EMBO/SourceData huggingface dataset:
Figure 3Configuration of the two-compartment model. The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell (orange), resulting in two separate compartments, defined as swarmbot and environment (chamber). Four major components involved in the population dynamics are explored in the model, including cell density (n), nutrient (s), carbenicillin (a) and BlaM concentration (b). Each component has a different transport rate, depending on the molecule size.Simulated microbial swarmbot dynamics under a static condition. a = 0.4, a microbial swarmbot with a low n0 cannot initiate growth (red lines) but one with a high n0 leads to growth both in the swarmbot and, after a delay, in the chamber (green lines). While (C) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic (a = 0). Solid lines: cell density inside swarmbots; dashed lines: cell density in the chambers.(D) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot. The device consists of an input channel leading to a culturing chamber and a trapping slot. The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot, while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through. Input flow patterns (static or pulsing) are controlled by a programmable syringe pump.(E) Experimental validation of model prediction. At 100µg/ml carbenicillin, a microbial swarmbot with a high initial density led to robust growth in the capsule and, after a delay, in the chamber (green lines). While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration (red lines). (F) In contrast, both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin, regardless of the initial cell densities.(G) Time-lapse images of microbial swarmbot dynamics. The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density. But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration (rightmost). The concentration of carbenicillin was 100 µg/ml and the scale bar represents 200 µm.(H) The control strain lacking BlaM circuit. The population could not survive at the same concentration of carbenicillin at the high initial density.
Figure 5Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4).Snapshots of safeguard performance at the end time point of (A). The red arrow in panel 6 indicates significant growth in the chamber; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population.
Figure 1(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm(b) Costaining with the NSC markers NESTIN, SOX2, and PAX6 reveals that TCF20 is expressed in NSCs.(c) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0. n=4 independently repeated tests.(d) Western blotting results show the changes in TCF20 protein expression level during brain development. (e) Statistical analysis of normalized band intensity in the western blot results. n= 4 samples for each group.(f) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution. These electroporated embryos were collected at E16. (g) Statistical analysis of the distributions of GFP-positive cells in different brain regions. The percentage of GFP-positive cells in the CP (cortical plate), IZ (intermediate zone), and VZ/SVZ (ventricle zone and subventricle zone) was analyzed. n=7 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.01(**), p<0.001(***). Scale bar in (F) 50 μm.
Figure 2(b) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm.(c) Statistical analysis of 2-hour BrdU labeling experiments. n=6 brains Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(d) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs.(e) Immunostaining with pax6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. (f) Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n=3 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(g) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains. White arrows represent GFP, sox2 double positive cells; Red arrows represent GFP, TBR2 double positive cells. area. Scale bar: 50 μm. (h) Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n=8 brains for each group. (i) Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n=5 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).
Figure 3(a) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. (b) Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: 25 μm(c) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. (d, e) Statistical analysis of CTIP2, GFP and TBR1, GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: (d) 50 μm(f) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: f) 25 μm
Figure 4(b) Western blotting analysis of TCF20 in TCF20 WT, HET, KO mice reveals that TCF20 was deleted. (c) Statistical analysis of the normalized band intensity of TCF20. n=3 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(d) RT-qPCR analysis of TCF20 in TCF20 WT, HET, and KO mice reveals that TCF20 was deleted. n=4 samples. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(e) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out. Scale bar: 50 μm. (f) Statistical analysis of the normalized fluorescence intensity of TCF20. n=6 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(g) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation.(h) Immunostaining of different cortical layer markers TBR1, CTIP2, and SATB2, White dash line represents the positive area. Scale bar: 50 μm. (i-k) Statistical analysis of different cells layers in TCF20 WT, HET and KO mice. n=7 brains of each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(l) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution. Scale bar: 50 μm. (m) Statistical analysis of GFP+ distribution in different zones. n=3 brains for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).
Figure 5(a) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms (b-d) (b) Total call durations, (c) Call durations and (d) Call numbers of WT, HET, and KO mouse pups. nWT=13 mice, nHET=16 mice, and nKO=18 mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(e) A sketch of the mouse trace in the open field test. (f-h) (f)Statistical analysis of time spent in the center area, (g) Statistical analysis of total mouse movement distance in 5 min, (h) The ratio of mice in the center area movement distance to the total distance. n for WT=15 adult mice, n for HET=12 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(i) Statistical analysis of the Y maze test. S, O, and N represent the start Arm, old Arm and new Arm. The percent of time that mice stayed in each arm was recorded and analyzed; n for WT=11 adult mice, n for HET=11 adult mice. Whiskers represent the minimum to maximum value; central lines represent median values; boxes represent the values between upper quartile and lower quartile. (j) Results of the elevated-plus maze. Time that mice spent in open arms; n for WT=8 adult mice, and n for HET=11 adult mice. (k) Results of elevated-plus maze. Time that mice spent in closed arms ; n for WT=8 adult mice, and n for HET=11 adult mice.Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(l) Numbers of marbles buried in the marble burying test; n for WT=10 adult mice, and n for HET=10 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(m) Diagram of the three-chamber social interaction test and mouse movement trace. HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases. (n) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1; n for WT=16 adult mice, n for HET=13 adult mice. (o) Comparison between the time that WT and HET mice spent in stranger1 chamber. n for WT=16 adult mice, n for HET=13 adult mice. (p)Time spent in each chamber when one contains familiar mouse stranger1, as well as new mouse stranger2. n for WT=16 adult mice, n for HET=13 adult mice. (q) Comparison between the time of WT and HET mice staying in the stranger2 chamber. n for WT=16 adult mice, n for HET=13 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).
Figure 6(c) Heat map of significantly different genes.(d) TDG is significantly decreased after TCF20 deletion.(e) Volcano map showing that TDG is significantly decreased.(f, g) Electroporation of GFP at E13.5 to E16.5 in TCF20 KO mice impairs cell distribution. n=3 brains. Scalebar 25 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).(h) Chip-qPCR of TCF20 binding to the TDG promoter. n= 4 biological repeats. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).
Figure 7(b) TDG ChIP-qPCR revealed that TDG mainly binds to TCF-4 CpG islands. Seven regions (from -5 kbp to -200 bp) of the promoter were analyzed. -500 bp and -200 bp regions were located in the CpG island of the TCF-4 promoter. (n=4 biological repeats). Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (c-f) MeDIP-qPCR showed that 5fC, 5caC, and 5mC are increased in the TCF-4 promoter, while 5hmC is decreased when TCF20 is insufficient. The MeDIP assay measure the region -500 bp from the TCF-4 promoter. (n=4 biological repeats) Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (g) NSCs were isolated from C57B6/J E13.5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids. Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm (h)Statistical analysis of 5fC fluorescence level. n=20 cells, and P<0.0001 Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). (k, l) Overexpression of TDG and TCF-4 rescued the TCF20 knockdown phenotype in brains; n=3 brains. Scalebar 50 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).
Figure 1B Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody. E-H Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280-440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co-immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280-440 (FIP∆) was quantified in comparison to that with wild-type FIP200 (H; signals normalized to that of the immunoprecipitated protein;
Figure 2To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D.J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments;
Figure 3A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01). CC HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).
Figure 4A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls. B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01). CHEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experimentsE HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).
Figure 5B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatmentLysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1-319 of ATG16L1, precisely deleting only the WD40 repeats residues 320-607) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry-ATG16L1 , or immunoprecipitated with anti-FIP200 or anti-HA antibodies Co-immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6±2.1% and 3.4±2.1% in HEK293A and HeLa cells, respectively, relative to that of full-length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± S.D. deviation of three independent experiments each).F Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co-immunoprecipitation of ATG16L1 with Rabaptin5-AAA triple mutant was reduced to 1.5±1.2% relative to that with wild-type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments).