Figure 3. Super-stoichiometric β-tubulin... | Rockefeller University Press

Figure 3.

$Super-stoichiometric β-tubulin creates aberrant tubulin assemblies. (A) Schematic for β-tubulin induction experiment. (B) Representative Western blot of β- and α-tubulin during TUB2-6xHIS induction. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (C) Quantification of TUB2-6xHis induction for both α- and β-tubulin across overexpression. Dots represent mean ± SD from four independent induction experiments. (D) Quantification of cells forming colonies after TUB2-6xHis induction or empty vector control. Dots represent mean ± SD from four independent induction experiments. (E) Example images of wild-type cells overexpressing β-tubulin for 0 or 2 h and stained for α- or β-tubulin. Arrows indicate astral microtubules; arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are maximum intensity projections of in focus z positions. (F) Quantification of astral microtubules (aMTs) across induction time for cells stained for either α- or β-tubulin. Dots represent mean ± 95% CI from three independent experiments with at least 100 cells in each experiment. (G) Quantification of tubulin assemblies across induction time for cells stained for either α- and β-tubulin. Dots represent mean ± 95% CI from three independent experiments with at least 100 cells in each experiment. (H) Example images of cells expressing Spc110-tdTomato and GFP-Tub1. TUB2-6xHis was induced for 3 h and then cells were shifted to 4°C for 2 h to depolymerize microtubules. Uninduced control cells are also shown. Arrows indicate microtubules; arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are maximum intensity projections of z series. (I) Quantification of the fraction of astral microtubules or tubulin assemblies in induced or uninduced cells after indicated time at 4°C. Dots are mean ± 95% CI from at least 100 cells in three independent experiments. (J) Example images of cells expressing Bim1-3GFP and mRuby-Tub1 with TUB2-6xHis induced or uninduced for 2 h. Confocal images were processed in Fiji using the Despeckle filter and stacks were converted to sum projections. Scale bar = 1 µm. (K) Quantification of the fraction of cells exhibiting a focus of Bim1-GFP that tracks a dynamic microtubule plus end in uninduced or induced cells. Each dot represents a single experiment. Bars are mean ± 95% CI. (L) Example images of cells expressing the indicated + TIP and mRuby-Tub1 with TUB2-6xHis induced or uninduced for 2 h. Confocal images were processed in Fiji using the Despeckle filter and stacks were converted to sum projections. Scale bar = 1 µm. (M) Quantification of the fraction of cells exhibiting a focus of +TIP that tracks a dynamic microtubule plus end in uninduced or induced cells. Each dot represents a single experiment. Bars are mean ± 95% CI. Source data are available for this figure: SourceData F3.$

Super-stoichiometric β-tubulin creates aberrant tubulin assemblies. (A) Schematic for β-tubulin induction experiment. (B) Representative Western blot of β- and α-tubulin during TUB2-6xHIS induction. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (C) Quantification of TUB2-6xHis induction for both α- and β-tubulin across overexpression. Dots represent mean ± SD from four independent induction experiments. (D) Quantification of cells forming colonies after TUB2-6xHis induction or empty vector control. Dots represent mean ± SD from four independent induction experiments. (E) Example images of wild-type cells overexpressing β-tubulin for 0 or 2 h and stained for α- or β-tubulin. Arrows indicate astral microtubules; arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are maximum intensity projections of in focus z positions. (F) Quantification of astral microtubules (aMTs) across induction time for cells stained for either α- or β-tubulin. Dots represent mean ± 95% CI from three independent experiments with at least 100 cells in each experiment. (G) Quantification of tubulin assemblies across induction time for cells stained for either α- and β-tubulin. Dots represent mean ± 95% CI from three independent experiments with at least 100 cells in each experiment. (H) Example images of cells expressing Spc110-tdTomato and GFP-Tub1. TUB2-6xHis was induced for 3 h and then cells were shifted to 4°C for 2 h to depolymerize microtubules. Uninduced control cells are also shown. Arrows indicate microtubules; arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are maximum intensity projections of z series. (I) Quantification of the fraction of astral microtubules or tubulin assemblies in induced or uninduced cells after indicated time at 4°C. Dots are mean ± 95% CI from at least 100 cells in three independent experiments. (J) Example images of cells expressing Bim1-3GFP and mRuby-Tub1 with TUB2-6xHis induced or uninduced for 2 h. Confocal images were processed in Fiji using the Despeckle filter and stacks were converted to sum projections. Scale bar = 1 µm. (K) Quantification of the fraction of cells exhibiting a focus of Bim1-GFP that tracks a dynamic microtubule plus end in uninduced or induced cells. Each dot represents a single experiment. Bars are mean ± 95% CI. (L) Example images of cells expressing the indicated + TIP and mRuby-Tub1 with TUB2-6xHis induced or uninduced for 2 h. Confocal images were processed in Fiji using the Despeckle filter and stacks were converted to sum projections. Scale bar = 1 µm. (M) Quantification of the fraction of cells exhibiting a focus of +TIP that tracks a dynamic microtubule plus end in uninduced or induced cells. Each dot represents a single experiment. Bars are mean ± 95% CI. Source data are available for this figure: SourceData F3.