The relationship between molecular size (and shape) and in vivo diffusion of free proteins. (A) Passively diffusing reporter macromolecules used in this study, showing their molecular mass, in vitro diffusion constants (D) measured using DLS, and sedimentation coefficients (S) measured from sucrose gradient centrifugation. (B) Measured diffusion constant (D) in solution plotted against molecular mass and sedimentation coefficients (inset), ±SEM. (C) Images of representative single cell’s FRAP experiments (bottom) of six different-sized substrates in WT yeast cells. Bars, 1 µm. The nucleus, cytoplasm, and vacuole of the top cell is indicated. Nuclear fluorescence is plotted after normalizing each exponential fit between 2 (at t = 0) and 1 (at t = ∞; top). (D) Diffusion curves from all such measurements of WT cells for the different-sized reporters, normalized as in C. (E) Population distributions of permeability coefficients for the different reporters as a function of their size. Each dot indicates the computed permeability coefficient for a single cell.