1 (vacuole). Fraction 2 is designated ER* to indicate that it contains membranes in addition to ER membranes. Data correspond to fractionation of a homogenate of WT cells prepared at the end of the labeling pulse. Middle, quantification of Dpm1 and Pma1 in fractions prepared from homogenates of WT and Δ-s-tether cells taken after a 30 min chase period. The blots were quantified by ImageJ. Bottom, quantification of ergosterol in the different fractions from the middle panel. <b>C.</b> WT and Δ-s-tether cells were processed as in panel A. The SR of ergosterol in each fraction ([³H]ergosterol [cpm] ÷ ergosterol mass) was normalized to the SR of the total homogenate at each time point to obtain an RSR. The figure shows RSR versus time (t = 0 min is the start of the labeling pulse). The lower portion of the graph (solid symbols) is based on 3–4 independent experiments; the upper portion (open symbols) is based on 2–5 independent experiments. The lines are mono-exponential fits of the data that plateau at RSR = 1. <b>D.</b> Transport of ergosterol in Δ-s-tether cells with block in vesicular transport. Mid-log cultures of WT, <i>sec18-1</i>^<i>ts</i> (CBY2859), Δ-s-tether (CBY5838), and Δ-s-tether <i>sec18-1</i>^<i>ts</i> (CBY5851) cells were grown at 24 °C, shifted to the restrictive temperature of 37 °C for 20 min, pulse-labeled with [³H]methyl-methionine for 4 min and chased for 15 min at the same temperature. The bar chart shows the RSR of the PM fraction from samples taken at the end of the pulse and chase periods. Data are mean ± SEM (<i>n</i> = 3). Numerical data presented in this figure may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003864#pbio.2003864.s003" target="_blank">S1 Data</a>. Δ-s-tether, Δ-super-tether; cpm, counts per minute; ER, endoplasmic reticulum; PM, plasma membrane; RSR, relative specific radioactivity; SR, specific radioactivity; WT, wild type.</p