Journal: Nature Communications

Article Title: Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes

doi: 10.1038/s41467-021-23530-0

Figure Lengend Snippet: a Preparation of N-MT hybrid NSF including its monomerization, labeling, re-hexamerization, and N-domain removal (PDB ID: 3J94 for NSF, 3KZZ for SNAP tag). b Experimental scheme of surface immunoprecipitation (IP) for the pulldown of N-MT hybrid NSF using two types of antibodies (N-domain and SNAP tag) and the distributions of deconvoluted photobleaching step(s) considering 90% labeling efficiency. c Experimental scheme of NSF pulldown via the SNARE–αSNAP complex and the distributions of deconvoluted photobleaching step(s). d Relative avidity of N-MT hybrid hexamers to the SNARE–αSNAP complex according to the number of N-MT subunits in single NSF hexamers. Data in ( c ) divided by those using the anti-N-domain antibody in ( b ) and normalized to the value for single-mutant hexamers. e Measurements of WT and N-MT hybrid NSF’s disassembly activity. Counts for SNARE complexes under ATP-non-hydrolyzing (1 mM ATP/1 mM EDTA) and inducing ATP hydrolysis (1 mM ATP/10 mM Mg 2+ ) for 5 min. f Normalized counts of Cy3-VAMP2 under ATP-hydrolyzing condition with two types of NSF at several time points. Inset shows a semi-log plot of early time points. τ represent the time constant of exponential decay. The data represent mean ± s.e.m. for four independent experiments ( b , c ). The data in ( d ) represent the normalized values obtained by dividing the data in ( c ) by the data using the anti-N-domain antibody in ( b ) ± error propagated by the calculations. The data represents mean ± s.d. for n = 12 (-ATP/Mg2+) or 10 (+ATP/Mg2+) images from two independent experiments ( e ) and mean ± s.e.m. for three independent experiments ( f ). Source data are provided as a Source Data file.

Article Snippet: Purified SNAP-tag-αSNAP was diluted to 5 μM and labeled with 10 μM benzylguanine (BG)-Alexa647 dyes (New England Biolabs) for 2 h at room temperature or overnight at 4 °C.

Techniques: Labeling, Immunoprecipitation, Mutagenesis, Activity Assay

Journal: bioRxiv

Article Title: Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration

doi: 10.1101/727008

Figure Lengend Snippet: A) Similar as , but testing the expanded G4C2r induced toxicity. dtau KO significantly rescued in vivo neurodegeneration induced by (G4C2) 30 expression. B) Representative images and quantifications of neurodegeneration in the neuromuscular junctions (NMJs) in flies with the indicated genotypes. NMJs of muscle 6/7 in abdominal segment A2 and A3 of third instar larvae were stained with anti-HRP (neuronal axon marker, red channel) and anti-Bruchpilot (Brp) (active zone marker, green channel). The Brp-positive active zones were reduced by (G4C2) 30 expression, and rescued by dtau KO. The white arrows indicated the buttons of NMJs. Scale bars, 10 μm. C-D) Similar as , but for G4C2 flies. dtau KO significantly rescued motor function deficits and shortened lifespan induced by (G4C2) 30 expression. For all plots in A-C, error bars indicate mean ± SEM. Asterisks denote statistically significant differences (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001).

Article Snippet: The tau-snap plasmid were generated by inserting the cDNA expressing one transcript of human tau (0N4R, cloned by PCR amplification from the UAS-htau4R Drosophila strain) into the snap-tag vector (NEB, cat. no. P9312S). (G4C2) 3 and (G4C2) 25 plasmids were generated by inserting (g4c2) 3 or (g4c2) 25 repeats into pTT-sfGFP vector.

Techniques: In Vivo, Expressing, Staining, Marker

Journal: bioRxiv

Article Title: Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration

doi: 10.1101/727008

Figure Lengend Snippet: A) The schematic picture of the qPCR primers for measurements of the (G4C2) 30 transcript level. B) qPCR measurements of (G4C2) 30 transcripts in brain tissues from flies of indicated genotypes using indicated qPCR primers. n indicates the number of individual flies. The statistical analysis was performed by two-tailed unpaired t tests. C) The schematic picture of the qPCR primers for measurements of the endogenous dtau transcript level. D) qPCR measurements of the indicated dtau transcripts in brain tissues from flies of indicated genotypes. The statistical analysis was performed by two-tailed unpaired t tests. Neither the dtau transcript levels nor their splicing were influenced by (G4C2) 30 compared to (G4C2) 3 . For all plots, bars indicate mean ± SEM. Asterisks denote statistically significant differences (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). For all the qPCR primers tested, the no reverse transcriptase control showed no amplification, validating that the signals were not contaminated by genomic DNA.

Article Snippet: The tau-snap plasmid were generated by inserting the cDNA expressing one transcript of human tau (0N4R, cloned by PCR amplification from the UAS-htau4R Drosophila strain) into the snap-tag vector (NEB, cat. no. P9312S). (G4C2) 3 and (G4C2) 25 plasmids were generated by inserting (g4c2) 3 or (g4c2) 25 repeats into pTT-sfGFP vector.

Techniques: Two Tailed Test, Amplification

Journal: bioRxiv

Article Title: Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration

doi: 10.1101/727008

Figure Lengend Snippet: A-C) Representative Western-blots and quantifications showing that the total tau levels were increased by (G4C2) 30 expression in the 3rd instar larva, pupa and adult stage flies, compared to the (G4C2) 3 expression controls. n indicates the number of individual flies. The statistical analysis was performed by two-tailed unpaired t tests. D) Representative immunofluorescence images of whole-mount fly brains showing increased transgenic htau levels in (G4C2) 30 flies. All the flies was dissected and stained at day 16. E) Representative Western-blots and quantifications showing that total tau levels were increased by (G4C2) 25 compared to (G4C2) 3 controls in transfected HEK293T cells. The statistical analysis was performed by two-tailed unpaired t tests. F) Representative immunofluorescence images and quantifications (measured by the number of tau puncta per cell) showing that (G4C2) 25 expression increased tau aggregates in HeLa cells, compared to the (G4C2) 3 expressing controls. The statistical analysis was performed by two-tailed unpaired t tests. The aggregates were tested in HeLa cells, because tau aggregates were hardly visible in HEK293T cells. G) Representative Western-blots and quantifications showing that the cleaved product (free GFP cleaved from GFP-mCherry-atg8) band is largely missing in motor neurons expressing (G4C2) 30 , suggesting an inhibition of autophagy activity in these neurons. n indicates the number of individual flies. The statistical analysis was performed by two-tailed unpaired t tests. H) qPCR showing a reduction of starvin ( stv ) mRNA levels by (G4C2) 30 expression compared to the (G4C2) 3 expressing controls. The results were consistent in both elav(I)-GAL4 or elav(III)-GAL4 driven flies. I) Representative Western-blots and quantifications of lysates from transfected HEK293T cells showing that BAG3 protein levels were reduced by (G4C2) 25 expression compared to the (G4C2) 3 expressing controls. For all plots, bars indicate mean ± SEM. Asterisks denote statistically significant differences (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001).

Article Snippet: The tau-snap plasmid were generated by inserting the cDNA expressing one transcript of human tau (0N4R, cloned by PCR amplification from the UAS-htau4R Drosophila strain) into the snap-tag vector (NEB, cat. no. P9312S). (G4C2) 3 and (G4C2) 25 plasmids were generated by inserting (g4c2) 3 or (g4c2) 25 repeats into pTT-sfGFP vector.

Techniques: Western Blot, Expressing, Two Tailed Test, Immunofluorescence, Transgenic Assay, Staining, Transfection, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration

doi: 10.1101/727008

Figure Lengend Snippet: A) Representative Western-blots and quantifications showing that the phosphorylated tau levels detected by the antibody pS262 or AT8 were increased by (G4C2) 30 expression in the adult fly heads, compared to the (G4C2) 3 expressing controls. n indicates the number of biological replicates. The statistical analysis was performed by two-tailed unpaired t tests. B) Similar as A, but in transfected HEK293T cells.

Article Snippet: The tau-snap plasmid were generated by inserting the cDNA expressing one transcript of human tau (0N4R, cloned by PCR amplification from the UAS-htau4R Drosophila strain) into the snap-tag vector (NEB, cat. no. P9312S). (G4C2) 3 and (G4C2) 25 plasmids were generated by inserting (g4c2) 3 or (g4c2) 25 repeats into pTT-sfGFP vector.

Techniques: Western Blot, Expressing, Two Tailed Test, Transfection

Journal: bioRxiv

Article Title: Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration

doi: 10.1101/727008

Figure Lengend Snippet: Representative Western-blots of ubiquitinated proteins showing an increase of these proteins in (G4C2) 30 expressing larva or pupae.

Article Snippet: The tau-snap plasmid were generated by inserting the cDNA expressing one transcript of human tau (0N4R, cloned by PCR amplification from the UAS-htau4R Drosophila strain) into the snap-tag vector (NEB, cat. no. P9312S). (G4C2) 3 and (G4C2) 25 plasmids were generated by inserting (g4c2) 3 or (g4c2) 25 repeats into pTT-sfGFP vector.

Techniques: Western Blot, Expressing