pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels - PubMed (original) (raw)

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

Amriya Naufer et al. J Cell Biol. 2018.

Abstract

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

© 2018 Naufer et al.

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Figures

Figure 1.

Figure 1.

Early endosomal markers are recruited to tPCs. (a and b) tPCs fuse with endocytic compartments acquiring features of mature phagolysosomes. Representative confocal imaging of RAW macrophages ingesting filamentous bacteria (blue) 5 min (a) and 30 min (b) after onset of phagocytosis. Images illustrate the recruitment of EEA1 (left set), GFP-Rab5 (center set), and LAMP1 (right set) to the tPC. Actin jackets, denoted by F-actin accumulation (red), delineate the top border of the tPCs. Images in main panels show merged z-stacks, and images to the right of main panels show magnified single planes from framed regions showing recruitments of early endosomal markers to the tPCs. RAW cells were either transiently expressing GFP-Rab5 or immunolabeled for EEA1 or LAMP1. (c) Number of tPCs positive for EEA1, Rab5, and LAMP1 recruitment over time from panels a and b. tPCs were scored positive for markers if labeling was observed along the entirety of the cup. Data shown are means ± SEMs of percentages from three independent experiments (n = 50 at each time point). Bars: 5 µm; (enlarged areas) 1 µm.

Figure 2.

Figure 2.

PtdIns(3)P coexists with late endosomal markers at tPCs. (a) p40PX-GFP (green) recruitment to the base of tPCs in RAW macrophages engulfing filamentous bacteria (blue) at indicated time points. Actin jackets, denoted by F-actin accumulation (red), delineate the top border of the phagocytic cup. Main panels show representative images of merged z-stacks, and images to the right of each main panel show magnified single planes from framed regions showing recruitments of p40PX-GFP to tPCs. (b) Recruitment of 2-FYVE-GFP and p40PX-GFP, indicative of the presence of PtdIns(3)P around tPCs. Cells transiently expressing either 2-FYVE-GFP or p40PX-GFP were scored for partially internalized filamentous bacteria positive for PtdIns(3)P. Data shown are means ± SEMs from three independent experiments (n = 30 for each time point). (c) RAW macrophages expressing 2-FYVE-GFP were challenged with filamentous bacteria, fixed, permeabilized, and immunostained for LAMP1. Left and middle panels show partially internalized bacteria. Right panel depicts fully internalized bacteria. Main panels are merged z-stacks, and images at the bottom show single planes from framed regions. Bars: 5 µm; (enlarged areas) 1 µm. (d) Number of tPCs positive for 2-FYVE, LAMP1, Rab7, and RILP-C33 recruitment at 30 min after the onset of phagocytosis. Cells expressing 2-FYVE-GFP, GFP-Rab7, or RILP-C33-GFP or immunostained for LAMP1 were scored for partially internalized filamentous bacteria. tPCs were scored positive for markers if labeling was observed along the entirety of the cup. Data shown are means ± SEMs of percentages from three independent experiments (n = 30 for each).

Figure 3.

Figure 3.

PtdIns(3)P synthesis at tPCs is driven by the class III PtdIns 3-kinase, Vps34. (a) RAW cells expressing 2-FYVE-GFP (green) were treated with 1 µM DMSO (vehicle), 100 µM

LY294002

, 1 µM ZSTK474, or 1 µM Vps34-IN1. After these treatments, cells were allowed to engulf filamentous bacteria for 30 min, followed by fixation and staining for F-actin jackets. (b) RAW cells expressing 2-FYVE-GFP underwent phagocytosis for 20 min, followed by treatment with 1 µM Vps34-IN1 and then fixed after 25 min of treatment. Cells were stained as in panel b_._ (c) Cells from experiments in panels b and c were scored for the presence of PtdIns(3)P, detected via 2-FYVE accumulation at tPCs. Data shown are means ± SEMs from three independent experiments (n = 35 for each). P < 0.05. Bars, 5 µm. (d) RAW cells expressing 2-FYVE-GFP (green) were treated with 10 nM apilimod for 1 h before the phagocytosis. Representative phenotype from 90 cells analyzed in three independent experiments.

Figure 4.

Figure 4.

PtdIns(3)P dynamics on the tPCs. (a) RAW macrophage expressing 2-FYVE-GFP (white), engulfing a filamentous bacteria (red), selected frames from Video 1. Images to the right of the main panels are magnifications of the framed regions in the main panels depicting 2-FYVE-GFP in tPCs. For the first two images, the area magnified corresponds to the most distal tip of the tPC containing filamentous bacteria. In the 4 last micrographs (times 3:15 to 14:45), panel i is the most distal tip of the tPC and ii is the proximal tPC. Bars: 5 µm; (enlarged areas) 1 µm. (b) RAW macrophages expressing 2-FYVE were challenged with filamentous bacteria and fixed after 5, 15, 30, and 45 min of phagocytosis. The length of filamentous bacteria positive for 2-FYVE was plotted against the bacterial length internalized at each time point. The corresponding correlation coefficients (r2) were calculated. (c) Regions of tPCs that surpass a length of 20 µm (red line) tend to be devoid of 2FYVE-GFP. The ratio of 2FYVE-GFP–positive length to the total length of the tPC is shown. A ratio of 1 indicates that the entire tPC is decorated with 2FYVE-GFP, a ratio <1 indicates that a portion is devoid of this probe, and a ratio of 0 indicates that the tPC is completely divested of the probe. Data from three independent experiments (n = 30 for each time point). (d) RAW macrophages expressing p40PX-GFP, treated with 10 nM apilimod or 0.1% DMSO (vehicle) for 1 h, were allowed to engage in phagocytosis of pHrodo-conjugated filamentous bacteria for 30 min of phagocytosis. The length of filamentous bacteria positive for p40PX-GFP was measured in three independent experiments, n = 30 for each condition, and plotted as described in panel b.

Figure 5.

Figure 5.

Loss of PtdIns(3)P is correlated with the acidification of distal tPCs. Evolution of p40PX-GFP and pHrodo fluorescence in growing tPCs. RAW macrophages expressing p40PX-GFP (top, green; middle, white) were challenged with pHrodo-conjugated filamentous bacteria and imaged live by simultaneous acquisition of both fluorescence emissions. Micrographs are selected frames from Video 2. pHrodo fluorescence is shown in red in the top row of micrographs and in false-rainbow palette in bottom one. Blue in the rainbow palette indicates neutral pH and red indicates acidic pH. Brackets in the panels from the left indicate position of external filamentous bacteria, detected in bright light field. Arrowheads in lower panels point to the distal tip of the tPCs. Asterisks indicate bacterial-associated pHrodo fluorescence extracted into cytoplasmic vesicles as the tPCs remodel during internalization. Bars, 5 µm.

Figure 6.

Figure 6.

Forced acidification divests PtdIns(3)P reporters from tPCs. (a) Selected frames from a time-lapse sequence from Video 4 showing a RAW macrophage expressing p40PX-GFP (white) engulfing a pHrodo-conjugated bacteria (turning red as it acidifies). Arrowhead indicates the time at which cells were exposed to culture media at pH 4.5. Images at the bottom of main panels show magnifications of the framed areas. Right panels show pHrodo signal (false-rainbow palette) and left panels show p40PX-GFP. Bars, 5 µm. (b) RAW cells expressing 2FYVE-GFP were challenged with filamentous bacteria in neutral or acidic conditions. After 10 and 20 min of phagocytosis, cells were imaged for accumulation of 2FYVE-GFP at tPCs. Data shown are means ± SEMs from three independent experiments (n = 20 for each; *, P < 0.05). (c) RAW cells expressing p40PX-GFP were challenged with pHrodo-conjugated filamentous bacteria. The phagosomal pH was determined for p40PX-GFP–positive and –negative phagosomes after 15 and 30 min of phagocytosis, respectively. (Ap+) cells were treated with 10 nM apilimod 1 h before phagocytosis and pH measured 30 min of phagocytosis. Data represent 15 phagosomal pH values ± SD from three independent experiments. ANOVA test was used to compare each group; ***, P < 0.001. (d) Binding of p40PX-GFP to PtdIns(3)P does not depend on pH. (Left) Protein-lipid overlay (PLO) using recombinant p40PX-GFP-Hisx6 and membranes containing 400 and 200 pmols of PtdIns(3)P. This is a representative of three independent experiments. (Right) Densitometry of spots from PLOs expressed relative to that observed at pH7, expressed as means ± SEMs of three independent experiments.

Figure 7.

Figure 7.

pH neutralization causes PtdIns(3)P to persists in phagosomes. (a) RAW cells expressing 2FYVE-GFP (rainbow) were treated with 0.1% DMSO (vehicle), 1 µM ConA, 10 mM NH4Cl, or both ConA and NH4Cl for 15 min before the phagocytosis of IgG-opsonized beads. White and magenta arrows depict examples of 2FYVE-positive and -negative phagosomes, respectively. (b) The phagosome/cytosol ratio of 2FYVE-GFP fluorescence for each time point indicated, normalized to 7 min, represented as relative units (R.U). Data shown are means ± SEMs from three independent experiments (n = 30 for each). ANOVA test was used to compare each treatment condition to vehicle. For each time point; *, P < 0.05; **, P < 0.01. Bars, 5 µm.

Figure 8.

Figure 8.

p40PX-mCh recruitment to zymosan-containing phagosomes is pH dependent. (a) Live-cell imaging of p40PX-mCh–expressing (white) RAW cells, internalizing pHrodo-conjugated zymosan particles (green), either in culture media (vehicle) or in acidic media (pH 4.0), with or without 1 µM ConA. The arrowhead indicates the switch to acidic media, 30 s after the onset of the experiment. Images were acquired every 15–30 s. Images to the right of the main panels show fluorescence intensities (rainbow) for pHrodo-zymosan (top) and p40PX-mCh (bottom) phagosomes framed in the main panels. Blue indicates low fluorescence levels for p40PX-mCh and pHrodo (neutral pH), whereas red indicates high-emission signal for p40PX-mCh and pHrodo (acidic pH). (b) Progression of pHRodo and p40PX-mCh fluorescence intensities in zymosan-containing phagosomes for the experimental conditions described in panel a. Each time point was normalized to 0 mins, represented as relative units (R.U). Data shown are normalized means ± SEMs from 3 independent experiments (n = 10–15 phagosomes for each). ANOVA test was used to compare each treatment condition to control; ***, P < 0.001. Bars, 5 µm. (c) RAW cells expressing p40PX-mCh were challenged with pHrodo-conjugated zymosan and pH determined for p40PX-mCh–positive and –negative phagosomes. Data represent 15 phagosomal pH values ±SD from three independent experiments. Each phagosome was internally calibrated (***, P < 0.001).

Figure 9.

Figure 9.

The Vps34 complex II association to phagosomes is controlled by pH. (a) Vps34 complex II subunits in isolated latex beads phagosomes. RAW macrophages in culture media (control), acidic culture media (pH 4.0), or cell-culture medium with 10 mM NH4Cl were allowed to internalize IgG-opsonized latex beads for 20 min. After this period, phagosomes were isolated and immunoblotted for the presence of Vps15, UVRAG, Vps34, Raptor, and Rab7. Human IgG was used as a control for number of phagosomes loaded. (b) Protein densitometric quantification for blots shown in panel a. Human IgG was used to normalize phagosomal loading. Data shown are means ± SEMs from four to six independent experiments; *, P < 0.05; **, P < 0.01. (c) Phos-Tag SDS-PAGE determination of the phosphorylation states of UVRAG, Vps34, and Vps15 in phagosomes isolated as described in panel a. (d) The phosphorylation coefficient was used to quantify the preferred states of phosphorylation. This was done by calculating an internal ratio of the most (top) to least (bottom) phosphorylated species. Data shown are means ± SEMs from three or four independent experiments. *, P < 0.05.

Figure 10.

Figure 10.

PtdIns(3)P on endosomes is controlled by pH. (a) The cytoplasmic pH of pHrodo-AM–loaded RAW macrophages over time. RAW macrophages were incubated in either culture media (vehicle) or acidic media (pH 4.0), with or without 1 µM ConA. pHrodo-AM fluorescence intensity is shown in rainbow palette. Bars, 5 µm. Graph illustrates the changes in fluorescence intensity over time. Each time point was normalized to 0 mins, represented as relative units (R.U). Data shown are means ± SEMs from three independent experiments (n > 50 cells for each condition). ANOVA test was used to compare each treatment condition to control; ***, P < 0.001. (b) RAW macrophages expressing 2FYVE-GFP (white) show PtdIns(3)P containing endosomes (arrows). 2FYVE-GFP–positive endosomes disappeared when macrophages were exposed to acidic cell-culture medium (pH 4.0) for the time points indicated (representative of the independent experiments). Bars, 5 µm. (c) Determination of PtdIns(3)P level in RAW macrophages. Cells were incubated with 3H-_myo_-inositol overnight and subjected to the conditions indicated for 20 min. After inositol isolation and separation, levels of PtdIns(3)P and PtdIns(4,5) were determined. For each treatment, PtdInsP levels were normalized to the parent PtdIns peak and compared with control. Data shown are means ± SEMs from four independent experiments. *, P < 0.05.

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