Figure 3. SSNA1 forms stretches on growing microtubule ends.
(A) A representative field of view of microtubules at 10 min post-SSNA1 wash-in. Microtubule extensions were pre-grown with 15 µM 647-tubulin and then 15 µM tubulin and 2.5 µM 488-SSNA1 (15% labeled) were introduced into the channel. (B) A kymograph of a microtubule from a wash-in experiment. The solid orange line indicates the time of SSNA1 introduction. The dashed vertical lines mark the boundary between the pre-existing lattice and the new lattice. (C) Quantification of the mean SSNA1 fluorescence intensity on the GMPCPP-stabilized microtubule seeds and pre-existing GDP microtubule lattices. A total of 17 microtubules were analyzed. Statistical significance was determined by Welch’s t-test. (D) Linescans showing the normalized fluorescence intensities of the microtubule seed (red), dynamic microtubule extension (magenta), and SSNA1 (cyan). The two dashed vertical lines mark the boundary between the pre-existing and new microtubule lattice, as indicated on the kymograph in (B). (E) An example kymograph showing stretches of SSNA1 forming at both microtubule ends. (F) Quantification of the SSNA1 fluorescence intensity toward and away from the direction of microtubule growth. The vertical dotted line indicates the position on the lattice at which the SSNA1 stretch initiated. N=16 SSNA1 stretches were analyzed. (G) Representative images of curved microtubules that were grown with 9 µM tubulin and 5 µM 488-labeled SSNA1 (7% labeled) for 60 min. The orange box indicates the zoomed region shown in inset. (H) Plots of curved microtubule extensions of microtubules grown in the conditions described for (G). A total of 17 curls were analyzed. (I) Corresponding quantification of SSNA1 intensity as a function of microtubule curvature on dynamic microtubules. Individual data points are in gray and the means and SD of binned data (0.1 µm–1 bin width) are in black. The solid line is a linear fit to the means and the slope is not significantly different from zero (slope= 0.04 [95% CI: –0.05 to 0.13] a.u. × µm, p-value=0.4). (J) Taxol-stabilized microtubules were incubated with 5 µM 488-SSNA1 and imaged for 60 min. Sum projection images of a representative field of view are shown. (K) Plots of curved Taxol-stabilized microtubules. A total of 59 microtubules were analyzed. (L) Corresponding quantification of SSNA1 intensity as a function of microtubule curvature on Taxol-stabilized microtubules. Individual data points are in gray and the means and SD of binned data (0.1 µm–1 bin width) are in black. The solid line is a linear fit to the means and the slope is not significantly different from zero (slope = −0.006 [95% CI: –0.118 to 0.105] a.u. × µm, p-value=0.9).
Figure 3—figure supplement 1. SSNA1 stretches can resolve and serve as stable rescue sites.
