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. 2020 Aug 3;219(9):e202003041. doi: 10.1083/jcb.202003041

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© 2020 Black et al.

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Figure 9. — Experimental sptPALM reveals bimodal dynamics of G1/S TFs. (A) Experimental individual molecule trajectories of Swi6-mEos3.2 in the nucleus of a live cell grown in SC + 2% glucose medium. Each individual trajectory is represented by a different color. (B) Histogram of the log of apparent diffusion coefficients, D_app, calculated from all individual trajectories of Swi6-mEos3.2 (red) for all cells in three FOV (∼5,000 trajectories per FOV). The distribution was fit to two Gaussians. The distribution for Swi6 Log D_app obtained from spt tracking in fixed cells (light gray, immobile, except for instrument jitter) and the distribution of Log D_app of mEos3.2-Gdh2 (glutamate dehydrogenase; also light gray) diffusing freely in the cytoplasm are plotted for comparison and indicated by the arrows. (C) Plots of the average mean squared displacements for all trajectories of mEos3.2-tagged Swi4-mEos3.2, Mbp1-mEos3.2, and Swi6-mEos3.2 are shown for SC + 2% glucose medium. Two classes of motion are apparent in all cases, one fast (green) and one slow (blue). (D and E) Distributions of diffusion coefficients from two component analysis of complete individual molecule trajectories for mEos3.2-labeled Swi4-mEos3.2, Mbp1-mEos3.2, and Swi6-mEos3.2 (left, middle, and right) in SC + 2% glucose (D) and SC + 2% glycerol (E) medium. A decrease in the fraction of the fast component is apparent for Mbp1 and Swi6 in the poor carbon source. The average reduced normalized root sum square (RSS) value for a two Gaussian distribution fitting model was 6.4, whereas a single distribution model yielded a reduced RSS value of 27.8, demonstrating that the dynamics involved have at least two modes of diffusion.