Quantitative mapping of averaged focal adhesion dynamics in migrating cells by shape normalization

Abstract

SummaryThe spatially ordered formation and disassembly of focal adhesions is a basic requirement for effective cell locomotion. Because focal adhesions couple the contractile actin-myosin network to the substrate, their distribution determines the pattern of traction forces propelling the cell in a certain direction. In the present study, we quantitatively analyzed the spatial patterning of cell-substrate adhesion in migrating cells by mapping averaged focal adhesion growth dynamics to a standardized cell coordinate system. These maps revealed distinct zones of focal adhesion assembly, disassembly and stability and were strongly interrelated with corresponding actin flow and traction force patterns. Moreover, the mapping technique enables precise detection of even minute responses of adhesion dynamics upon targeted signaling perturbations. For example, the partial inhibition of vinculin phosphorylation was followed by the reduced number of newly formed adhesions, whereas growth dynamics of existing adhesions remained unaffected.

Role of Molecular Determinants of Store-operated Ca2+ Entry (Orai1, Phospholipase A2 Group 6, and STIM1) in Focal Adhesion Formation and Cell Migration

“…To look deeper into the process of FA formation, we analyzed the age of FAs in polarized cells (Fig. 9), which was assessed by the level of tyrosine phosphorylation of Vinculin at position 1065, which is known to be high in newly formed FAs and decreases with their age (46). Consistent with all our other data, cells deficient in Orai1 turned out to have FAs significantly older (with about 32% less Vinculin phosphorylation) than control cells, which further supports the idea of Orai1 involvement in assembly rate and formation of the new FAs.…”

“…Newly formed FAs are known to exhibit a relatively high level of tyrosine phosphorylation that decreases upon FA maturation (45). It was shown recently that phosphorylation of Vinculin correlates with FA assembly rate at the leading edge of migrating cells (46). To verify that the observed reduction in FA assembly rate correlates with changes in Vinculin phosphorylation at position 1065, cells were transfected with scrambled RNA, siRNA against Orai1, STIM1, or PLA2g6, fixed and stained for total Vinculin (Vin, red) and phosphorylated Vinculin (pVin, green), and pVin to Vin ratio (pVin/Vin) was calculated as a direct indicator for the age of FAs (Fig.…”

Modulation of Glycosaminoglycans Affects PrPScMetabolism but Does Not Block PrPScUptake

“…Here, only cells detected as positive for PrP Sc uptake, defined by an intensity threshold, were selected for image analysis. To analyze HS and PrP Sc spatial distributions within the cell, we adapted a method for averaging of cell images, as previously described (49). In brief, the orientation of each cell was defined by the angle where the spatial spread of the fluorescence signal was maximal.…”

Measuring the regulation of keratin filament network dynamics

“…Cell shape normalization (optional). The cell shape in each frame was transformed into an idealized circular unit cell (39). First, the cell contour was determined by thresholding a smoothed version of the original image.…”