Figure 4

Talin A gradients correlate with front-rear gradients in blebbing activity. (a1) Dictyostelium cells chemotaxing under 0.7% agarose, with TalA-mNeon primarily enriched in the cell rear. (a2) Corresponding cell contours for 30 time points recorded at 2 frames per second, colour scaled according to fluorescent intensity normalised for each cell (blue: low, red: high). (b1,b2) Graded distribution of TalA-mNeon observed under 2% agarose. Bars: 5 microns. (c1,c2) Combined fluorescence profiles for the 3 time series shown in (a2,b2) plotted against angular position around the cell contour, with 0° defined as the cell front. TalA-mNeon fluorescence increases exponentially from cell front to rear (0.7% agarose, R² = 0.83, 2% agarose, R² = 0.77). (d–e) In the model we translate observed TalA-mNeon distributions into gradients in membrane to cortex linkage strength, along the polarization axis of the cells. We use a maximum of 60% increase in linker strength between the very front and rear of the cells, as measured in micropipette aspiration assays¹⁹. (f) Polar histograms of the angular distribution of experimental blebs, for the cell contours shown in figures (a2 and b2) demonstrate that blebs are indeed directed towards the cell front. (2% agarose data, blue; 0.7% agarose data, red.) (g) Histograms for the observed bleb frequency against normalised TalA-mNeon fluorescence clearly show that blebs do not nucleate in the regions of highest TalA-mNeon. (2% agarose rank correlation coefficient, −0.80, 0.7% agarose rank correlation coefficient, −0.85). (h) Example contours with automatically detected bleb sites indicated by (‘b1’) colour scaled according to fluorescent intensity normalised for each cell (top, 0.7% agarose cell, bottom, 2% agarose cell).