Figure 3. Reversibility of switching the FRET signature when relaxing Fn ﬁbers and the associated release of nonspeciﬁcally bound proteins. (a) Fn ﬁbers were manually deposited in parallel orientations onto prestrained silicone substrates and incubated with Alexa 633-labeled BSA or nothing as a control. The substrates were imaged for FRET as well as the relative BSA-633 intensity of the bound proteins before and after the indicated absolute strains factor values (b). After imaging at 5, 10, 20, and 30 min, the samples were restretched to their original conformation and again imaged. Each data point represents (a) the mean ( standard deviation of the average acceptor intensity divided by donor intensity (IA/ID) per pixel, or (b) the mean ( standard deviation of the normalized average BSA-633 intensity of all pixels that contained Fn to distinguish between protein binding to the background silicone substrate. Fn to stretched Fn ﬁbers could be increasingly mediated by not just speciﬁc but also nonspeciﬁc interactions. Physiological Implications. Since force-dependent cell signaling pathways regulate various cell functions13,17,47,49 me- chanical forces provide living systems with an additional dimension of functional regulation. While the stretching of proteins might allow cells to alter the structure-function relationship of proteins in very speciﬁc manners,4-7,12,13,48,49 stretch-induced unfolding also comes with a price. As shown here for Fn ﬁbers, protein stretching unravels secondary structure, leading to the exposure of cryptic and hydrophobic residues (Figures 1, 2, 4 and Supporting Information, Figure S1). Coping with the exposure of fouling hydrophobic patches upon stretch, which might lead to adverse down- Nano Lett., Vol. 9, No. 12, 2009 stream consequences in a physiological environment, such as the nonspeciﬁc adsorption of other proteins, requires their immediate functional rescue. Here, we show that albumin and casein, if present at physiological concentrations, can serve in this role as they rapidly block newly exposed hydrophobic sites (Figure 1). Our data might thus provide a previously unrecognized physiological explanation for why such high concentrations of these proteins are found in body ﬂuids. Since cells go through rapid cycles of contraction and spreading,49 each of which will stretch and release ECM ﬁbers, how fast can ECM ﬁbers recover their fully relaxed length when released? We recently showed that also freely suspended stretched Fn ﬁbers could fully relax and refold within minutes in buffer solution (Klotzsch et al., submit- ted), but no previous information was available that demonstrated whether stretched Fn ﬁbers could relax in the presence of serum proteins. Here, we show that stretched Fn ﬁbers deposited on silicone sheets have contracted within minutes (Figure 3a). Fiber relaxation appears to be possible through the release of nonspeciﬁcally bound proteins, as experimentally demonstrated by labeling albumin with a third color (Figure 3b). While demonstrated here for ECM ﬁbers, many other force-bearing proteins exist in tissues and include all of the proteins that physically link the cell exterior to the interior.48 Since force-induced protein unfolding has been demonstrated for intracellular proteins as well (for review, see refs 12, 13, and 48), we would like to propose that intra- and extracellular molecular chaperones might have dual functions. They might not just recognize misfolded or thermally denatured proteins, but one of their key functions could be to rapidly block and thereby pacify stretch-induced hydrophobic residues of extra- and intracellular proteins. In support of this hypothesis, it was shown already that mechanical stress induces the expression of heat shock proteins.50 In summary, deciphering the mechanisms by which proteins can be utilized as mechanochemical signal converters is of fundamental importance to many areas of research. While the molecular design principles by which force can activate or eliminate protein binding are slowly emerging for a variety of proteins (as reviewed in ref 48), we illustrate here that molecular chaperones might play an essential role in preventing unwanted hydropho- bic-hydrophobic interactions caused by mechanically stretched and unfolded proteins in physiological environ- ments. Thus, molecular chaperones might play an ad- ditional and not yet recognized role: our data suggest that they are essential and enabling players in mechanotrans- duction processes where proteins are employed by cells as molecular switches that convert mechanical into biochemical signals via force-induced unfolding processes. To be effective, mechanical chaperones must have a high relative concentration, a kon fast enough to compete effectively with other proteins, and the koff has to be fast enough to not inhibit refolding once the tension is released. 4163