Journal: bioRxiv
Article Title: Injury-induced tau pathology promotes aggressive behavior in Drosophila without neurodegeneration
doi: 10.1101/2025.11.22.689595
Figure Lengend Snippet: A. Schematic depicting an in vitro reconstituted microtubule dynamics assay with labeled parameters that we measured. A diagonal line represents tubulin (magenta) polymerizing off of a stable GMPCPP seed (blue), then undergoing a catastrophe and rapidly depolymerizing back to the GMPCPP seed. If the microtubule begins to polymerize again before reaching the GMPCPP seed, this is considered a rescue event. B. Representative kymographs of microtubule dynamics showing the polymerization of 10 μM tubulin (magenta) + 1 mM GTP from GMPCPP seeds (blue) in the absence or presence of human sfGFP-2N4R-tau (HsTau, green) expressed in bacteria or insect cells or Drosophila melanogaster sfGFP-tau (DmTau, green) expressed in insect cells at the indicated concentrations. Scale bars: y, 2 min; x, 2 μm. C-E Quantification of microtubule plus end growth rate ( C ), catastrophe frequency ( D ), and rescue frequency ( E ) for 10 μM tubulin + 1 mM GTP in the absence or presence of 10 nM bacterially-expressed HsTau, 50 nM insect cellexpressed HsTau, or 10 nM insect cell expressed-DmTau (n=33, 25, 31, and 39 analyzed kymographs from n=3 independent trials). For microtubule growth rate ( C ), tubulin alone vs. bacterial HsTau, p= 0.7113; vs. insect-cell HsTau, p= 0.8525; vs. DmTau, p= 0.0048. For catastrophe frequency ( D ), tubulin alone vs. bacterial HsTau, p< 0.0001; vs. insect-cell HsTau, p= 0.0803; vs. DmTau, p< 0.0001. For rescue frequency ( E ), tubulin alone vs. bacterial HsTau, p< 0.0001; vs. insect-cell HsTau, p= 0.0027; vs. DmTau, p< 0.0001. F. Images of BEAS-2B cells expressing EB1-tdTomato in conjunction with either GFP empty vector, GFP-HsTau, or GFP-DmTau visualized by spinning disk confocal microscopy, with associated EB1-tdTomato comet trajectories represented by colored lines (2.5 fps for 3 min) showing the growth pattern of microtubules under each transfection condition. Scale bar: 20 µm. G. Quantification of polymerization events per minute for each transfection condition, GFP empty vector, GFP-HsTau, and GFP-DmTau (n=45, 41, and 35 cells, respectively from 3 independent experiments). For GFP vs. HsTau, p= 0.0667; vs. DmTau, p= 0.0008. H. Magnified view of EB1 comets under each transfection condition, GFP empty vector, GFP-HsTau, or GFP-DmTau. Scale bar: 2 µm. I. Quantification of EB1 dwell time under each transfection condition, GFP empty vector, GFP-HsTau, or GFP-DmTau (n=63, 85, and 56 cells, respectively). For GFP vs. HsTau, p= 0.3398; vs. DmTau, p= 0.9308. Two-sided unpaired Student’s t -tests were used to determine p -values. All graphs display all datapoints with lines indicating means ± s.d.
Article Snippet: GMPCPP microtubules were polymerized by combining unlabeled tubulin, 650-tubulin, and biotin-tubulin (∼10:1:1) to a concentration of ∼60mM in BRB80 supplemented with 1mM DTT and 1mM GMPCPP seed (Jena BioScience NU-405S), then incubating at 37°C for 20-30 minutes.
Techniques: In Vitro, Labeling, Bacteria, Expressing, Plasmid Preparation, Confocal Microscopy, Transfection
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