Journal: bioRxiv

Article Title: β3 accelerates microtubule plus end maturation through a divergent lateral interface

doi: 10.1101/2024.07.17.603993

Figure Lengend Snippet: Individual β3 variant residues modulate response to microtubule targeting agents. (A) TUBB3 variant residues categorized by functional domain: lateral interface, longitudinal interface, GTP-binding pocket, paclitaxel (PTX) binding pocket, core, or the CTT. Expected number of variant residues was determined by multiplying the proportion of amino acids in each domain by 33 which is the number of β3 variant residues including the CTT. This represents the number of mutations that would be expected from a random distribution throughout the tubulin protein. Observed and expected variant residues were compared using a Chi-squared test to determine if the residues are randomly distributed across domains or enriched. (B) Positions of β3 variant residues on β-tubulin contained within a microtubule lattice. The 180° turn displays a view from inside the lumen looking out. Residues are colored according to the domains defined in panel A. (C) Liquid growth assay to determine the doubling time of mutants with β3 variant residues. Bars represent mean with 95% CI from triplicate experiments with biological replicates included in each experiment. No significant differences in doubling time were determined. (D) Growth delay factor (GDF) of each strain in either 15µM EpoA or 2.5µM Nocodazole. GDF was calculated by dividing the average doubling time in the presence of MTA by the doubling time in DMSO from same experiment. The grey shading represents 95% CI for wild-type GDF in 15µM EpoA and 2.5µM Nocodazole. Values that fell outside the 95% CI were statistically significant and p-values are reported in Figure S3 for the raw doubling times. (E) Serial dilution of yeast cells expressing β3 variant residues indicated to the left of the plate. Cells were spotted to rich media or rich media supplemented with 15 µg/ml benomyl and grown for 2 days at 30°C.

Article Snippet: The cultures were aliquoted into a 96-well plate, treated with 1% DMSO, Epothilone A (S1297, Selleck Chemicals, Houston, TX), or nocodazole (M1404, Sigma Aldrich, Burlington, MA) and incubated at 30°C with single orbital shaking in an Epoch2 microplate reader (BioTek; Winooski, VT).

Techniques: Variant Assay, Functional Assay, Binding Assay, Growth Assay, Serial Dilution, Expressing