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. 2021 Dec 31;10:e67282. doi: 10.7554/eLife.67282

Figure 2. The progressive slowdown in microtubule growth correlates with SSNA1 accumulation on microtubule ends.

(A) A representative kymograph of a microtubule grown with 9 µM Alexa-647-labeled tubulin and 5 µM Alexa-488-labeled SSNA1 (7% labeled) showing the progressive localization of 488-SSNA1 to the microtubule end region over time. The yellow arrow indicates SSNA1 enrichment at the microtubule end at the onset of growth slowdown. The dotted orange lines demarcate the position of the microtubule seed (not shown). (B) Microtubule end positions as a function of time for microtubules grown with 9 µM tubulin and 5 µM Alexa-488-labeled SSNA1, color-coded with 2-min segment velocity. (C) Microtubule end velocities over a 2-min segment as a function of time calculated from the end positions in (B) and color-coded with velocity for consistency. Plots of microtubule end velocities and corresponding SSNA1 fluorescence intensities at microtubule ends (D) as a function of time and (E) as a function of 2-min segment velocity. A total of 26 microtubules were analyzed. For the plots in (D) and (E), the median for each bin is shown as a bright red point with horizontal line.

Figure 2—source data 1. An Excel sheet containing numerical data for the quantification of microtubule end position and velocity over time, and SSNA1 intensity as a function of both time and microtubule end velocity, as presented in Figure 2 and Figure 2—figure supplement 1.
elife-67282-fig2-data1.xlsx (447.4KB, xlsx)

Figure 2.

Figure 2—figure supplement 1. Microtubule growth rate slows down over time in the presence of SSNA1.

Figure 2—figure supplement 1.

(A) A representative kymograph of a microtubule grown with 9 µM tubulin and 3 µM SSNA1 showing that microtubule growth rate slows down over time; the position of the microtubule tip over time is represented by the overlaid track (white line). (B) Microtubule end positions as a function of time for microtubules grown with 9 µM tubulin and 3 µM SSNA1, color-coded with 2-min segment velocity. A total of 27 microtubules were analyzed. (C) Microtubule end velocity in a 2-min segment as a function of time calculated from the end positions in (B) and color-coded with velocity for consistency. For each bin, the median is shown as a bright red point with horizontal line.
Figure 2—figure supplement 2. Labeled SSNA1 forms fibrils.

Figure 2—figure supplement 2.

647-SSNA1 fibrils were grown by incubating 58 µM 647-His-SSNA1 at 35°C for 4 hr. The reaction was then flowed onto coverslips coated with anti-His-antibody and imaged by TIRF microscopy.