Figure 2.

Force-induced exposure of cryptic cysteines on FnIII₇ and FnIII₁₅ evaluated with iodoacetamide and bis-ANS binding to Fn fibers physisorbed to silicone sheets. (a) Fibers were pulled from a drop of wild-type Fn (blue) and deposited onto a silicone sheet that was mounted into a uniaxial stretch device.(14) As a negative control, a drop of alkylated Fn (red) randomly labeled with Alexa 633 on lysines was positioned at the opposite side, and fibers were pulled and deposited in between fibers of alkylated Fn. (b) Differential interference contrast image of prepared fiber sample after it had been washed and stretched in BPS buffer containing 2% BSA. (c) Fluorescence emission of Alexa 633 conjugated to 5% of the fibrillar Fn molecules. (d) Alexa 488 maleimide binding to stretch-exposed cryptic binding sites. After stretching, 5 ng of Alexa 488 maleimide were added to the solution and allowed to react for 15 min. (e−i) Differentially strained Fn fibers that were deposited under various angles to the strain axis. After deposition, all exposed hydrophobic patches on these fibers were blocked with iodoacetamide. Data analysis was performed by calculating the Alexa 488 to Fn-633 ratios. (j−k) Stretch-dependent binding of bis-ANS to Fn fibers. Fn fibers containing 5% Alexa Fluor 633-labeled Fn (Fn-633) were manually deposited onto prestretched silicon sheets in various orientations and subjected to a 0 and a 300% absolute strain, as indicated. Bis-ANS was then adsorbed for 45 min to the substrates, and the ratio of bis-ANS over the Fn-633 intensity was calculated for each pixel ((j) inset histogram). The intensity ratio was converted into a false shade of blues, where lighter colors indicated a greater degree of bis-ANS binding, and was displayed in the spatially resolved representative image shown. (k) The intensity ratio of the bix-ANS over the Fn-633 fluorescence, computed pixel-by-pixel, for 48 fibers oriented in various directions versus their absolute strain. Scale bar in (b) is 100 μm.