EMBO/SourceData huggingface dataset:
Figure 2Localization of Ykt6 in multiple atg mutants. (A) Wild-type or the respective mutant cells expressing mCherry-tagged Ape1 and GFP-tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1-promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 (A)(B) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker. Cells (n≥250) and Ape1 dots (n≥70) were quantified.(C) Localization of Atg9 relative to Ykt6. Cells expressing GFP-tagged Ykt6 and mScarlet-tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9. Cells (n≥200) and Atg9 dots (n≥80) were quantified. Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 (C)Ykt6 localizes to the isolation membrane during starvation. (D) Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying the Ape1 encoding plasmid pRS315-CUP1pr-BFP-APE1 were grown in SGC medium containing CuSO4 and starved for 1 h, before to be analyzed by microscopy. Scale bar, 5 µm. (E) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view. Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying plasmid pRS315-CUP1pr-BFP-APE1, cells , images were analyzed by Imaris. Scale bar, 5 µm. Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 (D,E).(F) The only partial time points (90 s to 600 s) of images were assembled, and analyzed by Imaris and image J. Scale bar, 5 µm. 500-1000 cells were analyzed in each independent experiment. The 3D stacks were cropped by Imaris and different channels were split by Image J.
Figure 3(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.(C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection(D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Arrows in images indicate dots colocalization of Atg1 with Atg8 (D)(E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified. Arrows in images indicate dots colocalization of Ykt6 with Dsl1 (E).(G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.
Figure 4(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 and Atg8 (A)Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. (B) Percentage of Ykt6 puncta co-localizing with Atg8. Cells (n≥150) and Atg8 dots (n≥100) were quantified.(C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 with Sec63 (C).(D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.
Figure 5(A-B) Quantification of size and number of autophagosomes in the indicated ts mutant strains. Error bars represent standard deviation of 3 independent experiments. GFP-tagged Atg8 in the different mutants were grown at 23°C (ts mutant) or 30°C (atg1∆) in SDC and then shifted to SD-N for 2 h at 30°C (atg1∆) or 37°C (ts mutant), and analyzed by fluorescence microscopy. Scale bar, 1 µm.(C) Localization of Atg8 relative to Atg9 during nitrogen starvation. dsl3 cells expressing mCherry-tagged Atg9 and carrying a CEN plasmid expressing GFP-Atg8 were grown at 23°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.(D) Atg9 localization in dsl3 cells during starvation. dsl3 ts cells expressing mCherry-tagged Atg9, and carrying a CEN plasmid expressing GFP-Atg8 and plasmid pRS315-CUP1pr-BFP-APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure, and starved for 1 h. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.
Figure 6(B) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Atg1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Right, percentage of Ykt6 puncta co-localizing with Atg1. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg1 dots (n≥100) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg1 (B)(C) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex. Purified Ykt6 was incubated with the Atg1-Atg13 complex in the presence or absence of ATP, and Ykt6 band was cut out and analyzed by mass spectrometry.(D) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions. Wild-type and atg1D211A cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual focal planes are shown. Scale bar, 5 µm. (E) Percentage of Ykt6 puncta co-localizing with Atg8. Numbers in the column indicates the percentage. Cells (n≥300) and Atg8 dots (n≥60) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg8 (D).
Figure 7(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6.(B) Effect of Ykt6 phospho-mutants on autophagy. ykt6∆ cells expressing wild-type Ykt6 plus a pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutants with S to A mutations [SA; S182, S183A], or S to D mutations [SD; S182D, S183D]), and a plasmid expressing GFP-Atg8 were grown in SGC and then shifted to SG-N for 2 h. Cells were analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (C) Quantification of Atg8 dots per cell from images in (B). Cells (n≥100) and Atg8 dots (n≥50) were quantified.(D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.
Figure 2After 2 hour starvation, E. coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems, and optical density (OD) was measured over time (inset example figures). Grey dots are OD measurements and the black lines are an empirical fit (see Methods). For separate experiments (n = 18), the lag time is plotted against the frequency, represented as the time-integrated (TI) feedrate f (mmol glucose/g dry cell weight/hour). An empirical fit (grey solid line, see Methods) was used to separate the lag (non-dividing) and dividing phasesNormalized absolute cell counts versus time show linear increases after lag time for exemplary feedrates. Data are mean ± standard error of technical replicates (n = 2-3). Lag predicted from the empirical fit is indicated by vertical dotted linesThe average size of cells decreased after lag. Micrographs and cell length distributions (n > 400 per distribution) are shown for specific time points, with f = 0.6 mmol/g/hImmobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency. The labeled times indicate the period, time between pulses, for a given experimentTime course of the distribution of cellular DNA content. Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment (f = 0.6 mmol/g/h) The DNA content distribution over time is shown on the left side, and three specific time points are shown on the right. Within the first time point (t = 0), the highest distribution is taken to be high DNA content (2N), and the distribution at half of the 2N average was taken (1N) as medium DNA. DNA distributions were separated into medium (1N) and high DNA (2N). Distribution-specific estimated counts (see Methods) over time (f = 0.6 mmol/g/h) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD increase
Figure 4The spin flask system for glucose pulsing was connected to a real-time metabolomics platform. Traces of exemplary ions are shown that correspond to hexose phosphate, guanine, phenylalanine, and hypoxanthine for pulsing at non-division supporting frequencies of 0.06, 0.12, and 0.18 mmol/g/h. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Glucose pulses are indicated by the grey bars, and the pink region shows a no pulse control. Dots are ion intensity measurements. Solid lines are a moving average filter of the measured ion intensity. For clarity, dots are not shown for f = 0 mmol/g/h condition A metabolic scheme describing the propagation of fed glucose. Pulsed glucose is hypothesized to pass through central carbon metabolism and then be converted to downstream pathways including amino acid synthesis and nucleic acid synthesis. For nucleic acid synthesis, glucose is converted to the intermediate PRPP, which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis. Different pathways can be blocked with antibioticsInfluence of antibiotics that inhibit macromolecular synthesis at the non-division TI feedrate of 0.18 mmol/g/h. Antibiotics were added one minute after the second pulse (yellow region). Four different ions are shown corresponding to glutamate, phenylalanine, guanine, and thymine. Chloramphenicol (blue) inhibits protein biosynthesis, rifamycin (orange) inhibits RNA polymerase, and azidothymidine (AZT; red) inhibits DNA synthesis
Figure 5Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non-dividing cells. After 6 hours of pulsing uniformly labeled 13C-glucose, cultures were lysed, and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers. Labeling data are presented as the mean ± standard error of independent biological replicates (n = 3, all pairwise P < 0.02 as determined by one sided Student"s t-
Figure 7Pulsing experiment was repeated in the presence of protease inhibitor (PI) that reduced the lag time for a given TI feedrate (f = 0.28 mmol/g/h). The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line
Figure 9Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate (f = 0.38 mmol/g/h). Induction level corresponds to the amount of doxycycline (max - 50 ng/uL, half - 10 ng/uL, and zero 0 ng/uL) added at the onset of starvation. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line. Lag time reduced with synthesis levels of titrated FtsZ in the wild-type strain at f = 0.31-0.32 mmol/g/h. Lag time increased with titrated synthesis of ClpX in wild-type cells at f = 0.43-0.45 mmol/g/h
Figure 1E Quantification of mean fluorescence intensity (MFI) for CD80, CD86, CD206, CD163, CCR2, and MHC-Ⅱ in TAMEMs and typical macrophages subtypes via flow cytometry (*P < 0.05, ***P < 0.001, and ****P < 0.0001; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (E) in Graph Pad Prism 8.F Representative immunofluorescent (IF) staining images for CD86 and CD206 in TAMEMs and typical macrophages subtypes (scale bar: 10 μm; n=6, biological replicates).
Figure 3A Quantification of concentrations of 22 soluble proteins from the cytokine and angiogenesis array analysis (*P < 0.05, ***P < 0.001, and ****P < 0.0001; ns: not significan; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (A, in Graph Pad Prism 8.F, G Western Blotting analysis of phosphorylated IκB (F) and quantification of gray value (G) in fibroblasts (L929) that pre-treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the LPS-treated group; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G, in Graph Pad Prism 8.H Representative images and quantification of the Matrigel-based angiogenesis assay in mouse vascular endothelial cell (SVEC4-10) treated with CMs from TAMEMs and other controls, VEGF treatment as a positive control (****P < 0.0001 and ns: not significant (P > 0.05) vs. the normal medium group; scale bar: 100 μm; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in H) in Graph Pad Prism 8.
Figure 4E Representative images of the wound closure process were captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates).G H&E (day 7 and 14) and Masson's (day 14) staining for full-thickness skin samples containing the entire wound area (scale bar: 500 μm; n=8), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates), (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).H- J Quantitative analysis of regenerated granulation (H) on day 7 and Re-epithelialization of wound bed (I) and Masson's trichrome-stained tissue (J) on day 14 (blue for collagen) (***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (H, I, J, in Graph Pad Prism 8.
Figure 5E IF staining for CD31 (red; endothelial marker, for new capillary formation) at day 7 and α-SMA (green; smooth muscle marker, for vascular maturation) at day 14, with both the entire wound region and magnified yellow-box zone (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium).F Quantification of the CD31+ vessels at day 7 and CD31+ α-SMA+ vessels and CD31+ α-SMA- vessels at day 14 (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (F, in Graph Pad Prism 8.G Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 post-injury, uninjured skin was analyzed as positive control. Up: the images of the entire sampling area (yellow circled) containing the healed wound (green circled); Down: magnified zone (blue boxed in the upper images; scale bar: 100 μm; n=3, biological replicates).
Figure 6G The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK-8 kit (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G) in Graph Pad Prism 8.H Immunofluorescent staining for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (scale bar: 10 μm; n=6, biological replicates).
Figure 7B Gross view of the wound closure process was captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates). C The percentage of wound closure determined at each time point (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (C) in Graph Pad Prism 8.D Full-thickness skin samples containing entire wound sites were stained with H&E (day 7 and day 14) and Masson (day 14) (scale bar: 500 μm, n=8, biological replicates), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).F Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 postinjury, uninjured skin was analyzed as positive control (Up: the images of entire sampling sites (the yellow circular region) containing healed wound sites (the green circular region); Down: the images of higher magnification that indicates an area in the blue boxed region in the upper images; scale bar: 100 μm; n=3, biological replicates).
Figure 1Western blot analysis of total brain lysates of wild type, Tmem106b-/-, Grn-/- and Grn-/-/Tmem106b-/- mice using antibody 6F2 to detect TMEM106B. (4.5-month-old mice with the given genotype; n = 3 biological replicates per genotype).Hind limb clasping test wild type, Tmem106b-/-, Grn-/-, Grn+/-/Tmem106b-/- and Grn-/-/Tmem106b-/- mice. Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age.Rotarod performance of wild type, Tmem106b-/-, Grn-/-, Grn+/- /Tmem106b-/- and double knockout animals at 3 to 4 months of age. Number of animals used for analysis, 10 wild type, 12 Tmem106b-/-, 5 Grn-/-, 7 Grn+/-/Tmem106b-/-, 3 Grn-/-/Tmem106b-/- mice.Representative picture of a 4.5-month-old double knockout (Grn-/-/Tmem106b-/- ) mouse after manual flip.
Figure 2Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed brain mRNAs from Tmem106b-/- and wild type mice. From 680 detected genes 21 are significantly upregulated, while 10 are significantly reduced.Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of Grn-/- and wild type mice. From 680 detected genes 12 are significantly upregulated, while 6 are significantly reduced.Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot for Grn-/-/Tmem106b-/- and wild type mice. From 680 detected genes 79 are significantly upregulated, while 24 are significantly reduced.D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type. 7 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 15 significantly altered genes. Tmem106b-/- and Grn-/- mice share one significant altered gene. Data represent the mean ± SD.