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

Figure 1. Human SSNA1 promotes microtubule nucleation and suppresses microtubule dynamicity.

(A) Representative images of a templated microtubule nucleation assay in which microtubule extensions (gray) were nucleated from GMPCPP-stabilized seeds (red) in the presence and absence of SSNA1. Images shown are for the 6 µM tubulin condition with and without 2.5 µM 488-SSNA1 at 15 min after the introduction of the nucleation reaction. (B) Quantification of the fraction of seeds that nucleated in 15 min with tubulin alone (control, light gray) and 2.5 µM 488-SSNA1 (dark gray) as a function of tubulin concentration. Data are individual experimental replicates ±SE from six experimental days (N=30–68 microtubules for each concentration tested in the tubulin-alone control condition, N=33–77 microtubules for each concentration tested in the SSNA1 condition). The data were fitted to a sigmoidal curve of the form y(x)=xs/(C+xs) (solid lines). For tubulin alone, C=5.9 µM (95% CI: 5.5–6.2) and s=6.0 (95% CI: 3.4–8.6). For the SSNA1 condition, C=5.0 µM (95% CI: 4.7–5.3) and s=8.0 (95% CI: 4.3–11.9). (C) Representative kymographs of microtubules grown from GMPCPP-stabilized seeds with 9 µM Alexa-647 tubulin alone (control) and in the presence of 500 nM and 3 µM SSNA1. The microtubule plus ends are shown on the right and the minus ends are shown on the left. (D) Quantification of microtubule dynamicity as a function of the SSNA1 concentration. Dynamicity is calculated as the total length of growth and shrinkage over the observation time. (E) Quantification of the microtubule catastrophe frequency at the plus and minus ends of microtubules grown with 9 µM tubulin and concentrations of SSNA1 from 0 µM to 1 µM. (F) Quantification of the microtubule shrinkage rate at the plus and minus ends of microtubules grown with 9 µM tubulin and concentrations of SSNA1 from 0 µM to 1 µM. For the quantifications in panels (D–F), data are weighted means ± SE obtained from four independent experimental days (N=43–485 growth events for each concentration tested at microtubule plus ends; N=30–120 growth events for each concentration tested at microtubule minus ends). Plus end data are in dark purple; minus end data are in light purple.

Figure 1—source data 1. An Excel sheet containing numerical data for the quantification of microtubule nucleation and microtubule dynamics presented in Figure 1 and Figure 1—figure supplement 2.

Figure 1.

Figure 1—figure supplement 1. Human SSNA1 protein purification.

Figure 1—figure supplement 1.

(A) SDS-page gel showing purified His-SSNA1 protein (left) and a Western blot of purified SSNA1 protein probed with anti-SSNA1 Rabbit Polyclonal Antibody (ProteinTech; cat# 11797-1-AP) (right). (B) Mass spectrometry analysis of His-SSNA1 purified from Escherichia coli cells. The hits with more than 10 total peptides are listed.
Figure 1—figure supplement 2. SSNA1 modulates all parameters of microtubule dynamics.

Figure 1—figure supplement 2.

Quantification of the growth rate, shrinkage rate, catastrophe frequency, and rescue per shrinkage length at the plus ends (dark purple) and minus (light purple) ends of microtubules grown with 9 µM tubulin and concentrations of SSNA1 from 0 µM to 3 µM. Data are weighted means ± SE obtained from four independent experimental days (N=43–485 growth events for each concentration tested at microtubule plus ends; N=30–120 growth events for each concentration tested at microtubule minus ends). Note that no catastrophes were detected at the minus ends with SSNA1 concentrations greater than 1 µM; therefore, the shrinkage rate and rescue per shrinkage length could not be measured in those conditions.