microtubule gmpcpp seeds 5  (Cytoskeleton Inc)

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

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) KIF5C(1-560)-HaloTag motility on dynamic microtubules. Left panel shows full kymograph of dynamic microtubule channel. Insets: upper panel shows early time point when the microtubule is short; Left, overlay of dynamic microtubule (cyan) and GMPCPP-stabilized microtubule seed (magenta) channels, (−) and (+) represent the microtubule minus- and plus-end; Center, KIF5C channel; Right, overlay of a line representing the growing microtubule plus-end on the KIF5C channel. Horizontal scale bars: 10 μm; Vertical scale bars: 30 sec. The plot in the middle represents the location of KIF5C run terminations (blue circles) along a growing dynamic microtubule (dark blue line; microtubule plus-end), respective to the distance to the minus-end of that microtubule. The plot on the right shows the length of KIF5C runs (blue lines) that terminated at the plus-end (dark blue line). The numbers refer to the maximum KIF5C run-length observed in 120 seconds periods over 10 minutes. B) same as (A), but for KIF1A(1-393)-HaloTag. C) Percentage of runs initiating and D) terminating within 2μm of a microtubule plus-end (n=1035-1755 runs from 4-6 microtubules; average±95%CI). E) Circles represent KIF1A (red) and KIF5C (blue) run lengths observed on three representative microtubules and normalized to the maximum length of the microtubule they were observed on. The line traces show the dynamic profile of the representative microtubules along time. See also Figure S5 and S6.

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques:

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) Binding and B) quantification of KIF1A, KIF5B, and KIF5C to GMPCPP- and GDP-taxol-stabilized microtubules in the presence of AMP-PNP (n=1004-5855 microtubules per condition; n.s., non-significant, ***p<0.0001; one-way ANOVA with Sidak’s post-hoc test). C) Binding and D) quantification of KIF1A binding to GMPCPP- and GDP-taxol-stabilized microtubules under increasing ionic strength. Circles show mean intensity values; the 95% confidence intervals are too narrow and not visible in the graph. (n=398-2491 microtubules). E) Binding and F) quantification of KIF1A binding to fully tyrosinated and detyrosinated GMPCPP-stabilized microtubules G) under increasing ionic strength. Circles show mean intensity values; the 95% confidence intervals are too narrow and not visible in the graph. (n=2709-6737 microtubules). H) Binding and I) quantification of KIF1A binding to GMPCPP-stabilized microtubules in the presence or absence of EB3 (n=13569-14309 microtubules). J) Representative kymographs and stills depictingKIF1A motors rapidly detaching from a microtubule at the EB1 comet region (asterisks). The stills on the right show one of these events (each time point integrates a 150 msec interval).

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques: Binding Assay

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) KIF1A motor domain. The T258M mutation affects a residue located in KIF1A motor domain loop 11. B) Panels and quantification showing KIF1A-WT and -T258M(1-393)-HaloTag binding to GMPCPP- and GDP-taxol-stabilized microtubules. (n=2303-2351 microtubles per group; average±95%CI; **p<0.01,****p<0.0001; Kruskal-Wallis with Dunn’s post-hoc test). C) same as Figure 3A-B and D) same as Figure 3E but for KIF1A-T258M(1-393)-HaloTag, which is represented in orange. E) eCDF showing the location of KIF1A-WT (red) and KIF1A-T258M (orange) run initiation, respective to the plus-end tip. X-intercepts represent the location on the microtubule where run initiations starts to follow a random pattern. (KIF1A-WT, n=1035 runs from 6 microtubules; KIF1A-T258M, n=1628 runs from 5 microtubules). F) Correlation between GDP-taxol/GMPCPP microtubules binding ratio and distance of run initiation to the plus-end tip. (KIF5C: n=1755 runs from 4 microtubules; KIF1A-WT: n=1035 runs from 6 microtubules; KIF1A-A255V: n=702 runs from 4 microtubules; KIF1A-T258M: n=1628 runs from 5 microtubules; KIF1A-R350G: n=596 runs from 4 microtubules; GDP/GMPCPP MT binding ratio – KIF5C-WT: n=5855/4000; KIF1A-WT: n=2491/2303; KIF1A-A255V: n=2130/1046; KIF1A-T258M: n=2351/2351; KIF1A-R350G: n=2106/1162). G) KIF1A-WT rapidly detaches from microtubules once it reaches the microtubule plus-end, whereas KIF1A-T258M frequently lingers at the microtubule plus-end before detaching. See also Figure S5, S6, and Table S1.

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques: Mutagenesis, Binding Assay