Figure 5. Colocalization of NMIIA and NMIIB in lamellae is greater at early stage of contractile system assembly than in steady-state conditions.

(A) STED microscopy of cells in indicated conditions stained with NMIIA and NMIIB antibodies. White outlines indicate cell boundaries based on phalloidin staining (see Fig. S3). Yellow outlines are examples of regions used for determination of correlation coefficients in lamellipodia (Lp), lamellae (La) and cell bodies (Body). Bar, 10 µm.

(B) Square regions from lamellae and cell bodies of each cell (yellow boxes in A) are zoomed as separate channels (NMIIA and NMIIB) and merged images (NMIIA+NMIIB). Correlation coefficient for NMIIA and NMIIB immunofluorescence (r) is indicated for each example on merged panels. “Overlap” panels show pixels exhibiting colocalization (white) of NMIIA and NMII signals that were thresholded to discard the lowest 5% intensity levels.

(C) Average correlation coefficients for NMIIA and NMIIB immunofluorescence in lamellipodia, lamellae, and cell bodies for the same experimental conditions, as in A. Top and bottom of a box indicate 75th and 25th quartiles, respectively; whiskers encompass all data; dot is the mean; and the middle line is the median. Asterisks indicate statistical significance (**, p<0.0001; *, p<0.01, unpaired Student’s t-test); the number of analyzed regions (N) from 11–14 cells for each condition is indicated on the plot. Bar, 2 µm.

See also Figure S3.