Protein accumulation in the nuclear/spindle region is a consequence of different molecular crowding states. (A) Cells with different diameters (λ) and different spatial positions of the FCS measurements were made comparable by expressing measurement positions relative to the cell size (red and orange bullets). Dashed circles indicate the nuclear space; gray-shaded objects symbolize condensed chromatin. (B) FCS analysis of GFP–α-tubulin, Megator-mCherry, and mRFP-Mad2 during mitosis after MT depolymerization with colchicine. Depicted are the mean or median values of the dwell time τD and the anomaly degree (α) of the diffusional motion within the focus in both the nuclear region (N) and cytoplasm (C). ***, P < 0.001. (C–E) FCS measurements were performed at 10 loci per cell (tubulin, 11 cells; Megator, 15 cells; Mad2, 15 cells). Position data were rescaled (nuclear region: λ = −0.2, . . . , +0.2). The number of fluorescent molecules (N) within the focus was relativized to the maximum number of fluorescent molecules (Nmax) detected in each cell to account for varying expression levels. (F and G) Modeling of protein diffusion into/out of the nuclear region. Diffusion of soluble tubulin (F) into and Mad2 (G) out of the nuclear region progresses within few seconds to a steady state, as indicated by the apparent fluorescence in the nuclear region Fn(t) and the cytoplasm Fc(t). Representative fluorescence images of the initial condition, an intermediate state, and the steady state are shown in the top panels. Dashed white lines indicate the respective line scans used for quantification of fluorescence (inset graphs).
