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

Figure 6. SSNA1 protects microtubules against severing by spastin.

(A) Representative images of a mixed population of Taxol-stabilized microtubules that were either coated or not coated with 647-SSNA1 and treated with 25 nM GFP-spastin in the presence of 1 mM AMPPNP. (B) Quantification of the SSNA1 fluorescence intensities on SSNA1-coated (N=79) and non-coated (N=72) microtubules, p<0.001, unpaired t-test. (C) Quantification of the spastin fluorescence intensities on SSNA1-coated (N=79) and non-coated (N=72) microtubules, p=0.4, unpaired t-test. The colors in (B) and (C) represent different experimental repeats, the larger markers are mean ± SD for each experimental day. X marks the overall mean. (D) Representative images of a mixed population of microtubules (red) that were either coated or not coated with 2 µM 647-SSNA1 (cyan) prior to the introduction of 25 nM GFP-spastin (blue). The images are from 0 min, 2 min, and 4 min post-addition of spastin and 1 mM ATP. The cyan arrows indicate microtubules that were coated with SSNA1 and the blue asterisks were non-coated. (E) Representative kymographs of a non-coated (left) and SSNA1-coated (right) microtubules. (F) Overlay of the tubulin signal for the field of view in (D) at 0 min (green) versus 4 min (magenta), showing the loss of non-SSNA1-coated microtubules. (G) An example trace of tubulin fluorescence intensity over time for non-coated (blue) and SSNA1-coated (gray) microtubules in one field of view. Error bars represent SE. (H) Tubulin intensity for a time point where control (non-coated) microtubules reach an average of 20% of initial intensity. p<0.001, unpaired t-test. The colors represent different experimental repeats, the larger markers are mean ± SE for each experimental day. X marks the overall mean. N=66 microtubules per condition. Data are from three independent experimental repeats.

Figure 6—source data 1. An Excel sheet containing numerical data for the quantification of the SSNA1, GFP-spastin and tubulin fluorescence intensities as presented in Figure 6.

Figure 6.

Figure 6—figure supplement 1. SSNA1 protects microtubules against full-length human spastin-mediated severing.

Figure 6—figure supplement 1.

Representative images of control (left) and SSNA1-coated (right) microtubules incubated with 200 nM full-length human 6His-MBP-spastin. Microtubules were pre-incubated with either SSNA1 storage buffer (control) or 1 µM 488-SSNA1 for 10 min prior to the addition of spastin. Spastin was introduced at t=0 min.