EMBO/soda-nel huggingface dataset:
Mobile late endosomes modulate peripheral endoplasmic reticulum network architecture (2020)
Figure 2

Figure 2. ER hitchhiking and other processes of ER movement.

A. Schematic representation of TAC-mediated (I) ER movement and ER sliding (II).

B. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing mCherry-KDEL (green), transiently expressing EB3-GFP (yellow) and SiR-lysosome-stained LE/Lys (magenta). Zoom insets show regions of interest containing a mobile LE/Lys as denoted by the white arrow. First row shows ER (mCherry-KDEL) (green), the second row shows SiR-lysosome-stained LE/Lys (magenta), the third row shows EB3-GFP (yellow) and the fourth row shows the merge of the three preceding channels. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV6.

C. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) marked ER and SiR-lysosome-stained LE/Lys (orange) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either DMSO or 2µm paclitaxel. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz.Images related to movie EV7 and 8

D. Kymograph of panel (C).

E. MDA quantification of panel (C) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n = 20 cells per condition from 3 independent experiments). Significance two-tailed student t-test **** P<0.0001; ns = not significant.

F. Representative images of n = 3 of COS7 cells stably expressing GFP-TMD (green) to mark the ER and SiR-lysosome stained LE/Lys (orange) merged. Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either siControl or siKTN1. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV9 and 10

G. Kymograph of panel (F).

H. MDA quantification of panel (F) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions, respectively. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=20 cells per condition from 3 independent experiments). Significance two-tailed student t-test. **** P<0.0001; ns = not significant.
Mobile late endosomes modulate peripheral endoplasmic reticulum network architecture (2020)
Figure 3

Figure 3. RAB7-positive endosomes and ER mobility.

A. Representative time-lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry-KDEL (green) and transiently expressing GFP-RAB7 (orange). Zoom insets shows a region of interest containing a mobile RAB7-positive endosome as denoted by the white arrows. First row represents the ER (mCherry-KDEL) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and GFP-RAB7 (orange). Scale bar: 10µm, zoom insets 3µm. Images related to movie EV11. Images were acquired for 90 frames at 1Hz.

B. Kymograph of panel (A).

C. MDA quantification of panel (A) and figures EV2A-G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. *P<0.05, ** P<0.01, **** P<0.0001 ns = not significant.

D. Violin plots of fractional distance analysis showing the average distribution of endosomes in cells from panel (E) and fig. EV3A. Graph represents mean ±s.d. of n=15 cells per condition from 3 independent experiments. Dashed lines represent median and solid lines represent interquartile range.

Cartoons representing the overexpression and control LE/Ly phenotype. Significance two-tailed student t-test. **** P<0.0001

E. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) and in lower panels transiently expressing mScarlet-RILP (magenta) and SiR-lysosome-stained LE/Lys (orange). Zoom insets shows region of interest containing ER (GFP-TMD). First row represents ER (GFP-TMD) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels. Scale bar: 10µm, zoom insets 3µm. Images related to movie EV19 and movie EV20. Images were acquired for 90 frames at 1Hz.

F. MDA quantification of panel (E) and figure EV3A normalized to 1 showing total average ER movement. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired 90 frames at 1Hz. Significance two-tailed student t-test. **** P<0.0001 ns = not significant.
Mobile late endosomes modulate peripheral endoplasmic reticulum network architecture (2020)
Figure 4

Figure 4. Endosomal clustering reduces peripheral ER junctions.

A. Schematic flow overview of the process for ER junction quantification.

B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange). COS7 cells in the middle panels overexpress mCherry-RILP (magenta). The COS7 cells in the lower panels overexpress mCherry-TMEM55B (magenta). Zoom insets show region of interest containing the ER (TMD-GFP) (green) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average. Scale bars: 15µm. zoom insets: 3µm.

C. Quantification of the number of ER junctions per µm2 from panel (B) as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell (n=15 from 3 independent experiments). Significance two-tailed student t-test. ***P<0.001.
Mobile late endosomes modulate peripheral endoplasmic reticulum network architecture (2020)
Figure 5

Figure 5. ER tethers support ER hitchhiking.

A. Representative images of time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (green) and SiR-lysosome-stained LE/Lys (orange) treated with either siControl or co-treated with siVAPA, siVAPB and siMOSPD2. Zoom insets show regions of interest containing mobile LE/Lys as denoted by the white arrow. First row represents ER (TMD-GFP) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and SiR-lysosome stained LE/Lys (orange). Scale bar: 5µm, zoom insets 3µm. Images related to movie EV40 and 47. Images were acquired for 90 frames at 1Hz.

B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange) treated with siControl or with siVAPA, siVAPB and siMOSPD2. Zoom insets show region of interest containing ER (TMD-GFP) (green) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average. Scale bars: 10µm. zoom insets: 3µm.

C. MDA quantification of ER of panels (A, B) and fig. EV4B-G normalized to control cells showing average ER movement surrounding mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=30 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. **P<0.01, ****P< 0.0001, ns=not significant

D. MDA quantification of SiR-lysosome-stained LE/Lys of (A, B) and fig. EV4B - G normalized to 1 showing total average endosomal movement. Graph represents mean ±s.d. of n = 15 cells per condition from 3 independent experiments. Significance two tailed student t-test. ns= not significant.

E. Quantification of number of ER junctions per µm2 from (B) and fig. H - M as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell of n = 15 from 3 independent experiments. Significance two-tailed student t-test. *P<0.05, **P<0.01 ***P<0.001, ****P< 0.0001, ns=not significant.
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 2

Figure 2. MepM is essential for YcbB-mediated β-lactam resistance. Growth was tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) on BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. (A) BW25113 ΔrelA derivatives harboring plasmids pKT2(ycbB), pKT8(relA') and the vectors pHV6 and pHV7 used to construct these plasmids, respectively. Expression of β-lactam resistance requires induction of both ycbB and relA'. (B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 3

Figure 3. MepM endopeptidase activity is required for YcbB-mediated β-lactam resistance. Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 5

Figure 5. Hydrolysis of 4→3 and 3→3 cross-links by purified MepM. (A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit). (B) Structure of the muropeptides as determined by mass spectrometry (supplementary Fig. EV2). (C) Hydrolysis of the four types of dimers by MepM. Sacculi were incubated with lysozyme and MepM at various concentrations. The relative abundance of the muropeptides was estimated by calculating the relative peak areas. (D) Hydrolysis of dimers containing 4→3 and 3→3 cross-links by MepM. The relative peak areas of Tri-Tri and Tri-Tetra containing 3→3 cross-links and that of Tetra-Tri and Tetra-Tetra containing 4→3 cross-links were combined. The inset shows variations in the relative peak areas of the Tri and Tetra monomers. (E) Relative abundance of Tri and Tetra stems in all muropeptides.
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 6

Figure 6. Complementation of the mepM deletion by plasmids encoding L-rhamnose-inducible copies of the eight endopeptidase genes. Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 8

Figure 8. Minimal complement of endopeptidases required for growth in the context of the formation of 4→3 cross-links by PBPs. (A) BW25113 Δ8EDs harboring a plasmid carrying the mepM gene under the control of the PrhaBAD L-rhamnose-inducible promoter with the "weak" TIS2 translation initiation signal (plasmid pHV53) was grown on BHI agar in the absence or presence of 0.2% L-rhamnose. Growth was dependent upon induction of the mepM copy carried by pHV53. (B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 9

Figure 9. Complementation of the mepK deletion by plasmids encoding L-rhamnose-inducible copies of the eight endopeptidase genes. (A) Induction of ycbB under the control of the ParaBAD promoter in BW25113 ΔmepK pHV63(ycbB) was studied by the disk diffusion assay. The clear zone around the disk containing L-arabinose indicates that production of YcbB inhibited growth. (B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 10

Figure 10. Complementation of mepK deletion with catalytically inactivated endopeptidases. (A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 11

Figure 11. Growth phenotype of sltY mutants. (A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives. (B) Ceftriaxone-resistance depends upon ycbB and relA' expression. The disk diffusion assay was performed with BW25113 ΔrelA ΔsltY harboring pKT2(ycbB) and pKT8(relA') or pKT8(relA') only. Induction was performed with 40 µM IPTG and 1% L-arabinose for ycbB and relA', respectively. Disks were loaded with 30 µg of ceftriaxone.
Microglial PD-1 modulates neuroinflammation and Alzheimer's disease pathology by  astrocytic PD-L1 (2021)
Figure 1

Fig. 1: Expression of PD-L1 in AD and in APP/PS1 transgenic mice

A) ELISA quantification of PD-L1, pTau 181 and Aβx-42 in the CSF of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18, Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).

B, C) (B) Colocalization of PD-L1 and GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm).

D) Immunohistochemical detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM).

E) Immunohistochemical detection of PD-L1 in 4-month-old female APP/PS1 mice (bar = 20 µm).

F) Induction of PD-L1 on astrocytes using 1 μM aggregated Aβ1-42 or 10 μg/ml TNFα / 100 U/μl IFNγ analyzed by flow cytometry.
Microglial PD-1 modulates neuroinflammation and Alzheimer's disease pathology by  astrocytic PD-L1 (2021)
Figure 2

Fig. 2: Secretion of soluble PD-L1 from astrocytes

A) C6 cells expressing PD-L1-myc treated with the γ-secretase inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.

B) Immunoblot of HEK293 cells expressing PD-L1-myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).

C) Evaluation of three independent experiments of B (n=3, mean +SEM, Student's t-test, * p=0.012, t = 3.4803, df = 4).

D) 18 h 1 μM PMA treatment of C6 cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.

E) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNFα / 100 U/μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019.

F) Scheme of PD-L1 secretion: PMA induced S2 cleavage results in the formation of sPD-L1 and a C-terminal fragment (PD-L1-CTF), that is cleaved by γ-secretase.
Microglial PD-1 modulates neuroinflammation and Alzheimer's disease pathology by  astrocytic PD-L1 (2021)
Figure 3

Fig. 3: PD-1 expression in APP/PS1 and AD

A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid deposits) in AD by immunohistochemistry (bar=50 µm).

B) Colocalization of PD-1 and Iba1 or PD-1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar=20 µm).

C) PD-1 mRNA quantification in laser-dissected microglia from CX3CR1-EGFP+/- mice (wt) or mice plaque-associated (plaque) and plaque-distant (distant) microglia in APP/PS1 CX3CR1-EGFP+/- mice (n=6, Mann Whitney test, ** p=0.0043, W =35).

D) Induction of PD-1 on CD11b+-microglia in vitro using 1 μM Aβ or 100 ng/ml LPS, analyzed by flow cytometry.

E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05).

F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type mice (represented by dashed lines).

Data information: (C, E-H) central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.
Microglial PD-1 modulates neuroinflammation and Alzheimer's disease pathology by  astrocytic PD-L1 (2021)
Figure 4

Fig. 4: Deletion of PD-1 in the APP/PS1 mouse model aggravates plaque pathology and behavioral deficits

A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).

C) Thioflavin T histochemistry from sagittal brain sections in the hippocampus (Hc), and cortex (Cx) of female APP/PS1 and APP/PS1 PD-1-/- mice. Nuclei were stained using Hoechst 33342 (bar = 500 μM).

D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001).

F) Western blot analysis of brain lysates from female APP/PS1 and APP/PS1 PD-1-/- mice for the expression of APP and APP C-terminal fragments using antibodies 6E10 (transgene expression) and CT15 (transgene and endogenous expression). Tubulin was used as a loading control.

G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001).

H) Analysis of the area under the curve (mean +SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=12 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, ANOVA (DF=3, F=6.98, p=0.001), * p<0.05, ** p<0.01).