Figure 4. Conformational gradient in the GTP cap revealed by spatially resolved single EB3 dwell time distributions.

(a) TIRF microscopy kymograph of single mGFP-EB3 molecules (green) at 25 pM in the plus end region of an E254D microtubule (E254D MT) growing at 10 µM E254D tubulin (v_g = 58.5 nm/s) in the additional presence of 1 nM Alexa647-EB3 (magenta) (for end region visualization). (b) Dwell time distribution of single mGFP-EB3 molecules plotted as a survival function (1- CDF, cumulative density function). Number of microtubules analyzed - 150, number of mGFP-EB3 binding events - 1834. (c) Local dwell time distributions at distinct distances from the growing microtubule plus end. Dashed magenta lines are mono-exponential fits. The distance bins are 0–0.59, 1.18–1.77 and 3.84–5.02 µm; bin centers shown in the legend. Experimental data as in (b). (d) Local mean EB3 dwell times as a function of distance from the growing E254D microtubule end. Filled symbols correspond to the local mean dwell times calculated based on data in (c). The solid magenta line is a mono-exponential fit with a decay length of 850 nm. (e) TIRF microscopy kymograph of single mGFP-EB3 molecules (green) at 25 pM in the plus end region of a wildtype (WT) microtubule growing at 19 µM wildtype tubulin (v_g = 59.8 nm/s) in the presence of additional 1 nM Alexa647-EB3 (magenta). (f) mGFP-EB3 dwell time distribution. Number of microtubules analyzed - 548, number of mGFP-EB3 binding events - 2425. (g) Local mGFP-EB3 dwell time distributions. Dashed cyan lines are mono-exponential fits. Experimental data as in (f). (h) Local mean EB3 dwell times in the wildtype microtubule plus end region. Filled symbols correspond to the local mean dwell times calculated based on data in (g). The solid cyan line is a mono-exponential fit to the data with a decay length of 450 nm. (i) Schematic of high affinity EB3 binding to the GTP lattice of a GTPase-deficient microtubule. (j) Schematic of a growing end of a GTPase-competent microtubule displaying a gradually decreasing EB3 binding affinity (illustrated by a color gradient) as the lattice conformation changes as a consequence of GTP hydrolysis.

Figure 4—figure supplement 1. Flow chart of single molecule localization and spatially-resolved dwell time analysis.

For single molecule analysis of mGFP-EB3 binding to microtubules, TIRF microscopy movies were recorded in the presence of 25 pM mGFP-EB3 and additional 1 nM Alexa647-EB3 to visualize the growing microtubule end region. (a) Left: Single TIRF microscopy frames of a movie showing the Alexa647-EB3 channel. Right: Microtubule (MT) positions were marked by hand on a maximum-intensity projection of all frames. (b) Kymographs were generated for each marked microtubule in (a), and the growing plus-end position traced by hand in the kymographs (white line). (c) The x-y position of each microtubule end was calculated for each frame in the original movie (red crosses). The moving end position and the positions of the static microtubule projection from (a) were used to create a dynamic binary mask movie. (d) For each frame of the original movie, the mGFP-EB3 channel was analyzed using a Single Molecule Localization (SML) plugin, to determine the coordinates of each potential single molecule event (red circles). The mask from (c) was then used to exclude all events from outside the microtubules of interest. (e) Events were linked together in time and space using specific linking parameters. The resulting kymograph corresponding to (b) shows automatically detected and linked single molecule events, color-coded by duration. (f) For each linked event, the dwell time and initial distance from the nearest microtubule end were calculated. (g) Data from all movies were binned at specific distances from the growing microtubule end (braces in f), to give spatially-resolved dwell time survival functions (1- CDF, cumulative density function), or binned over all distances to give a total survival function. The example plots in (g) are the same as displayed on Figure 4b–c.

Figure 4—figure supplement 2. Spatially-resolved EB3 dwell time distributions at the ends of E254D and wildtype microtubules.

(a) Local dwell time survival functions at specific distance bins from the microtubule end for E254D microtubules. Dashed black lines are mono-exponential fits. The global reduced χ² value for all the fits is half that of a mono-exponential fit to the pooled data in Figure 4b. (b) Reconstructed comet for all single molecule events, from summing the complete emission of each binding event in each distance bin from the microtubule end. The total intensity is normalized to the number of microtubules analyzed. (c) and (d) depict the same information as (a) and (b) but for wildtype (WT) microtubule ends. Intensities in (b) and (d) can be compared. Good agreement of the reconstructed comets based on single molecule data with the measured comets at higher EB3 concentrations (Figure 3i and j) demonstrate equivalence of single molecule and ensemble analysis.

Figure 4—figure supplement 3. Wildtype and E254D microtubules grow with similar speeds under single molecule experiment conditions.

Quantification of microtubule growth speeds at 19 µM wildtype (WT) and 10 µM E254D tubulin concentrations in the presence of 1 nM Alexa647-EB3 and 25 pM mGFP-EB3. The boxes extend from 25^th to 75^th percentiles, the whiskers extend from 5^th to 95^th percentiles, and the mean value is plotted as a line in the middle of the box. The experimental data is the same as analysed and presented in Figure 4.