Fig. 2: 17β-estradiol preferentially binds to free tubulin heterodimers and this complex is incorporated into the growing microtubule tip.

A Schematic of the TIRF microscopy-based binding assay in which anti-estradiol antibody is bound to the coverslip, and then a mixture of Alexa-488-labeled tubulin either without estradiol (left) or with estradiol (right) is exposed to the coverslip. Coverslip binding of tubulin is then assessed. B Top: Raw data showing fluorescent tubulin (Alexa-488, green) bound to the coverslip in control (left) and estradiol (right) conditions. Bottom: Binding areas automatically detected by MATLAB script (white) from above raw images in control (left) and estradiol (right) conditions. C Top: Raw data showing polymerized microtubules (Alexa-488, green) bound to the coverslip in control (left) and estradiol (right) conditions. Bottom: Binding areas automatically detected by MATLAB script (white) from above raw images in control (left) and estradiol (right) conditions. D Left: Quantification of the binding area detected for fluorescent tubulin in the presence of increasing estradiol concentrations (n = 120 in each experiment, N ≥ 3 experiments (error bars: mean ± SEM)). Right: Quantification of the binding area detected for fluorescent microtubules in the presence of increasing estradiol concentrations (n = 120 in each experiment, N ≥ 3 experiments (error bars: mean ± SEM)). E Schematic of estradiol-GLOW cell-free TIRF microscopy experiment in which estradiol-GLOW (red) bound to Alexa-488-labeled tubulin dimers (green) could be visualized when polymerized from stable seeds (blue). F Left: Typical TIRF microscopy images of stabilized microtubule seeds (blue), growing microtubule tips (green), and estradiol-GLOW (red). Right: Quantification of the area fraction of estradiol-GLOW bound to the growing microtubule tip (green), and to the stabilized seed (blue), for each growing microtubule. G Confocal microscopy time-series of E2-GLOW (red) associated with a paused microtubule tip (green) in LLC-Pk1 cells (cyan arrows). Shown images are band-pass filtered to reduce noise. Ultimately, the estradiol-GLOW is incorporated into the microtubule lattice (yellow arrows). See also Supplementary Movie 4.