a) When depleted of ATP, myosin is in a rigor state. The FLNa within the network
is not stressed and PA-GFP β7 integrin fluorescence decays with a
characteristic time constant k(s) of 1.6 seconds (blue). After
caged ATP is released myosin reactivates, straining FLNa crosslinks. The decay
time constant increases to 2.5 seconds as the integrin dissociates more slowly
from FLNa under stress. b) PA-mCherry alone as a control shows no significant
difference in the unstrained or strained state. c) Fluorescence intensity in
time of PA-GFP FilGAP after photoactivation. In the ATP depleted state
FilGAP’s fluorescence decay time k is 1.5 s, and after
releasing the caged ATP (red) k decreases to 0.9 s. PA-GFP
V734Y FilGAP, a non-FLNa binding mutant as a control, shows no significant
difference in the decay times of unstrained (0.7 s) or strained state (0.8 s)
(n=20).