Tubulin Dimers Oligomerize before Their Incorporation into Microtubules
Figure 4
Fluorescence correlation spectroscopy analysis.
(A) Cross-correlation functions of the mix of free fluorophores (solid blue curve) and of the mix of differently labeled tubulins in the presence of GMPCPP (solid red curve), GDP (short-dashed red curve), and GTP (long-dashed curve) normalized as described in Supplementary Material S1. For the tubulin mixes, we obtained amplitudes of ratioG = 0.33 (GMPCPP), 0.22 (GDP), and 0.20 (GTP; see Eq. S4) well above the baseline value of 0.09. (B) Measured and normalized autocorrelation functions of free Cy5 fluorophore (solid blue curve) and Cy5-labeled tubulin at 25 nM (dashed red curve) and 500 nM (solid red curve). For tubulin at 500 nM, a diffusion coefficient of Dtubulin = 23.4±2.2 µm2s−1 was found. This is 2-fold smaller than for the bona fide free tubulin dimers measured at 25 nM due to oligormer formation.