Figure 1.
Single Cell adhesion Force Spectroscopy assay.
A. Force was measured from bending of the cantilever and a typical force-separation curve is shown for an approach-retraction cycle highlighting the two assessed parameters: maximum adhesion force FMax.Adh. and the work of adhesion WAdh. (integral of the hatched area). I–IV refers to the four panels shown under B. B. Side view of a cycle of approach and retraction of a cell attached to a cantilever. Panels I and II: A cell can be seen hanging below the cantilever as it approaches the glass slide (light colored surface, Dictyostelium contour in red under I). Panels III and IV: The cell can be seen to remain on the cantilever as it is retracted and it maintains a rounded shape. Arrows highlight cell position. Scale bar is 20 µm.
Figure 2.
Decrease in adhesion during early development of Dictyostelium discoideum.
A. Microfluidic assay: the remaining fraction of cells after 40 minutes in chambers 2–8 is shown for cells that had developed for varying lengths of time. latB refers to 0 hr cells that were treated with 10 µM latrunculin B for 30 minutes to disrupt the actin cytoskeleton and assayed in the presence of the drug. Average of at least 5 independent experiments. The bars indicate the S.E.M. B. Single cell adhesion force assay of cells during early development. Box plot of the distribution of maximum adhesion forces FMax.Adh of individual single cells, where the bottom and the top of the box represents the first and the third quartiles, and the band corresponds to the median. Cells were developed for 0 h (n = 33), 3 h (n = 47), 6 hr (n = 31). Top whiskers are at 90% and bottom whiskers are at 10% of the distribution. Latrunculin B treated 0 hr cells (Lat) (n = 27) were assayed in the presence of 10 µM latrunculin B to disrupt the actin cytoskeleton. C. Work of adhesion WAdh. measured for the same cells. Nonparametric statistical hypothesis test Wilcoxon rank-sum test were used for significance.
Figure 3.
Developmental regulation of the decrease in adhesion.
A Wild type (AX3) and carA− cells lacking the cAMP receptor after 0 and 5 hr of development were analyzed with the microfluidic assay for the remaining fraction of cells after 40 minutes. B Work of adhesion (WAdh.) of wild type (AX3), cells lacking the cAMP receptor (carA−) or cells lacking talin (talA−) was measured after 0 hr (veg n = 36) and 6 hr (dev n = 30 for carA−, n = 34 for talA−) of development. Nonparametric statistical hypothesis test Wilcoxon rank-sum test was used for significance.
Figure 4.
Substratum adhesion in cells lacking sibA or sadA.
A Cell-substratum adhesion of wild type Ax3, sibA and sadA null cells at either 0 or 5 hr of development as measured by the microfluidic assay from the remaining fraction of cells after 40 minutes. B Work of adhesion WAdh. of wild type (AX3) and cells lacking either sibA or sadA was measured after 0 hr (n = 29 and 41) and 6 hr (n = 45 and 41) of development. Significance was judged from the Wilcoxon-rank-sum test.