gmpcpp  (Jena Bioscience)

Journal: bioRxiv

Article Title: Asgard archaeal origin of microtubules

doi: 10.64898/2026.02.15.705738

Figure Lengend Snippet: A) Coomassie-stained SDS PAGE gels showing strong soluble untagged AtubAB expression in E. coli C41(DE3) cells (left), purified His 8 and Strep-tagged AtubAhBs (middle) and purified untagged AtubAB (right), as used in this study. B) Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) reveals that purified untagged AtubAB is a stable heterodimer. C) AtubAB is a slow GTPase: 0.192 +/- 0.004 nmol phosphates released/nmol AtubAB/min (25°C), n = 3. D) Critical concentration of AtubAB polymerisation in BRB80 + 500 mM potassium glutamate Polymerisation Buffer (used throughout this study) is ∼3 g/L (62 µM dimer). E) Pelleting assay revealing that AtubAB polymerisation is GTP-dependent and can also be triggered by the non-hydrolysable GTP analogue GMPCPP. L, loaded; S, supernatant; P. pellet. F) 90° 360 nm light scattering assay showing the same GTP/GMPCPP-dependence of AtubAB polymerisation as in E. GTP hydrolysis leads to depolymerisation, adding GTP again leads to re-polymerisation. G) Pelleting assay showing the inhibition of GTP-induced AtubAB polymerisation by the tubulin inhibitor maytansine (MAY). DMSO and MAY reactions also contained GTP. (see also ) H) 90° 360 nm light scattering assay showing the same maytansine inhibition of GTP-induced AtubAB polymerisation as in G.

Article Snippet: The proteins were mixed to make two different stocks: AtubAB seed stocks were prepared at 50 μM final concentration (20% fluorescent-AtubAB, 10% biotinylated-AtubAB) in Polymerisation Buffer supplemented with 5 mM GMPCPP (Jena Bioscience NU-405S); free (un-polymerised) AtubAB-dimers were prepared at a final concentration of 50 μM (20% fluorescent-AtubAB) in Glutamate-free Buffer.

Techniques: Staining, SDS Page, Expressing, Purification, Size-exclusion Chromatography, Multi-Angle Light Scattering, Concentration Assay, Scattering Assay, Inhibition

Journal: bioRxiv

Article Title: Asgard archaeal origin of microtubules

doi: 10.64898/2026.02.15.705738

Figure Lengend Snippet: Polymerisation performed in BRB80 + 500 mM potassium glutamate Polymerisation Buffer with GDP, GTP and GMPCPP nucleotides (scale bars = 5 µm). GTP and GMPCPP-containing reactions show many filaments. See also the corresponding pelleting experiment in .

Article Snippet: The proteins were mixed to make two different stocks: AtubAB seed stocks were prepared at 50 μM final concentration (20% fluorescent-AtubAB, 10% biotinylated-AtubAB) in Polymerisation Buffer supplemented with 5 mM GMPCPP (Jena Bioscience NU-405S); free (un-polymerised) AtubAB-dimers were prepared at a final concentration of 50 μM (20% fluorescent-AtubAB) in Glutamate-free Buffer.

Techniques:

Journal: bioRxiv

Article Title: Asgard archaeal origin of microtubules

doi: 10.64898/2026.02.15.705738

Figure Lengend Snippet: (A) Inset: assay design; biotinylated, GMPCPP-stabilised AtubAB dimers (labelled with 20% Alexa Fluor 647, shown as red) were anchored via NeutrAvidin to PEG Silane. Free AtubAB dimers (4 µM 20% Atto 488-labelled, cyan) were added, and filament polymerisation was observed by TIRF (total internal reflection fluorescence) microscopy. Field view revealing red GMPCPP seeds with plus- and minus-end growth (cyan). Only this used 20% tagged (unlabelled) AtubAhBs to observe dynamics at high concentrations needed for field views. Scale bar = 10 µm. B) Time-lapse of plus-end growth of untagged AtubAB filament. The 10 th image down shows the moment just after catastrophe, the ultra-rapid depolymerisation of the mini microtubule. Scale bar 5 µm. C) Kymograph (time vertical, filament axis horizontal) showing an untagged AtubAB filament growing and undergoing catastrophe at the plus-end. One growth episode (line) and one catastrophe event (arrowhead) are highlighted - it is the same as in panel B. Scale bars, 2 min / 2 µm. See also Supplementary Figures S4A & B and Movie M5. D) Asgard archaeal 4-pf mini microtubules show all the hallmarks of eukaryotic 13-protofilament microtubules - but are much thinner. We therefore suggest it is possible that Asgard mini microtubules are the evolutionary precursors of eukaryotic microtubules. Arrows do not indicate evolutionary distances or complexity.

Article Snippet: The proteins were mixed to make two different stocks: AtubAB seed stocks were prepared at 50 μM final concentration (20% fluorescent-AtubAB, 10% biotinylated-AtubAB) in Polymerisation Buffer supplemented with 5 mM GMPCPP (Jena Bioscience NU-405S); free (un-polymerised) AtubAB-dimers were prepared at a final concentration of 50 μM (20% fluorescent-AtubAB) in Glutamate-free Buffer.

Techniques: Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Asgard archaeal origin of microtubules

doi: 10.64898/2026.02.15.705738

Figure Lengend Snippet: TIRF microscopy, AtubAB polymerisation and kymographs as in . Red: GMPCPP-stabilised seeds (not depolymerising), cyan: AtubAB with GTP/GDP, dynamic . i) Field of view using untagged proteins, only (4 µM final concentration). Scale bar 5 µm. ii) and iii) Further examples of dynamic untagged AtubAB filaments (4 µM final concentration), similar to , showing slow growth at the minus ends (left) and fast growth and dynamic instabilities at the plus ends (right). iv) Same as ii and iii for a sample with 20% tagged, unlabelled AtubAhBs added (Methods), to increase turnover at higher concentrations needed to get highly populated field views (as in ) (5 µM, see Methods). Scale bars = 2 min and 2 µm.

Article Snippet: The proteins were mixed to make two different stocks: AtubAB seed stocks were prepared at 50 μM final concentration (20% fluorescent-AtubAB, 10% biotinylated-AtubAB) in Polymerisation Buffer supplemented with 5 mM GMPCPP (Jena Bioscience NU-405S); free (un-polymerised) AtubAB-dimers were prepared at a final concentration of 50 μM (20% fluorescent-AtubAB) in Glutamate-free Buffer.

Techniques: Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: Asgard archaeal origin of microtubules

doi: 10.64898/2026.02.15.705738

Figure Lengend Snippet: Relate to and . i) & ii) AtubAB GMPCPP seeds were polymerised and filament growth rates (i) (minus ends, median speed of growth 0.48 µm/min; plus ends, median speed of growth 1.80 µm/min) and maximal lengths before undergoing catastrophe (ii) were measured. p values from two-sided Kruskal-Wallis tests for non-parametric samples are indicated; n, number of individual growth events quantified from at least three independent experiments. Thick lines, median; thin lines, quartile. iii) Cumulative AtubAB filament lifetime distributions of mini microtubules grown at the minus (blue) and plus ends (green). Mean lifetime estimates +/- error (lifetime at half cumulative distribution): 451.9 +/- 55.5 s (minus ends) and 45.1 +/- 1.6 s (plus ends). Line, gamma distribution fits.

Article Snippet: The proteins were mixed to make two different stocks: AtubAB seed stocks were prepared at 50 μM final concentration (20% fluorescent-AtubAB, 10% biotinylated-AtubAB) in Polymerisation Buffer supplemented with 5 mM GMPCPP (Jena Bioscience NU-405S); free (un-polymerised) AtubAB-dimers were prepared at a final concentration of 50 μM (20% fluorescent-AtubAB) in Glutamate-free Buffer.

Techniques:

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