EMBO/SourceData huggingface dataset:
Figure 3(A) Fluorescence microscopy demonstrates that the overexpression of Cherry-Lam6 (OE-Cherry-LAM6) results in an expansion of the following three contact sites: ERMES (Mdm34-GFP), NVJ (Nvj1-GFP), and vCLAMP (GFP-Vps39). This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion. The numbers represent the average contact site size (120 cells per sample). Scale bar represents 5 μm.(B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).
Figure 4(A) Fluorescent microscopy shows that the ERMES contact (as measured by the number of Mdm34-GFP puncta per cell) expanded in the Δvps39 background relative to WT. However, the expansion did not occur on the background of Δvps39 Δlam6, demonstrating that Lam6 is necessary for ERMES expansion under these conditions. Scale bar represents 5 μm.(B) Quantitation of (A). Bars represent the percentage of cells containing the specific number of puncta/cell out of total cells counted for the strain (for WT, n = 234 cells; for Δvps39, n = 246; for Δvps39 Δlam6, n = 271).(C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4).
Figure 1C. Quantification of Cre activity by dual luciferase assays (mean±SD, n=3). Data information Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence.D. Quantification of tTA activity by dual luciferase assays (mean±SD, n=3 (N and C) or n=7 (N/C)). Data information: Npu: Nostoc punctiforme CDS: coding sequence.
Figure 2A-B. Scheme of engineered Spc and Ccsp loci after integration of split-Cre and split-tTA effectors (red mark = stop codon) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections. Blue: DAPI. Scale bar: 100 µm.C-D. β-galactosidase and H&E staining of lung sections from BASC tracer and BASC viewer animals. Scale bars: 500 µm (overview) and 20 µm (boxed magnification).E. Localization of β-gal+ cells in adult BASC viewer animals. BADJ-associated and "alveolar" β-gal+ cells are expressed as percentage of all lineage-labeled cells (means±SD n=3).F. Number of β-gal+ cells in adult BASC tracer and BASC viewer animals, normalized to the number of terminal bronchioles (TBs) (means±SD n=3).
Figure 3A. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of adult BASC viewer animals. Arrow highlights labeled BASC at BADJ (magnified in box). Blue: DAPI. Scale bars: 100 µm (overview) and 20 µm (boxed magnification).B. β-galactosidase staining of AT2 cells (YFP+), Club cells (mCherry+) and BASCs (mCherry+/YFP+) isolated from BASC viewer mice by fluorescence-activated cell sorting (FACS). Scale bar: 50 µm.C. Doxycycline-dependent reversible labeling of BASCs in BASC viewer mice. Scale bar: 200 µm.
Figure 4A. Heatmaps for Spearman correlation.C. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and Club cells. D. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and AT2 cells. E. Heatmap of RNA-seq z-scores computed for the Top50 DEGs in BASC, AT2, and Club cells (FDR<0.05, Log2FC>±0.585, Means>5).
Figure 5B. Quantification of β-gal+ cells in FACS-purified AT2 and Club cell fractions isolated from 3 months old BASC v-race mice (mean±SD, n=3) and example of β-galactosidase label distribution in lung sections. Scale bar: 100 µm.C. BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life. β-galactosidase staining of lung sections derived from BASC v-race animals collected at indicated time points. Arrow highlights β-gal+ cell at P6. Scale bar: 200 µm.
Figure 6A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification).B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm.C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).
Figure 7A. β-galactosidase and H&E staining of lung sections from control (non-infected) and injured BASC viewer mice 7 days post infection (7 dpi) with influenza virus. Scale bar: 200 µm. B. Abundance of β-gal+ cells in control (non-infected) and injured (7 dpi) BASC viewer animals. Data depicted as mean±SD, n = 4, *p = 0.0286 (Mann-Whitney test), TB - terminal bronchiole. C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm.D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm.E. Abundance of β-gal+ cells in control and injured BASC viewer animals. The percentage of terminal bronchioles containing 0, 1, 2, 3, 4, ≥ 5 β-gal+ cells is shown. Data depicted as mean±SD, n=3.
Figure 8A. Quantification of lineage-traced Club cells in distal TBs and β-galactosidase staining of cleared accessory lobe preparation (scale bar: 250 µm) isolated from injured BASC v-race mice at 21 dpn (mean±SD, n=4). Bottom panel: example of sequential microscopic imaging to quantify β-gal+ mCherry+ cells in distal TBs. Arrows denote last airway bifurcation before bronchioalveolar duct junction (BADJ). Scale bar: 200 µm.B. β-galactosidase staining of FACS-purified Club cells from control and injured BASC v-race animals. Scale bar: 50 µm. The percentage of lineage-traced Club cells is shown (mean±SD, n=4 (oil ctrl.) or n=5 (21 dpn), *p = 0.0159 (Mann-Whitney test).C. Combined β-galactosidase (blue) and acetylated tubulin staining (cilia, brown color) of lung sections from BASC v-race animals at 21 dpn. Red: DAPI (pseudocolor). Examples of lineage-traced ciliated cells (arrowheads) and Club cells (asterisks) are highlighted. Scale bar: 50 µm.D. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and quantification of β-gal+ bronchioles in lung sections from adult BASC v-race (ctrl.) and BASC-ablated mice (DTA). Data depicted as mean±SD, n=3. E. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and fluorescence microscopy of lung sections from uninjured and naphthalene-exposed BASC v-race (ctrl.) and BASC-ablated (DTA) mice 12 days post naphthalene (dpn). Dotted lines depict bronchiolar boundaries. Scale bar: 100 µm. F. Quantification of Club cell recovery in injured BASC v-race (ctrl.) and BASC-ablated (DTA) animals, expressed as percentage of bronchiolar circumference re-populated by mCherry+ cells at 12 dpn. Data depicted as mean±SD, n=5, **p = 0.0079 (Mann-Whitney test). DTA - diphtheria toxin fragment A.
Figure 1Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.
Figure 2Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm.Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.
Figure 3PINK1 KO HeLa cells stably expressing mito-Keima were infected with control or shATAD3B. Five days later, cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were then analyzed and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561nm/458nm) of the cells described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.HeLa cells were treated with DMSO, 4 mM 3-NPA, 8 mM 3-NPA, or 10 µM CCCP for 2 h, and cell lysates were then analyzed by Western blotting using the indicated antibodies.PINK1 KO HeLa cells stably expressing ATAD3A-Flag (3A-Flag) or ATAD3B-Flag (3B-Flag) were mixed with control PINK1 KO cells respectively, and incubated for 24 h. Cells were treated with 4 mM 3-NPA (D) or 200 µM H2O2 (F) for 2 h, and then washed with fresh medium and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and anti-Flag antibodies and analyzed by confocal microscopy. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (E) or H2O2 (G) was quantified by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by individual two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. Scale bar, 10 µm.Control (GFP+) and shATAD3B (GFP-) PINK1 KO HeLa cells were mixed and incubated for 24 h. Cells were then treated with 4 mM 3-NPA (H) or 200 µM H2O2 (J) for 2 h, and washed with fresh medium, and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and DAPI, then analyzed by confocal microscopy. Control or shATAD3B cells are circled by white dashed lines. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (I) or H2O2 (K) was quantified using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, **p < 0.01. Scale bar, 10 µm.
Figure 4293T cells were treated with H2O2 for 2 h, and cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with rabbit IgG (control) or anti-ATAD3 antibody, followed by Western blotting with anti-LC3B or anti-ATAD3 antibodies.ATAD3 DKO HeLa cells expressing GFP-LC3B were transiently transfected with control, ATAD3A-Flag or ATAD3B-Flag. 48 h after transfection, cell lysates were immunoprecipitated (IP) with anti-Flag M2 affinity gel, and analyzed by Western blotting using anti-Flag or anti-GFP antibodies.HeLa cells were treated with 200 µM H2O2 for 2 h, and cell lysates were incubated with the indicated GST-ATAD3A or GST-ATAD3B protein fragments (expressed in E.coli) coupled to glutathione agarose beads (Pierce) for GST pull-down assay. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.The indicated GST-ATAD3A or GST-ATAD3B protein fragments and His-LC3B were expressed in E.coli and purified. GST pull-down assay was performed using glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using anti-GST or anti-His antibodies.The indicated GST-ATAD3B(WT) or GST-ATAD3B (LIR mutations) and His-LC3 were expressed in E.coli and purified. GST pull-down assay was performed using the glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.HeLa expressing GFP-LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B. Five days later, cells were treated with DMSO, or 200 µM H2O2 for 2 h. Cells were fixed and immunostained with anti-Tom20 and imaged by confocal microscopy. The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 (mitochondria). Scale bar, 10 µm.Quantification of the GFP-LC3 puncta colocalized or contacted with Tom20 (mitochondria) in cells described in (G). Data are presented as mean ± SD (n =30 cells from 3 coverslips), statistical significance was assessed by a two-way ANOVA, N.S., not significant, ***p < 0.001.293T cells transiently transfected with control or ATAD3B-Flag were treated with or without 200 µM H2O2 for 2 h. Cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with anti-Flag antibody, followed by Western blotting with anti-Flag or anti-LC3B antibodies. Relative protein levels of LC3B and ATAD3B-Flag were further evaluated by densitometry
Figure 5Mic10 KO COS7 cells stably expressing ATAD3A-Flag or ATAD3B-Flag were treated with DMSO or 200 µM H2O2 for 2 h, and then fixed and immunostained with anti-Tom20 and anti-Flag antibodies, and analyzed by confocal microscopy. Scale bar, 10 µm.293T cells treated with DMSO (B) or H2O2 (200 µM, 2 h) (D), were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix). Relative protein levels of proteins in (B) or (D) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from DMSO- or H2O2-treated cells were shown (C and E). Data are presented as mean ± SD (n = 3 independent experiments).Control, ATAD3A KO, ATAD3B KO, or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min, and then were analyzed by Western blotting with the indicated antibodies.293T cells were treated with DMSO, 200 µM H2O2, 10 mM NAC, or 200 µM H2O2 plus 10 mM NAC for 2 h. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies. Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in (F). Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.143B, 143B ρ0 cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies.293T cells were treated with DMSO or ddC (50 µM) plus EB (0.5 µg/ml) for 10 days. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37℃, and then were analyzed by Western blotting with the indicated antibodies.ATAD3A KO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix) (K). Relative protein levels of proteins in (K) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown (L). Data are presented as mean ± SD (n = 3 independent experiments).
Figure 6ρ0206_A and ρ0206_B cells were stained with mitoSOX and analyzed by confocal microscopy. DIC, Differential Interference Contrast. Scale bar, 25 µm. Quantification of the MitoSOX fluorescence intensity described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by student's t-test, **p < 0.01.Mitochondria carrying the 3243A>G mutation in the tRNA Leu(UUR) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206_A (100% wild-type mtDNA) and ρ0206_B (~90% m.3243A>G mtDNA) cell lines. ρ0206_A and ρ0206_B cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.ρ0206_A, ρ0206_B, and ρ0206-B cells pretreated with ddC (50µM) plus EB (0.5µg/ml) for 10 days, and then harvested and lysed. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-GAPDH antibodies.ρ0206_B cells were infected with control, ATAD3B-Flag or ATAD3B (mLIR)-Flag. After two weeks, total DNA from cells was isolated and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.ρ0206_B cells were infected with control, ATAD3A-Flag, ATAD3B-Flag, or ATAD3B (mLIR)-Flag. After two weeks, the cellular ATP levels were measured. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.MELAS patient fibroblasts were infected with control, ATAD3A-Flag, ATAD3B-Flag or ATAD3B(mLIR)-Flag. After two weeks, total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, **p < 0.01.
f1Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a-f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.