Measuring Microtubule Expansion Dynamics

Microtubule immobilization was carried out similar to the binding assay (see the Binding assay section) but slightly modified to fit the expansion assay. After GMPCPP-MT was added, 28 μM Cy5-labeled tubulin (10% labeled) was used for a 15-min polymerization period to lengthen and brighten the microtubules. Subsequently, 8 μM Alexa Fluor 488–labeled tubulin (2% labeled) with 1 mM GMPCPP was loaded into the chamber and polymerized for 20 min. Polymerization was terminated by washing free tubulin out with washout buffer. Images were acquired before and after 4 μM D2-mCherry in imaging buffer was added to the chamber followed by the dissociation of D2 by 1-min incubation in high-salt buffer and washout by imaging buffer for 30 s.
To calculate expansion, a background correction was performed in the same way as the data analysis for the binding assay (see the Data analysis of binding assays section). Microtubule lengths were measured using napari-filaments as follows (Fig S5): first, the Cy5 channel and Alexa Fluor 488 channel of the image were added with 1:1 weight to create a total intensity image. This image was used for 2D spline fitting as mentioned in the data analysis of the binding assay (see the Data analysis of binding assays section). Next, the spline curves were clipped at the Cy5-labeled microtubule edges by fitting to the error function: $\begin{array}{l}f\left(x\right)=\frac{a-b}{2}\left(1+erf\left(\frac{x-x_0}{\sqrt{2}\sigma }\right)\right)+b \\ \mathrm{where}: erf\left(x\right)=\frac{2}{\sqrt{\pi }}{\int }_0^x{e}^{-t^2}dt\end{array}$
The parameters of the error function indicate that intensity increases from $b$ to $a$ , where the inflection point of the intensity change is $x_0$ . Cy5-labeled microtubule lengths were obtained by fragmenting the spline curve into 1,024 pieces and summing the lengths of all fragments.