Fig 1.
LPS-mediated activation of phagocytes augments lysosome volume and holding capacity.
(a) Lysosomes in BMDMs, BMDCs, and in RAW macrophages before and after 2 h of LPS stimulation, the latter causing lysosome tubulation. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 5 μm. (b) Relative lysosome volume between counterpart resting and LPS-treated phagocytes acquired by live-cell spinning disc confocal imaging. (c) Relative lysosome volume in resting and LPS-treated RAW macrophages fixed with a mixture of GF to preserve tubules. (d) Relative lysosome area from the midsection of resting and LPS-activated phagocytes using images acquired by SIM-enacted super-resolution microscopy. (e) Lysosome volume in resting and LPS-treated cells that are live or fixed with 4% PFA. (f) Image compilation of 6 representative fields in false colour showing changes in intensity of LY acquired by endocytosis over the indicated time in resting primary macrophages or macrophages stimulated with LPS. Scale = 250 μm. Colour scale: 0 to 4,095 (low to high). (g) Accumulation of LY continuously endocytosed over indicated time frame in resting, activated with LPS for 2 h, or co-activated with LPS continuously. (h) Rate of pinocytosis of LY in primary macrophages treated as indicated. (i) Retention of LY in resting or LPS-treated primary macrophages after 0.5 h internalization and chase in probe-free medium over indicated times. All experiments were repeated at least 3 independent times. For panels b through e, data are based on 30 to 40 cells per condition per experiment and are shown as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and unpaired post hoc test, in which the asterisk indicates a significant increase in lysosome volume relative to resting phagocytes (*p < 0.05). For panels g through i, fluorescence measurements were acquired by fluorimeter plate imager. Data are shown as the mean ± SEM, in which statistical analysis was performed using an analysis of covariance, whereby controlling for time as a continuous variable. An asterisk indicates a significant increase in LY for that series relative to resting phagocytes (*p < 0.05). See S1 Data for original data in Fig 1. BMDC, bone marrow–derived dendritic cell; BMDM, Bone marrow–derived macrophage; GF, glutaraldehyde-formaldehyde; LPS, lipopolysaccharides; LY, Lucifer yellow; PFA, paraformaldehyde; SIM, structured illumination microscopy.
Fig 2.
Lysosome remodelling requires protein biosynthesis.
(a) Western blot analysis of whole-cell lysates from resting primary macrophages or macrophages exposed to the indicated combinations and time of LPS and CHX. (b) Quantification of Western blots showing the levels of LAMP1, cathepsin D CtsD, and the V-ATPase V1 subunit H and D normalized to β-actin. Data are shown as the mean ± standard error of the mean from at least 3 independent experiments. For panels a and b, ‘2/4’ indicates cells stimulated with 2 h of LPS, followed by a 4 h chase, whereas ‘2 h’ and ‘6 h’ represent cells continuously exposed to LPS for those time periods. (c) Endogenous LAMP1-positive structures in resting and activated primary macrophages. (d) Quantification of total LAMP1 fluorescence levels in macrophages per μm3. (e) Live-cell spinning disc confocal micrographs of prelabelled lysosomes in resting primary macrophages or those stimulated with LPS and/or CHX. (f) Relative lysosome volume between resting primary macrophages and those exposed to specified conditions. Shown is the mean ± standard error of the mean from 30 to 40 cells for each condition and experiment, across at least 3 independent experiments. Scale bars = 5 μm. Statistical analysis was done with ANOVA and unpaired post hoc test. The asterisk indicates a significant difference (*p < 0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S2 Data for original data in Fig 2. CHX, cycloheximide; CtsD, cathepsin D; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; TBP, Tata-box binding protein.
Fig 3.
Lysosome remodelling is independent of TFEB and TFE3 activation.
(a) TFEB and TFE3 subcellular localization in resting primary macrophages (vehicle) or those treated with LPS for 2 or 6 h, or with torin1. Green = TFEB or TFE3 immunofluorescence signal; white = nuclei stained with DAPI. Areas within dashed boxes are magnified as insets. (b) Nuclear-to-cytosolic ratio of TFEB or TFE3 fluorescence intensity. Shown is the mean ± standard error of the mean from 30 to 40 cells per condition per experiment across at least 3 independent experiments. (c) Lysosomes in wild-type, tfeb−/−, tfe3−/−, and tfeb−/− tfe3−/− RAW strains before and after 2 h of LPS stimulation. Images were acquired by live-cell spinning disc confocal microscopy. Yellow arrowheads illustrate tubular lysosomes. (d) Average lysosome tubulation index in resting and LPS-activated strains. Shown is the mean ± standard error of the mean from 40 to 50 cells per condition, across 3 independent experiments. Lysosome tubules longer than 4 microns were scored, from which tubulation index was determined following normalization to the average number of tubular lysosomes in resting wild-type cells for each experiment. (e) Relative lysosome volume between LPS-treated and resting counterpart RAW strains acquired by live-cell spinning disc confocal imaging. The average lysosomal voxel counts for LPS-activated strains were normalized to resting wild-type cells. Shown is the mean ± standard error of the mean from 30 to 40 cells per condition per experiment across 3 independent experiments. (f, g) Relative mRNA levels of select lysosomal genes (f) or interleukin-6 (g) in activated primary macrophages relative to Abt1 housekeeping gene and normalized against resting cells. Quantification was done with qRT-PCR by measuring the ΔΔCt as described in methods. Shown is the mean ± standard error of the mean from 4 independent experiments. All statistical analysis was done with ANOVA and unpaired post-hoc test. The asterisk indicates a significant difference relative to resting condition (*p < 0.05). For panels a and c, scale bar = 5 μm. See S3 Data for original data in Fig 3. Abt1, activator of basal transcription 1; ATP6V1D, ATPase H+ transporting V1 subunit D; ATP6V1H, ATPase H+ transporting V1 subunit D; BMDM, Bone marrow–derived macrophage; IL-6, interleukin-6; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; qRT-PCR, quantitative real-time-polymerase chain reaction;; TFE3, transcription factor E3; TFEB, transcription factor EB; TRPML1, transient receptor potential cation channel, mucolipin subfamily; ΔΔCt, change in threshold cycle.
Fig 4.
mTOR stimulates lysosome volume and holding capacity.
(a) Lysosomes in primary macrophages were pretreated with a vehicle (DMSO), Akti or torin1, followed by 2 h LPS stimulation where indicated. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 5 μm. (b) Lysosome volume in primary macrophages treated as indicated and normalized to resting macrophages. Shown is the mean ± standard error of the mean from 30 to 40 cells per condition per experiment across 3 independent experiments. (c) Western blot analysis of whole-cell lysates from resting primary macrophages or macrophages exposed to the indicated combinations and time of LPS, torin1 and Akti. (d) Quantification of Western blots showing the levels of LAMP1, CtsD, and the V-ATPase V1 subunit H and D normalized to β-actin. Data are shown as the mean ± standard error of the mean from at least 3 independent experiments. For panels a and b, ‘2/4’ indicates cells stimulated with 2 h of LPS followed by a 4 h chase, whereas ‘2 h’ and ‘6 h’ represent cells continuously exposed to LPS for those time periods. (e) Quantification of pinocytic capacity in macrophages treated as indicated. Shown is the mean ± standard error of the mean from 4 independent experiments. For panels b and d, data were statistically analysed with ANOVA and unpaired post hoc test (*p < 0.05). For panel e, data were statistically assessed using an analysis of covariance, whereby controlling for time as a continuous variable. An asterisk indicates a significant increase in LY for that series relative to resting phagocytes (*p < 0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S4 Data for original data in Fig 4. Akti, Akt inhibitor; CtsD, cathepsin D; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; LY, Lucifer yellow; mTOR, mechanistic target of rapamycin; V-ATPase, vacuolar H+ ATPase pump.
Fig 5.
S6K is required for the LPS-mediated lysosome expansion.
(a) Western blot analysis of protein puromycylation in resting and activated primary macrophages. LPS increases the amount of puromycylated proteins that is blocked by p70S6K inhibitor (LY2584702) or cycloheximide. Lane 1 is control lysates from cells not exposed to puromycin. The band indicated by the arrow is a nonspecific band recognized by the anti-puromycin antibody. p-S6 and β-actin were used to monitor p70S6K activity and as a loading control, respectively. (b) Normalized puromycylation signal (excluding nonspecific band) normalized over β-actin signal. Data are shown as the mean ± standard deviation from 3 independent experiments. Statistical analysis was done with an ANOVA, in which an asterisk indicates conditions that are statistically distinct from control (*p < 0.05). (c) Lysosomes in primary macrophages were pretreated with LY2584702 (LY2) followed by 2 h of LPS where indicated. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 5 μm. (d) Lysosomal tubulation was scored for each condition as shown, in which a tubule was defined as longer than 4 μm in length. Tubulation index was determined by normalizing scores to resting cells. (e) Total lysosome volume in primary macrophages treated as indicated. Panels d and e show the mean ± standard error of the mean from 30 to 40 cells per condition per experiment, across 3 independent experiments. (f) Western blot analysis of whole cell lysates from resting and activated primary macrophages with or without LY2584702. (g) Quantification of Western blots showing the levels of LAMP1 and the V-ATPase V1 subunits H and D, normalized to β-actin. p-S6 and total S6 blots are shown to support effectiveness of LY2584702 treatment. Shown is the mean ± standard deviation of the mean from 5 independent blots. For panels b, c, and e, data were statistically analysed with ANOVA and unpaired post hoc test (*p < 0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S5 Data for original data in Fig 5. CHX, cycloheximide; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; S6K, S6 kinase; V-ATPase, vacuolar H+ ATPase pump; LY, Lucifer yellow.
Fig 6.
Active 4E-BP1 suppresses LPS-mediated lysosome expansion.
(a) Lysosomes in resting or LPS stimulated (2 h) RAW cells stably expressing the 4E-BP1 (4Ala) phosphorylation mutant or the empty pBabe vector. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 10 μm. (b) Lysosomal tubulation was scored for each, in which a tubule was defined as longer than 4 μm in length. Tubulation index was determined by normalizing scores to resting. (c) Total lysosome volume in engineered RAW macrophages treated as indicated. Panels b and c show the mean ± standard error of the mean from 30 to 40 cells per condition per experiment across 3 independent experiments. (d) Western blot analysis of whole-cell lysates from stable cell lines. (e) Quantification of Western blots showing the levels of LAMP1 and the V-ATPase V1 subunits H and D, normalized to β-actin for both cell lines. Anti-HA blot demonstrates expression of 4E-BP14Ala. Shown is the mean ± standard deviation of the mean from 3 independent blots. For panels b, c, and e, data were statistically analysed with ANOVA and and unpaired post hoc test (*p < 0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S6 Data for original data in Fig 6. HA, hemagglutinin; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; V-ATPase, vacuolar H+ ATPase pump; 4E-BP1, 4E binding protein-1.
Fig 7.
LPS increases translation of mRNAs encoding lysosomal proteins in an mTOR-dependent manner.
(a–g) Percent of target mRNA—(a) LAMP1, (b) ATP6V1H, (c) ATP6V1D, (d) CtsD, (e) β-actin, (f) PPIA, and (g) B2M—associated with each ribosome fraction in resting, LPS- or LPS/torin1-treated RAW cells. Left and middle panels show 2 h and 6 h treatments, respectively. Shown is a representative experiment from 4 independent experiments, each of which contained 3 technical replicates. Right panels: Pooled percent of mRNA in subpolysomal (fractions 7–10), light polysosome (fractions 11 and 12), and heavy polysomes (fractions 13–16). Shown is the mean percent ± standard deviation from 4 independent experiments with each point in triplicate for each experiment and mRNA. Heavy fractions were statistically analysed by ANOVA and Tukey’s post hoc test, in which a single asterisk indicates statistical difference from resting conditions, whereas two asterisks indicate differences between LPS and LPS+torin1 conditions within 2 and 6 h exposure. See S7 Data for original data in Fig 6. ATP6V1D, V-ATPase V1 subunit D; ATP6V1H, V-ATPase V1 subunit H; B2M, β2-microglobulin; CtsD, cathepsin D; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; mRNA,; mTOR, mechanistic target of rapamycin; PPIA, peptidylpropyl isomerase A.
Fig 8.
Transcriptome analysis of heavy polysomes during LPS-mediated activation of RAW cells.
(A) Distributions (kernel density estimates) of FDRs from comparisons of gene expression between 6 h treatments with LPS to control (left) and LPS in presence or absence of torin1. Differences in polysome-associated mRNA (orange), total mRNA (purple), translation (red), and buffering (blue) were assessed using polysome profiling. (B) Scatter plot of polysome-associated mRNA versus total mRNA log2 fold changes (6 h LPS to control). The number of transcripts exhibiting changes in translation (red), buffering (blue), and mRNA abundance (green) stratified into increased (light shade) or decreased (dark shade) expression are indicated. (C) Scatter plot of polysome-associated mRNA versus total mRNA log2 fold changes (left) together with cumulative distribution plots for polysome-associated mRNA (middle) and total mRNA (right) log2 fold changes from the comparison between 6 h LPS treatment to control. Translationally activated (light red) and translationally suppressed (dark red) mRNAs are indicated together with background transcripts (i.e., not in either of sets; grey). (D) Same plots and subsets of transcripts as in panel c but using gene expression data originating from the comparison between 6 h LPS treatment in presence or absence of torin1. (E–G) Heat map of log2 fold changes for total mRNA (E), polysome-associated mRNA (F), and changes in translation efficiencies (G) following 6 h treatments with LPS relative to resting (green) and LPS in presence relative to absence of torin1 (orange) for genes annotated to the lysosme pathway. The sidebars indicate genes with significantly changed expression in their associated analysis separately for the 2 comparisons (i.e., green or orange). See S1 Table and deposited data in Gene GEO with accession number GSE136470 for original data in Fig 8. FDR, false discovery rate; GEO, Gene Expression Omnibus; LPS, lipopolysaccharides.
Fig 9.
mTOR and S6K control Eα52–68 peptide presentation in activated BMDCs.
BMDCs were incubated with Eα52–68 peptide for 4 or 6 h in the presence or absence LPS with or without torin1 and LY2584702. Cells were then fixed and stained with Y-Ae antibodies to detect I-Ab::Eα52–68 complex formation, and DAPI to stain nuclei. (a) I-Ab::Eα52–68 complexes (displayed in pseudocolour) and DAPI (greyscale) are shown for BMDCs treated as indicated. (b, c) Anti-I-Ab::Eα52–68 antibody signal was quantified by fluorescence intensity associated with each cell. Shown is the mean of the total fluorescence intensity of I-Ab::Eα52–68 complexes ± standard deviation from 3 experiments, in which 50 to 100 cells were quantified for each. Data were analysed using ANOVA, whereby a single asterisk indicates a difference compared with unstimulated BMDCs exposed to Eα52–68 and two asterisks indicates a difference compared with LPS-stimulated BMDCs fed Eα52–68 (**p < 0.05). Scale bar = 30 μm. Colour scale: 0 to 2,500 (low to high). S10 Fig show similar data for HEL presentation. (d) BMDCs were fed Eα52–68 peptide for 4 or 6 h in the presence or absence LPS with or without torin1 and LY2584702. Following mild fixation, APCs were co-incubated with T cells as described in Materials and methods to measure I-Ab::Eα52–68 complex induced T-cell activation. T-cell–secreted Il-2 was measured using an ELISA system. All data were analysed using ANOVA, whereby two asterisks indicates a difference compared with unstimulated BMDCs exposed to Eα52–68 (**p < 0.05). (e) Western blot analysis of whole-cell lysates from APCs. p-S6 and β-actin were used to monitor mTOR-p70S6K signalling axis activity and as a loading control, respectively. (f) Quantification of Western blots showing the levels of MHC-II (I-A/I-E) normalized over β-actin signal. Data are shown as the mean ± standard deviation from 4 independent experiments. Statistical analysis was done with an ANOVA, in which a single asterisk and two asterisks indicates a significant difference of 2 h and 6 h conditions, respectively, from resting cells (p < 0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S8 Data for original data in Fig 9. APC, antigen-presenting cell; BMDC, bone marrow–derived dendritic cell; ELISA, enzyme-linked immunosorbent assay; HEL, Hen Egg Lysozyme; IL-2, interleukin-2; LPS, lipopolysaccharides; LY2, LY2584702; MHC-II, major histocompatibility complex-II; mTOR, mechantistic target of rapamycin; S6K, S6 kinase.