Fig. 4. Nucleation and branching of microtubules mediated by CrSSNA1 under various conditions.

(A) Aster-like formation of microtubules (20% HiLyte488 tubulin) occurs within 3 min after mixing tubulin with lower concentration (200 nM) of SSNA1 (upper) under conditions mimicking molecular crowding (7.5% PEG, typically used as a crowding agent), where tubulin alone does not form any polymers. Microtubules propagate out from tubulin concentrate, serving as a nucleation center. These experiments were performed three independent times with similar results. (B) 200 nM SSNA1 and 8 μM tubulin self-associate forming clusters in the presence of PEG with the concentration >~5%. (C) SSNA1 antibody recognizes the microtubule nucleation center. (D) Plot of the percentage of the concentrates growing into asters with microtubules as a function of time (min) in the presence of 50, 100 and 200 nM CrSSNA1. Error bars are mean +/- SD from n=3 independent experiments. As little as 50 nM of CrSSNA1 is sufficient to observe aster formation in the presence of 7.5% PEG. (E) Counts of microtubules observed per field of view, in the presence of different concentrations of CrSSNA1. Box plots cover 50% of the entire population (boxes) and 90% (whiskers) with median values indicated as lines within the boxes. Sample Size: 0 nM: n=42 fields of view, 50nM: n=29, 100nM: n=30, 200nM: n=25. Data were pooled from 3 independent experiments except for the first point (0 nM) for which data were pooled from 4 independent experiments. (F) Green colored dynamic microtubules on red-microtubule GMPCPP seeds in the presence of higher concentration of SSNA1 (30 μM) without molecular crowding agents to achieve globally concentrated conditions. ‘branch-like’ nucleation is observed. Branches were categorized as ‘splitting’, ‘end-joining’, ‘side-branching’ or ‘dynamic branching’ and their ratio (n = 895 branches, mean +/- S.D. pooled from 3 independent experiments) are shown in (G). “?” shows the bundled microtubules, making it difficult to categorize. “X” shows microtubules without branching. Branch-like nucleation can be seen from the locally concentrated SSNA1, condition described in (A-E), however observations of individual microtubules are challenging due to the high local protein concentrations. (H) A negative-stain EM image of SSNA1-mediated branched microtubules in the presence of 200 nM SSNA1 and 7.5% PEG, representative of three independent experiments. See supplementary table 3 for source data.