EMBO/soda-nel huggingface dataset:
SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 3

Figure 3. MAP4K3/GLK induces ACE2-containing exosomes from epithelial cells.

A ZetaView nanoparticle tracking analysis of particle numbers and sizes of CD63+ extracellular vesicles (EVs) isolated from the supernatants of HCC827 lung epithelial cancer cells, which were transfected with GLK wild-type or GLK kinase-dead (K45E) mutant. EVs were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns.

B Immunoblotting of ACE2 and CD63 proteins in exosomes isolated from GLK wild-type- or GLK (K45E) kinase-dead-overexpressing HCC827 cells. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.

C Immunoblotting of ACE2 and CD63 proteins in serum exosomes isolated from the sera of 4 healthy controls (HC) and 7 COVID-19 patients from NHRI Biobank for COVID-19 patients in Taiwan (Cohort #3). Serum collection days from the onset are shown; treatment of oxygen therapy on the patient is also indicated. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.

D Confocal microscopy analysis of Flag-tagged ACE2 proteins (red) in recipient cells after incubation with exosomes for 72 h. Exosomes were isolated sequentially from the supernatants of Flag-ACE2-overexpressing or GFP-GLK plus Flag-ACE2-coexpressing HCC827 cells using ExoQuick kits and then ExoQuick ULTRA columns. Arrows denote Flag-tagged ACE2 proteins. Original magnification, ×630; scale bars, 10 μm.

E Confocal microscopy analysis of SARS-CoV-2 spike (S) protein (green) and RFP-labeled early endosomes (red) in exosome-recipient HCC827 epithelial cells. After incubation with exosomes for 72 h, recipient cells were treated with the spike protein (S) for another 24 h. Yellow color suggests the localization of S protein in early endosomes. Original magnification, ×630; scale bars, 25 μm.

F Cell entry eﬃciencies of SARS-CoV-2 pseudovirus into exosome-recipient HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates).

Data information: In (F), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test). Data shown are representative results of two (A) or three (B, D, E, F) independent experiments.

Source data are available online for immunoblotting data of this figure.
SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 4

Figure 4. MAP4K3/GLK interacts with and stabilizes ACE2 proteins.

A The identified peptide sequences of the endogenous ACE2 proteins by mass spectrometry analyses using GLK immunocomplexes.

B Co-immunoprecipitation of Flag-tagged ACE2 with Myc-tagged GLK proteins from lysates of HEK293T transfectants. NS, normal serum.

C In situ PLA assays of the interaction between ACE2 and Flag-tagged GLK proteins in HEK293T cells. Nuclei were stained with DAPI. Red dots represent direct interaction signals. Original magnification, x200. Scale bars, 50 μm.

D Immunoblotting analyses of the endogenous ACE2, ACE1, CD147, and tubulin proteins, as well as the transfected Flag-GLK proteins in HCC827 lung epithelial cells, which were transfected with either GLK wild-type or kinase-dead (K45E) mutant. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.

E Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged GLK (anti-GLK), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 plus increasing amounts of Myc-GLK plasmids.

F Immunoblotting of ACE2, GLK, and tubulin proteins in the lung tissues of wild-type or GLK-deficient mice. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.

G Cycloheximide pulse-chase experiments in HEK293T cells. Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged GLK (anti-GLK), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 and Myc-GLK. Transfected cells were treated with 100 μg/ml cycloheximide (CHX) for up to 24 h.

H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 and Myc-GLK, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. NS, normal serum.

I, J Cell entry eﬃciencies of SARS-CoV-2 pseudovirus (I) or VSV-G pseudovirus (J) into GLK wild-type- or GLK (K45E)-overexpressing HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates).

Data information: In (I, J), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test); §§, P value < 0.01 (Dunnett's test). Data shown are representative results of three (B, C, D, E, F, G, H, I, J) independent experiments.

Source data are available online for immunoblotting data of this figure.
SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 5

Figure 5. MAP4K3/GLK phosphorylates and stabilizes ACE2 proteins.

A Immunoblotting analyses of ACE2 and Flag-tagged GLK in HCC827 cells transfected with ether Flag-GLK or Flag-GLK (K45E) kinase-dead mutant. For immunoblotting of phosphorylated ACE2, Phos-tagged SDS-PAGE gel (Phos-tagTM) was used, followed by immunoblotting with anti-ACE2 antibody. Asterisk denotes unglycosylated ACE2 proteins.

B In vitro kinase assays using purified proteins. ACE2 serine phosphorylation, Flag-tagged GLK (anti-GLK), Flag-tagged GLK (K45E) kinase-dead mutant (anti-GLK), and Flag-tagged ACE2 proteins (anti-ACE2) were detected by immunoblotting.

C Mass spectrometry analysis of GLK-phosphorylated ACE2 proteins after in vitro kinase assays. ACE2 Ser776 and Ser783 residues were phosphorylated by GLK wild-type but not GLK (K45E) kinase-dead mutant.

D Mass spectrometry analysis of the ACE2 peptides from the serum exosomes of COVID-19 patients. The ACE2 protein peptide sequences containing phospho-Ser776 or phospho-Ser783 residue of ACE2 proteins detected in the serum exosomes of COVID-19 patients (Cohort #3) are shown. Exosomes were isolated using ExoQuick kits, and soluble proteins were removed by ExoQuick ULTRA columns. Exosomes were further purified by immunoprecipitation using a combination of anti-CD9, anti-CD63, and CD81 magnetic beads.

E In vitro kinase assays using immunoprecipitated Flag-tagged ACE2 or Flag-tagged GLK immunocomplexes. ACE2 serine phosphorylation, ACE2, and GLK were detected by immunoblotting using anti-phospho-serine, anti-ACE2, and anti-GLK antibodies, respectively.

F Immunoblotting analyses of Myc-tagged GLK, Flag-tagged ACE2, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual ACE2 (S776/783A, S776A, or S783A) mutants plus either empty vector or Myc-GLK.

G Immunoblotting analyses of Flag-tagged ACE2 and tubulin proteins in HEK293T cells transfected with Flag-ACE2 wild-type or individual phosphomimetic ACE2 (S776/783E, S776E, or S783E) mutants.

H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells transfected with Flag-ACE2 wild-type or a phospho-mimetic ACE2 (S776/783E) mutant, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested.

Data information: Data shown are representative results of three (A, B, E, F, G, H) independent experiments.

Source data are available online for immunoblotting data of this figure.
SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19 (2022)
Figure 6

Figure 6. The E3 ligase UBR4 induces ACE2 ubiquitination and proteasomal degradation.

A The protein scores of UBR4 by mass spectrometry analyses using Flag-tagged ACE2 immunocomplexes from cells either treated with MG132 or co-transfected with Flag-ACE2 and Myc-GLK plasmids. The protein score is the sum of the highest ions score of MS/MS search for each distinct peptide.

B Immunoblotting of Myc-tagged ACE2 (anti-ACE2), Flag-tagged UBR4 (anti-Flag), and tubulin proteins from HEK293T cells co-transfected with Myc-ACE2 and Flag-UBR4. Arrowhead denotes Myc-tagged ACE2 proteins; asterisk denotes endogenous ACE2 proteins.

C Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged UBR4 (anti-Myc), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 and Myc-UBR4. The co-transfected cells were treated with 25 μM MG132 for 2 h before being harvested.

D Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 and Myc-UBR4, followed by immunoblotting with anti-Lsy48-linked ubiquitination, anti-Myc, or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested.

E In situ PLA assays of the interaction between Flag-tagged ACE2 (wild-type or mutant) and Myc-tagged UBR4 proteins in HEK293T cells treated with 25 μM MG132 for 2 h. Nuclei were stained with DAPI. Red dots represent direct interaction signals. Original magnification, x200. Scale bars, 20 μm.

F Mass spectrometry analysis of the ACE2 peptides containing ubiquitination residues from Flag-ACE2-transfected HEK293T cells treated with 25 μM MG132 for 2 h.

G Immunoblotting analyses of Flag-tagged ACE2, Myc-tagged UBR4, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual (K26R, K94R, K112R, or K114R) mutants plus either empty vector or Myc-UBR4.

H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 wild-type or Flag-ACE2 (K26/112/114R) mutant plus Myc-UBR4 plasmids, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested.

Data information: Data shown are representative results of three (A, B, C, D, E, G, H) independent experiments.

Source data are available online for immunoblotting data of this figure.
Queuosine-modified tRNAs confer nutritional control of protein translation (2018)
Figure 6

Fig 6 - ER stress and UPR response upon Q depletion

A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm.

B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm.

Data information: S (standard medium), SF (serum free medium), q (queuine).
Queuosine-modified tRNAs confer nutritional control of protein translation (2018)
Figure 7

Fig 7- Q-dependent phenotypes in mice.

A APB Northern blot using a tRNAHis probe. m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue. Both queuosinylation and methylation levels could be restored by the addition of q to the feed. The results are shown for three biological replicates.

B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q-free synthetic diet. Addition of q to the q-free diet significantly rescued the translation rate. n=3. Asterisks indicate statistically significant (p<0.05, t-test) differences.

C Box plot showing the body weight of mice fed a q-free synthetic diet for 60 days, center lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range; n=3. Addition of q to the q-free diet significantly rescued body weights.

D Dimethyl-labeling analysis of -q versus +q liver tissue. Top 10% of deregulated proteins are indicated in red and blue. UPR effectors: HSPA5/BiP, HSP90b1 and CALR are in orange.

E Gene ontology analysis of deregulated proteins. Signaling pathways and biological functions are presented

F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm.

Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.

Expanded View legends
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 1

Figure 1. Efa6 is required for dendrite pruning of ddaC sensory neurons.

A A schematic representation of dendrite pruning in ddaC neurons.

B-H Live confocal images of ddaC neurons expressing mCD8-GFP by ppk-Gal4 at WP stage or 16 h APF. Neurons expressing efa6 RNAi #1-#3 (C-E), efa612 mutant MARCM clones (F), and efa612/GX6w- transheterozygous mutant neurons (G) showed consistent dendrite pruning defects at 16 h APF, as compared to the wild-type neurons (B). The dendrite pruning defects were fully rescued when overexpressing full-length Efa6 in the efa612 mutant clones (H). Red arrowheads point to the ddaC somas.

I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.

Data information: In (I-J), the number of samples (n) in each group is shown above the x-axis. *p<0.05, ***p<0.001, as assessed by one-way ANOVA with Bonferroni test. All error bars represent SEM. Three independent experiments were conducted. The scale bars in (B-H) represent 50 µm. Source data are available online for this figure.
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 2

Figure 2. The MTED domain is essential for Efa6 to regulate dendrite pruning.

A A schematic illustration of the protein structure of Drosophila Efa6, and various truncated Efa6 proteins.

B-H Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6FL (C), Efa6Nterm (D), Efa6ΔPH (F) and Efa6Nterm+CAAX (H), but not Efa6Cterm (E) or Efa6ΔMTED (G), rescued the pruning defects in efa612/GX6w- mutant ddaC neurons (B). Red arrowheads point to the ddaC somas.

I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.

Data information: In (I-J), the number of samples (n) in each group is shown on the bars. ns, not significant, ***p<0.001, as assessed by one-way ANOVA with Bonferroni test. All error bars represent SEM. Three independent experiments were conducted. The scale bars in (B-H) represent 50 µm.

Source data are available online for this figure.
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 3

Figure 3. Overexpression of Efa6 induces premature dendrite pruning.

A-D Live confocal images of ddaC neurons at WP stage and 7 h APF. Efa6FL or Efa6Nterm-overexpressing neurons (B, C) showed precocious pruning phenotype at 7 h APF, compared to the control and Efa6ΔMTED-overexpressing neurons (A, D) in which most dendrites remained attached to the soma at the same time point. Red arrowheads point to the ddaC somas. Dorsal dendrites are marked by red arrows.

E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF.

F-I Confocal images of da sensory neurons expressing Efa6-Ki-GFP (F), or UAS-Efa6-GFP driven by ppk-Gal4 (H) were co-stained with GFP (Green) and Nrg (Magenta) at wL3 stage. (G, I) Confocal images of the epidermal cells expressing Efa6-Ki-GFP (G) or UAS-Efa6-GFP driven by A58-Gal4 (I) were co-stained with GFP (Green) and Dlg (Magenta) at wL3 stage. The ddaC soma is marked by dashed lines.

Data information: In (E), the number of samples (n) in each group is shown on the bars. Three independent experiments were conducted. The scale bars in (A-D) and (F-I) represent 50 µm and 10 µm, respectively.

Source data are available online for this figure.
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 4

Figure 4. Efa6 inhibits microtubule polymerization in the proximal dendrites of ddaC neurons.

A-F Representative kymographs of EB1-GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL. The horizontal arrow indicates the direction toward the soma, and the vertical arrow indicates the time. In contrast to the wild type (A), the number of EB1-GFP comets was significantly increased in efa612/GX6w- mutant neurons (B). The comet length and speed were significantly decreased in Efa6FL- and Efa6Nterm-overexpressing neurons, but not in Efa6ΔMTED-overexpression neurons (C-F).

G-J Quantification of EB1-GFP orientation, number, track length and speed.

Data information: In (G-J), the number of samples (n) in each group is shown above the x-axis. ns, not significant, **p<0.01, ***p<0.001, as assessed by two-tailed Student's t-test or one-way ANOVA with Bonferroni test. All error bars represent SEM. Three independent experiments were conducted. The scale bars in (A-F) represent 10 µm.

Source data are available online for this figure.
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 5

Figure 5. Efa6 promotes microtubule turnover in the proximal dendrites of ddaC neurons prior to dendrite pruning.

A-C Live confocal images of ddaC dendrites expressing tdEOS::α-tubulin driven by ppk-Gal4. The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 (A, right panels) and WP stages (B, right panels), and increased in Efa6-overexpressing neurons at wL3 stage (C, right panels), compared to the control neurons (A-C, left panels).

D-F Quantification of microtubule turnover rate as indicated by the remaining fluorescence intensity of converted EOS after 30 min.

Data information: In (D-F), the number of samples (n) in each group is shown above the x-axis. ***p<0.001, as assessed by two-tailed Student's t-test. All error bars represent SEM. Three independent experiments were conducted. The scale bars in (A-C) represent 10 µm.

Source data are available online for this figure.
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 6

Figure 6. Efa6 promotes dendrite pruning via microtubule destabilization.

A-H Live confocal images of ddaC neurons at WP stage and 16 h APF. Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons (A, B), while taxol treatment enhanced the pruning defects (C, D). Removal of one copy of γTub23C (F) or expression of γTub23C RNAi (H) almost fully rescued the dendrite pruning defects in efa6 mutant (E) or RNAi neurons (G), respectively. Red arrowheads point to the ddaC somas.

I-K Quantification of unpruned dendrite lengths at 16 h APF.

Data information: In (I-K), the number of samples (n) in each group is shown above the x-axis. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, as assessed by two-tailed Student's t-test or one-way ANOVA with Bonferroni test. All error bars represent SEM. Three independent experiments were conducted. The scale bars in (A-H) represent 50 µm.

Source data are available online for this figure.
A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila (2021)
Figure 7

Figure 7. Stathmin regulates dendrite pruning via inhibiting microtubule polymerization.

A-F Live confocal images of ddaC neurons at WP stage and 16 h APF. stai RNAi neurons (B), staiSK7 (C), staiKO (D) mutant MARCM clones showed similar dendrite pruning defects, compared to the wild-type ddaC neurons (A). Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects (E), compared to their individual RNAi knockdown. Expression of stai RNAi in efa612/GX6w- mutant background also significantly enhanced the dendrite pruning defects (F), compared to the RNAi or mutant alone. Red arrowheads point to the ddaC somas.

G-H Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.

I-L Representative kymographs of EB1-GFP comets driven by Gal44-77 in the proximal dendrites of ddaC neurons at 96 h AEL.

M-P Quantification of EB1-GFP comet orientation, number, track length and speed.

Data information: In (G-H) and (M-P), the number of samples (n) in each group is shown on the x-axis. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, as assessed by one-way ANOVA with Bonferroni test or two-tailed Student's t-test. All error bars represent SEM. Three independent experiments were conducted. The scale bars in (A-F) and (I-L) represent 50 µm and 10 µm, respectively.

Source data are available online for this figure.

Expanded View Figure Legends
Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP (2022)
Figure 1

Figure 1. Bassoon deletion affects the release competence of SV at glutamatergic synapse.

A. Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation.

B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy in WT (black) and BsnGT (red) neurons as described in A. Intensities were normalized to the peak of NH4Cl response.

C. Quantifications of mean RRP fraction in WT and BsnGT.

D. Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta (from 6 independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT.

E. Quantifications of mean TRP fraction in WT and BsnGT.

F. Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes.

G, Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and BsnGT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels).

H. Quantification of normalized IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in G.

I. Quantification of fraction of active (Syt1Ab-labelled) excitatory and inhibitory synapses on experiments illustrated in G.

J. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in G.

K. Quantification of normalized IF of Syt1Ab uptake on experiments from J.

L. Quantification of fraction of active excitatory and inhibitory synapses in J.

Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The dashed lines in H and K depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) or to the number of analysed images (G, J). In D and F, the data of 1050 synapses per genotype were processed. Data is obtained from 6 (A-F) or 3 (G-L) independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 2 µm in A and 5 µm in G and J.