microtubule gmpcpp seeds 5  (Cytoskeleton Inc)

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

Journal: bioRxiv

Article Title: Microtubule dynamic instability is sensitive to specific biological viscogens in vitro

doi: 10.1101/2024.05.27.596091

Figure Lengend Snippet: (A) Schematic of the bio-viscogens used in this study drawn to scale. Glyerol (blue), trehalose (red), and BSA (green) were used to increase the viscosity of solutions of αβ-tubulin (teal, labeled). Scale bar 2 nm. (B) Schematic of the single molecule assay based on interference reflection microscopy. TAMRA-labeled, GMPCPP-stabilized microtubule tempates are adhered to a cover glass surface using antibodies against TAMRA (see labels). Dynamic microtubule extensions are visualized with IRM. (C) Kymographs showing microtubule dynamic instability at 10 μ M tubulin in the presence of each viscogen ( η values indicated). (D) Plot of microtubule growth rate versus viscosity for the glycerol titration at 10 μ M tubulin. (E) Plot of microtubule growth rate versus viscosity for the trehalose titration at 10 μ M tubulin. (F) Plot of microtubule growth rate versus viscosity for the BSA titration at 10 μ M tubulin. (G) Plot of microtubule shrinkage rate versus viscosity for the glycerol titration at 10 μ M tubulin. (H) Plot of microtubule shrinkage rate versus viscosity for the trehalose titration at 10 μ M tubulin. (I) Plot of microtubule shrinkage rate versus viscosity for the BSA titration at 10 μ M tubulin. All data from (D) to (I) include n ≥ 3 replicates.

Article Snippet: GMPCPP-stabilized microtubule seeds were prepared by polymerizing a 1:4 molar ratio of tetramethylrhodamine (TAMRA, ThermoFisher Scientific) labeled:unlabeled tubulin ( ) in the presence of GMPCPP (Jena Biosciences) in two cycles, as described previously ( ).

Techniques: Viscosity, Labeling, Microscopy, Titration

Journal: bioRxiv

Article Title: Microtubule dynamic instability is sensitive to specific biological viscogens in vitro

doi: 10.1101/2024.05.27.596091

Figure Lengend Snippet: (A) Plot of cumulative frequency distribution of microtubule lifetimes with glycerol at 5 μ M tubulin. Each line represents the total distribution of lifetimes across n = 3 replicates. (B) Plot of cumulative frequency distribution of microtubule lifetimes with trehalose at 5 μ M tubulin. Each line represents the total distribution of lifetimes across n = 3 replicates. (C) Plot of cumulative frequency distribution of microtubule lifetimes with BSA at 5 μ M tubulin. Each line represents the total distribution of lifetimes across n = 3 replicates. (D) Plot of mean lifetime against viscosity for all three bio-viscogens (blue: glycerol, red: trehalose, green: BSA) at 5 μ M tubulin (E) Plot of rescue frequency against viscosity with glycerol at 10 μ M tubulin. (F) Plot of rescue frequency against viscosity with trehalose at 10 μ M tubulin. (G) Plot of rescue frequency against viscosity with BSA at 10 μ M tubulin. (H) Plot of rescue frequence against viscosity for all three bio-viscogens (blue: glycerol, red: trehalose, green: BSA) at 10 μ M tubulin. All data from (E) to (H) include n ≥ 3 replicates.

Article Snippet: GMPCPP-stabilized microtubule seeds were prepared by polymerizing a 1:4 molar ratio of tetramethylrhodamine (TAMRA, ThermoFisher Scientific) labeled:unlabeled tubulin ( ) in the presence of GMPCPP (Jena Biosciences) in two cycles, as described previously ( ).

Techniques: Viscosity

Journal: bioRxiv

Article Title: Microtubule dynamic instability is sensitive to specific biological viscogens in vitro

doi: 10.1101/2024.05.27.596091

Figure Lengend Snippet: (A) Schematic of a growing microtubule showing the GTP cap (dark blue) and EB3-GFP bindins (yellow) (B) Kymographs showing EB3-GFP comets during growth for each bio-viscogen (C) Schematic representation of the influence of tubulin concentration and growth rate on comet intensity. (D) Plot of comet intensity as a function of growth rate for all three bio-viscogens. Tubulin concentrations are 10 / 20 / 25 / 30 μ M for control conditions and 10 / 20 / 30 μ M with each bio-viscogen. Data from the three bio-viscogens were fit to a common line (black dashed line).

Article Snippet: GMPCPP-stabilized microtubule seeds were prepared by polymerizing a 1:4 molar ratio of tetramethylrhodamine (TAMRA, ThermoFisher Scientific) labeled:unlabeled tubulin ( ) in the presence of GMPCPP (Jena Biosciences) in two cycles, as described previously ( ).

Techniques: Concentration Assay, Control

Journal: bioRxiv

Article Title: Microtubule dynamic instability is sensitive to specific biological viscogens in vitro

doi: 10.1101/2024.05.27.596091

Figure Lengend Snippet: (A) Templated nucleation: Plot of the probability that a microtubule template nucleated a microtubule within a 15 min time window at 5 μ M tubulin in the presence of 3 bio-viscogens (glycerol: blue; trehalose: red; BSA: green). (B) Spontaneous nucleation with glycerol: plot of the tubulin signal in the pellet versus the total tubulin concentration with glycerol (blue) and control (black) (C) Spontaneous nucleation: plot of the critical concentration for spontaneous nucleation as a function of viscosity for all three bio-viscogens (glycerol: blue; trehalose: red; BSA: green).

Article Snippet: GMPCPP-stabilized microtubule seeds were prepared by polymerizing a 1:4 molar ratio of tetramethylrhodamine (TAMRA, ThermoFisher Scientific) labeled:unlabeled tubulin ( ) in the presence of GMPCPP (Jena Biosciences) in two cycles, as described previously ( ).

Techniques: Concentration Assay, Control, Viscosity

Journal: Nature Communications

Article Title: Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules

doi: 10.1038/s41467-022-32421-x

Figure Lengend Snippet: A Schematic of barcoded porcine-tubulin microtubule with the GMPCPP-, GTPγS- and GDP-tubulin sections (top), along with corresponding fluorescence channels of GDP- (HiLyte647), GTPγS- (HiLyte488) tubulin sections (unlabelled GMPCPP-tubulin, see “Methods”) and TagRFP-HURP (bottom); scale bars = 1.5 µm. B Boxplots of the TagRFP-HURP intensity/µm normalised to its median intensity on GMPCPP microtubules ( N = 3 flow chambers; n = 171 (GMPCPP), 300 (GTPγS) and 238 (GDP); P = Kruskal–Wallis test). C Representative image of porcine-tubulin GMPCPP seeds extended with either human WT- (green) or E254A tubulin (magenta), labelled with HiLyte488 and HiLyte647 porcine brain tubulin (1:7 labelled:unlabelled ratio) respectively (right panel) and incubated with TagRFP-HURP (left panel); scale bars = 3 µm. D Boxplots of HURP intensity/µm normalised to its median intensity on GMPCPP seeds ( N = 3 flow chambers; n = 121 (GMPCPP), 43 (WT) and 87 (E254A tubulin); P = Kruskal–Wallis test). E Immunofluorescence images of metaphase hTERT-RPE1 cells transducted either with RFP-wt-α-tubulin or RFP-E254A-α-tubulin and stained for EB1. Scale bars = 5 µm. F Live-cell imaging of hTERT-RPE1 EGFP-HURP/HaloTag-CENP-A cells transducted either with RFP-wt-α-tubulin or RFP-E254A-α-tubulin mutant (left), Z-projections of 5 × 0.5 µm, Scale bar = 5 µm; and boxplots of in vivo relative HURP intensities (right). N = 2; n = 38 and 42 cells. Boxplots indicate the 25th and 75th percentiles, the bars are medians, and the whiskers indicate values within 1.5 times the interquartile range in B , D , and minima and maxima in F ; P = two-sided Mann–Whitney test. Source data for all graphs are provided as a Source Data file.

Article Snippet: Barcoded microtubules were created by adding 16 μM of pig brain tubulin (1:15 labelled:unlabelled) to stabilised GMPCPP seeds (final ratio of 1:1 v:v), in the presence of 1 mM GTPγS (Jena Bioscience; NU-412-2), and incubating the solution at 37 °C for 1 h. The mix was diluted fivefold in BRB80 supplemented with 2 μM taxol (BRB80 + Tx), spun in an airfuge at 20 psi for 10 min at room temperature, before resuspending the pellet in BRB80 + Tx.

Techniques: Fluorescence, Incubation, Immunofluorescence, Staining, Live Cell Imaging, Mutagenesis, In Vivo, MANN-WHITNEY, Fluorescence Microscopy