EMBO/soda-nel huggingface dataset:
Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia (2022)
Figure 1

Fig. 1 | Autophagy during epidermal wound healing.

A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. For statistical hypothesis testing, anindependent and non-parametric (Kruskal-Wallis) t-tests were performed for the mean number of spots in control and experimental conditions. P values are indicated as follows: *P < 0.04; ***P < 0.0002 and lack of an asterisk means non-significant (P > 0.123).

E, F Effect of suppressing autophagy on wound healing and actin cable formation. Time-lapse series of single-cell wound healing in larvae expressing (E) Src-GFP (green) and DsRed2-Nuc (magenta) to mark cell membrane and nuclei and (F) endogenously GFP-tagged E-cadherin (DE-cad-GFP; green) and mCherry-marked myosin regulatory light chain (Sqh-mCherry; magenta) to visualize adherens junctions and actomyosin cables.

Data information: (A, B, E, F) Z-projections of time-lapse series in early L3 larvae, n=9-15 larvae each genotype. Scale bars, A, E, F, 20 μm and B, 10 μm. Pre W: pre wounding. Images from Movies EV1-4. Genotypes of all images are listed in Table 2.
Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia (2022)
Figure 3

Fig. 3 | Autophagy in unwounded epidermis.

A-E Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A). (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. For statistical hypothesis testing, anindependent and non-parametric (Kruskal-Wallis) t-tests were performed for the mean number of spots in control and experimental conditions. P values are indicated as follows: *P < 0.04; **P < 0.003; ***P < 0.0002 and lack of an asterisk means non-significant (P > 0.123). (D, E) Transmission electron micrographs of sections through the epidermis of larvae with elevated autophagy at two different magnifications. AP: autophagosome; Ax: cross-section of an axon; BL: basal lamina; C: cuticle; HD: hemidesmosome; M: mitochondrion; N: nucleus.

F Disruption of epidermal morphology after upregulation of autophagy. The membrane marker Src-GFP is lost from large areas of the epidermis and nuclei have lost their regular spacing. Image in control and rictori panels are from the time laps in Appendix Fig S5B and Movie EV12.

G Schematic for temporally controlled transgene expression and imaging in (H). Gene expression is induced at the end of the second larval instar, live imaging started 6 h later and continued for an additional 6 hours.

H Example of membrane dynamics after time-controlled Atg16B expression. Src-GFP containing material appears to be taken out of and eventually detached from lateral cell membranes (arrows). Over the period of observation, abnormal accumulation of GFP is seen in the nuclei (cyan asterisks). t = 0 is 6h after A58-Gal4 activation. Image from Movie EV9.

Data information: A, B, F, H, z-projections; n=20-50 larvae each genotype. Scale bars: A, F, H, 20 μm, B, 10 μm, D, 1 μm and E, 500 nm.
Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia (2022)
Figure 5

Fig. 5 | Loss of lateral membrane integrity.

A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols. E-F Electron micrographs showing morphological defects or absence of lateral cell membranes in epithelia with upregulated autophagy. (E) Membrane domains with tight apposition between neighbouring cells are marked in magenta (adherens junctions), green (septate junction) and orange (gap junctions). (F) Nuclei (yellow) and lateral membranes in healthy epidermal cells cannot be shown in one image because they are too far apart, whereas when autophagy is upregulated, nuclei are often found close together and not separated by plasma membranes. C: cuticle (blue).

Data information: A-D n=5-9 larvae for each FLIP protocol. Scale bars: A, 20 μm and E, F, 2 μm. Images from Movies EV13-15.
Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia (2022)
Figure 8

Fig. 8 | Epistasis of TOR and autophagy.

A The indicated overexpression and RNAi constructs were co-expressed with markers for the plasma membrane (Src-GFP, green) and nuclei (DsRed2-Nuc, magenta) in the larval epidermis. The effects of upregulating autophagy (top row) are suppressed when autophagy is blocked by simultaneously downregulating Atg1 or Atg5. n=20-40 larvae each genotype.

B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.

Data information: Scale bars: A, B, 20 μm. Images from Movie EV17.
A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae (2021)
Figure 3

Figure 3: VcdRP regulates central metabolism genes

A. A schematic representation of the three VcdRP and VcdR/P variants used for the transcriptome analysis. Left to right: first, the native VcdRP variant which harbors both the small protein as well as the base-pairing region. Next, a STOP codon was introduced in the 3rd codon of vcdP ORF as a readout for VcdR devoid of VcdP. Finally, the sequence of the VcdP codons was scrambled such that the correct small protein is translated but the nucleotide sequence of the scrambled vcdP variant is significantly altered when compared to native vcdRP. This variant also carries an artificial 5'UTR and a terminator. The arrows indicate the TSS.

B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%.

C. A Venn diagram of the differentially expressed genes (DEG) among VcdRP (red), VcdR (yellow) and VcdP (blue). RNA samples were harvested from V. cholerae ΔvcdRP strains carrying pBAD-based plasmids depicted in (A), including an empty vector control. Genes with a total count cutoff >10 in all samples, with an absolute fold-change ≥ 2.0 and a FDR adjusted p-value ≤ 0.05 were considered to be differentially expressed.

D. Gene enrichment analysis of the DEG shown in (C) using gene ontology analysis.

Data information: The differentially expressed genes in (C) are shown in Appendix Table S1 that is color-coded.
A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae (2021)
Figure 5

Figure 5: VcdP interacts with and increases the activity of citrate synthase

A. Immunoprecipitation of chromosomally tagged GltA::HA combined with either an empty vector control (pCtrl) or SPA-tagged vcdP over-expression plasmid, grown in LB medium to exponential phase (OD600 of 0.5). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti-Flag antibody were loaded on a SDS-PAGE gel. Western blotting with anti-HA and anti-Flag antibodies confirmed the interaction of GltA with VcdP in vivo. RNAP served as loading control. The solid triangles indicate the corresponding protein sizes.

B. Citrate synthase enzyme activity measurements performed on cellular lysates. An empty vector control (pCtrl) or vcdP expression plasmids (pVcdP and pVcdP*) were conjugated into V. cholerae wild-type, ΔvcdRP, ΔgltA or F383A GltA backgrounds. Cells were grown in LB medium to stationary phase and subsequently lysed. The protein concentrations of these cellular extracts were measured, and served as input for the assay. The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present. The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities.

C. Citrate synthase enzyme activity measurements performed in vitro. V. cholerae GltA and F383A GltA proteins were purified using a 6xHis tag in E. coli. Both the proteins were treated without the synthesized VcdP or VcdP* peptide, or with increasing concentrations of the two peptides (1x, 2x, 5x, 10x, 15x or 20x with respect to the protein monomer). The citrate synthase activity per milligram of protein as a result of its interaction with the peptide was determined from the initial velocities.

Data information: Data in (B and C) are presented as mean ± SD, n = 3. Statistical significance was determined using one-way ANOVA and post hoc Tukey's or Dunnett's multiple comparisons test. The p-value is summarized as follows - ns for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, and **** for p ≤ 0.0001. Source data are available online for this figure.
Dynamic control of endogenous metabolism with combinatorial logic circuits (2018)
Figure 3

Figure 3: Eliminating protein activity using a combination of CRISPRi, sRNA, and proteases. (a) Schematic showing three levels of repression. A small guide RNA (sgRNA) directs deactivated Cas9 (dCas9) to block transcription from a promoter. sRNA binds to the mRNA and promotes degradation by recruiting Hfq. The mf-Lon protease targets a tag (blue) added to the protein. The protease is also targeted to itself to reduce toxicity (Methods). (b) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth (Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf-LON). sgRNA and mf-LON are co-transcribed on a single transcript, processed by ribozymes (Appendix Figure S13). (c) The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 µM DAPG) at the 2-hour time point (dashed line) (Methods). (d) Metabolic pathways to acetate in E. coli are shown along with the targeted enzymes. The impact of either knocking out these enzymes or knocking them down by the various mechanisms after 20 hours are shown. On the left are shown empty strains containing either no acetate pathway modifications (WT; MG1655∆glnL), a double deletion (∆pta∆poxB), or single knockouts and protease tag modifcations (∆poxB pta::pdt3, ∆pta poxB::E170). On the right are shown knockdowns of pta (blue) and poxB (red), tested in strains ∆poxB pta::pdt3 and ∆pta poxB::E170, respectively. (e) Design of a PoxB mutant that can be targeted by the SuMMV protease. The structure of PoxB is shown (PDB: 3ey9) (Neumann et al, 2008), highlighting the amino acid sites selected for degron insertion (in red). (f) The impact on acetate production for a double mutant where poxB is complemented on a BAC (Methods). Note that complementation causes a ~3-fold reduction in acetate compared to WT in part d. N-ter and E170 refer to the location of the SuMMV tag. (g) Reduction of acetate production by the expression of SuMMV from an DAPG-inducible promoter (- no inducer, + 25 µM). The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the genome. Representative cytometry fluorescence distributions for Figure 3b/c are shown in Appendix Figure S11. Error bars represent one standard deviation of three biological replicates done on different days. Bars with single (*) and double (**) asterisks indicate a statistically significant difference with p-values less than 0.01 and 0.001, respectively, as assessed by an unpaired t-test.
Dynamic control of endogenous metabolism with combinatorial logic circuits (2018)
Figure 4

Figure 4: Dynamic control of acetate production. (a) The genetic circuit designed to control poxB is shown. The strain genotype is shown, including the positions targeted by the sgRNA and the SuMMV protease. (b) The transcription of poxB over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/acetate sensors will be on based on metabolite measurements. It should be noted that these are right-shifted compared to Figure 2 due to slower growth of the tested strains. (c) The production of acetate is shown over time for E. coli MG1655∆glnL∆pta poxB::E170 (black) as compared to the same strain containing the complete circuit (green). A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆pta containing an untagged poxB as a control (red). Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB. (d) The genetic circuit design to control pta is shown. (e) The transcription of pta over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/oxygen sensors will be on based on metabolite measurements. (f) The production of acetate is shown over time for E. coli MG1655∆glnL∆poxB pta::pdt3 as compared to a strain containing the complete circuit (green) tested in same strain. A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆poxB containing an untagged pta as a control (red). Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB. In Figure 4c/f, three experiments were performed and all individual data points are plotted. The lines represent averages of these data. Growth curves for each strain are shown in Supplementary Figure S16. The corresponding plasmid maps and parts sequences for Figure 4a/d are provided in Supplementary Figure S17 and Table S2.
Young microglia restore amyloid plaque clearance of aged microglia (2016)
Figure 1

Figure 1. Characterization of the ex vivo model.

(A) Representative image of a typical young WT cortico-hippocampal slice (350 µm) from co-culture (14 DIV) stained with DAPI (gray) to visualize nuclei (left). Dentate gyrus (DG), cornus ammonis 1 (CA1) and cortical (CTX) regions are indicated. An example of the co-culture dish is shown on the right. Co-culture includes 2 young WT (*, young co-culture) and 2 old APPPS1 (+, old co-culture) brain slices.

(B) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone (young alone, old alone) or in co-culture (young co-culture, old co-culture) at 7 days in vitro (DIV) stained with propidium iodide (PI, red) reveals reduced cell viability in old brain slices compared to young.

(C) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone (white and black bars, respectively) or in co-culture (grey and white squared bars, respectively) at 7 and 14 DIV. The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells. The values represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (n.s.= not significant; ***P < 0.001).

(D-F) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co-culture at 7 DIV and immunostained using neuronal (NeuN), astrocytic (GFAP) and microglial (CD68) markers (green) reveals reduced NeuN and GFAP and increased CD68 immunosignal. Scale bar: 75 µm (B, D, E, F).
Young microglia restore amyloid plaque clearance of aged microglia (2016)
Figure 2

Figure 2. Amyloid plaque clearance is enhanced in the ex vivo co-culture model of old and young brain slices.

(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture using antibody 6E10 to detect amyloid plaques (green) and Thiazine Red (TR, red) to visualize fibrillar amyloid cores. At 7 DIV, the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ. In contrast, at 14 DIV, the majority of amyloid plaques are core-only plaques co-stained with 6E10 and TR without detectable 6E10 positive halo Lower panels are higher magnification images of boxed regions. Scale bars: 50 µm.

(B) Quantitative analysis of core-only plaques in old APPPS1 slices cultured alone (white bars) or in co-culture (gray bars) at 7 and 14 DIV reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young WT slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).

(C) Western blot analysis of Aβ levels (triplicates) in the old APPPS1 tissue cultured alone or in co-culture with the young WT (14 DIV) using 2D8 antibody.

(D) Quantification of Western blot signals reveals decreased levels of Aβ upon co-culturing compared to old tissue cultured alone. The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments, each experiment was performed in triplicates (**P < 0.01).
Young microglia restore amyloid plaque clearance of aged microglia (2016)
Figure 3

Figure 3. Recruitment and clustering of CD68 positive cells at amyloid plaques is paralleled by a decrease in plaque size.

(A, B) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) using antibodies 6E10 (green) and CD68 (red). Nuclei were counterstained using DAPI (blue). Image of boxed region in A is depicted at higher magnification in B and reveals clustering of several CD68 positive cells around amyloid plaques. Scale bars: 100 µm (A) and 10 µm (B).

(C) Quantitative analysis of plaque-associated CD68 positive cells in co-culture samples at 7 and 14 DIV reveals a trend towards increased number of CD68 positive cells. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (n.s.= not significant).

(D) Quantitative analysis of co-culture samples at 7 and 14 DIV reveals a decrease in plaque size. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 replicates (***P < 0.001).

(E) Quantitative analysis of plaques surrounded by CD68 positive cells (clustered plaques) in co-culture samples at 7 and 14 DIV reveals an increase in the number of clustered plaques. The values are expressed as percentages of clustered plaques from the total number of amyloid plaques. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (***P < 0.001).

(F) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) treated with cytochalasin D (CytoD) and vehicle control (Ctr) and immunostained with M3.2 (green) and CD68 (red). Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance. Scale bar: 50 µm.

(G) Quantitative analysis of core-only plaques in the old APPPS1 slice in co-culture (14 DIV) treated with CytoD and Ctr reveals a decreased number of core-only plaques upon CytoD treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).
Young microglia restore amyloid plaque clearance of aged microglia (2016)
Figure 4

Figure 4. Amyloid plaque clearance depends on both young old microglia.

Either young WT (A, B) or old APPPS1 (C-E) tissue was treated with clodronate (Clo) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV. Treatment was stopped and subsequently old (A, B) or young (C-E) tissue was added to the culture as schematically indicated in A and C. and analyzed 14 days after. Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co-culture model.

(A) Immunofluorescence analysis of the old APPPS1 slice co-cultured with the young WT slice pre-treated with Clo and Ctr and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.

(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue co-cultured with the young WT tissue pre-treated with Clo and Ctr as indicated in A reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).

(C) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co-cultured with the young WT slice and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.

(D) Area of CD68 positive cells (CD68 coverage) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C. CD68 coverage is reduced upon Clo treatment. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).

(E) Quantitative analysis of core-only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (**P < 0.01).