gmpcpp-stabilized microtubule seeds  (Thermo Fisher)

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: PLoS ONE

Article Title: Nicotinamide adenine dinucleotides and their precursor NMN have no direct effect on microtubule dynamics in purified brain tubulin

doi: 10.1371/journal.pone.0220794

Figure Lengend Snippet: (A) Schematic of an IRM in vitro microtubule assay. Dynamic, label-free microtubules were grown from TAMRA-labeled GMPCPP-stabilized microtubule seeds attached to a silanized coverslip through anti-TAMRA antibodies. Microtubule dynamics was observed by time-lapse IR microscopy. Microtubule plus ends (marked with gray arrows) and minus ends were analyzed in this assay. Illumination light (blue line) is reflected from the glass/water interface and water/microtubule interface. Microtubule image is formed by the interference of reflected light. I IRM : interference intensity; I 0 : incident light intensity; I 1 : intensity of light reflected of glass/sample interface; I 2 : intensity of light reflected from water/microtubule interface (B) Representative kymographs depicting dynamic behavior of individual microtubule polymers in the presence or absence of NADs or NMN (as specified in the top right corner). Dashed lines indicate the position of TAMRA-labeled microtubule seeds. For all kymographs, microtubule plus-end is positioned to the right of the seed and corresponds to the orientation of microtubule on the illustration in panel (A) Horizontal scale bars, 3μm. Vertical scale bars, 5 minutes. (C) Scatter plots representing the effect of NADs and NMN on parameters of microtubule dynamic instability for an individual experimental repeat. Points on the diagrams depicting microtubule growth and shrinkage rates correspond to the average growth or shrinkage rate of individual microtubule within the sample. Time to catastrophe represents the lifetime of all analyzed events within the repeat. Red line indicates the average, blue error bars correspond to standard deviation (SD). The exact values for parameters of microtubule dynamic instability plotted here can be found in in bold. (D) Plots representing averages for all individual experiments. Same shape symbols correspond to the experiments performed side-by-side on the same day. Detail values for depicted averages are combined in . (E) Plots representing average growth rates and time to catastrophe measured for microtubule minus-ends. Averages for all individual experiments are combined in . Shaded boxes in panels D and E represents 95% confidence intervals with middle lines corresponding to the averages calculated based on the experimental repeats.

Article Snippet: GMPCPP-stabilized microtubule seeds labeled with TAMRA dye (ThermoFisher Scientific, Waltham, MA) were used to initiate the growth of dynamic microtubule extensions from unlabeled GTP-tubulin.

Techniques: In Vitro, Labeling, Microscopy, Standard Deviation