Mechanical proofreading: A general mechanism to enhance the fidelity of information transfer between cells

The cells and receptors of the immune system are mechanically active. Single molecule force spectroscopy, traction force microscopy, and molecular tension probe measurements all point to the importance of piconewton (pN) molecular forces in immune function. For example, forces enhance the ability of a T cell to discriminate between nearly identical antigens. The role of molecular forces at these critical immune recognition junctions is puzzling because mechanical forces generally facilitate bond dissociation, potentially increasing the difficulty for a receptor to recognize its cognate antigen. The advantage molecular forces confer in the process of immune recognition is not clear. Why would cells expend energy to exert force on the critical, but tenuous bonds that mediate immune surveillance? Do molecular forces provide some advantage to the immune system? The premise of this review is that molecular forces provide a specificity advantage to immune cells. Inspired by the recent discovery that receptor forces regulate immune signaling in T cells and B cells, we dub this notion “mechanical proofreading,” akin to more classic kinetic proofreading models. During the process of mechanical proofreading, cells exert pN receptor forces on receptor-ligand interactions, deliberately increasing the energy cost of the immune recognition process in exchange for increased specificity of signaling. Here, we review the role of molecular forces in the immune system and suggest how these forces may facilitate mechanical proofreading to increase the specificity of the immune response.


Introduction
Immune cells must detect and respond to rare traces of malignancies or infection.Accordingly, immune response must display extraordinary sensitivity and specificity.The Further complicating the issue of T cell antigen recognition is the observation that T cells are mechanically active.The pMHC-TCR interaction forms only when a T cell physically touches a target cells; thus, it is likely that the TCR-pMHC complex experiences force.In support of this notion, soluble, monovalent pMHC can bind to the TCR but fails to activate T cells 12,13 , while pMHC attached to a planar lipid bilayer does activate T cells 14 .Collectively, these results suggest that mechanical forces may regulate TCR triggering.A seminal optical tweezer study by Reinherz, Lang, and colleagues demonstrated that T cells trigger in response to forces exerted on the TCR-pMHC complex, positioning the TCR as a mechanosensor 15 .Furthermore, the TCR is not a passive recipient of external forces.Our group pioneered the development of molecular probes to map pN forces applied by cells [16][17][18][19] .In particular, DNA-based probes revealed that the TCR transmits defined piconewton forces to the pMHC, and that these forces facilitate TCR antigen discrimination (Fig 1A -D) 20,21 .Traction force microscopy and micropillar measurements also demonstrate that T cells transmit forces through the TCR-pMHC interaction and through the CD3 complex 22,23 .Additionally, biomembrane force probe and optical tweezer measurements have revealed that TCR-pMHC bond lifetimes increase under force (catch bond behavior) for agonist but decrease under force for non-agonist pMHC [24][25][26][27] (Fig 1E, F).Additional studies have revealed roles for molecular forces in regulating the pore-forming capability of cytotoxic T cells 28 and mediating the antigen recognition by CD8 + T cells 20 .The many contexts in which receptor forces regulate immune processes have been reviewed elsewhere 29,30 .Many mechanisms to explain T cell antigen recognition have been proposed 2 , but vital questions remain unanswered.Why would a T cell expend energy to exert force on an already tenuous, low affinity interaction?How do molecular forces aid in immune recognition?
Receptor forces have many potential effects on receptor-ligand binding which have been excellently reviewed elsewhere 31 .Many effects of molecular forces might hinder antigen recognition (Fig 2).For example, receptor forces alter equilibrium by shifting the energy landscape of bound and unbound receptor-ligand pairs (Fig 2A).Cell-generated forces may therefore destabilize receptor-ligand complexes critical to immune recognition events, potentially dampening cellular activation.Additionally, as predicted by the Bell model, tensile forces on receptor ligand interactions typically increase the bond dissociation rate in a force-dependent manner 32 , a phenomenon termed a slip-bond (Fig 2B).The increased dissociation rate reduces the number of bound receptors and is expected to dampen signaling.Individual slip bonds do not provide a specificity benefit under constant forces for pure affinity-based discrimination.For example, if a cognate antigen displays a k off,cognate = 1s −1 while a noncognate antigen has k off,noncognate = 10s −1 then the binding error rate (assuming identical on rates) is approximately the ratio of the off rates, k off,cognate / k off,noncognate = 1/10.The Bell model (see equation, Fig 2B) dictates that force alters the off rate of both the noncognate and cognate antigen by the same factor of exp(FΔx/k B T), leaving the ratio k off,cognate and k off,noncognate unchanged when both antigens experience the same cellular force and assuming Δx is similar for both antigens.This simple analysis is valid for constant forces, but dynamic forces 33 will have more complex effects that are difficult to treat in the context of this review.In rare cases, forces increase the lifetime of receptor-ligand interactions under force, a phenomenon known as a catch bond 34,35 .Catch bonds could conceivably stabilize receptor-ligand recognition under force, facilitating signaling.Finally, pN forces alter protein energy landscapes, which may facilitate conformational shifts (Fig 2C).Force-induced conformational changes may lead to recruitment of accessory proteins to mechanically strained receptors, amplifying the signal of mechanically strained receptor-ligand complexes.Given the many possible effects of molecular forces, it is crucial to define the precise role of pN molecular forces in the immune system.
The purpose of this review is to suggest that pN molecular forces provide a benefit to receptor-ligand interactions by increasing the specificity of signaling.In effect, cells expend energy in the form of pN receptor forces in exchange for increased specificity.With kinetic proofreading as a hallmark example, literature precedent exists for biological systems expending energy to purchase specificity.DNA replication and protein synthesis both leverage kinetic proofreading, increasing fidelity by using far-from-equilibrium intermediate states driven by triphosphate hydrolysis.These irreversible intermediate steps enable discrimination based on the off rates of "correct" versus "incorrect" substrates 36 .In addition to kinetic proofreading, the serial engagement model has also been widely discussed as a mechanism to facilitate TCR antigen discrimination 37,38 .These models have been reviewed elsewhere 2,39 , and are not the focus of this review.Instead, we focus on how molecular forces open many new possibilities to explain the specificity of immune recognition events.
Here, we review evidence for the importance of molecular forces in the immune system.Inspired by the work of Tolar 40 , Zhu 24 , Reinherz and Lang 15,27 , and by our recent work 20 , we interpret how forces might enable a cell to expend mechanical work to increase signaling accuracy.In an analogy to kinetic proofreading, we term the concept that cells harness mechanical work to increase the specificity of signaling "mechanical proofreading."

Mechanisms of mechanical proofreading
We highlight three potential mechanisms for mechanical proofreading.First, bond lifetime may be altered by molecular forces to facilitate mechanical proofreading.Most biological bonds decrease in duration under force (slip bonds), but a subset of bonds exhibit increased lifetime under force, a phenomenon known as catch bonds 34,35 .The change in bond lifetime due to molecular forces may provide a potent means of mechanical proofreading (Fig. 2B, Fig. 3A).Through the alteration of bond lifetime, mechanical proofreading may enable immune receptors to select for the rare ligands that produce catch bond behavior under force, increasing the fidelity of signaling.Second, mechanical proofreading could occur via a "stress test."Stress is defined as force per unit area.Analogously, cells may apply forces too great for a single receptor-ligand interaction to withstand.Only clusters of proteins sharing cellular forces among many bonds reduce the applied stress below a threshold level, enabling longer, more stable bonds (Fig. 3B).The mechanical proofreading stress test selects for multivalent interactions and does not require catch bonds to function.A third mechanism of mechanical proofreading is a "strain test."Strain is defined as the change in length of a material due to mechanical stress divided by the original length of the material.Analogously, if a receptor-ligand bond can withstand forces applied to it, the force may produce a conformational change (loosely analogous to strain) in one or both binding partners.This conformational change could result in adaptor protein recruitment, or exposure of cryptic binding sites, producing downstream signaling (Fig. 3C).The mechanical proofreading strain test selects for, and subsequently amplifies, the signal from mechanically stable individual receptor-ligand bonds that can survive the mechanical forces..In special cases, bond lifetime increases under force, a phenomenon known as a catch bond 34 .AFM measurements provided the first direct evidence for a catch bond between P-selectin and P-selectin glycoprotein ligand-1 35 .Since this initial observation, catch bonds have been demonstrated in selectins, integrins, the platelet glycoprotein GPIbα, E-cadherin, and recently in the TCR-pMHC interaction 24,35,[41][42][43] .
Qualitatively, catch bonds provide a means to spend mechanical energy (from cyotoskeletal and motor protein forces) in exchange for specificity.Most molecular bonds exhibit slip bond behavior; therefore, if a cell expends mechanical energy via applying force to a receptor-ligand bond, and bond lifetime increases, the interaction is very likely to be "correct."This enhancement of bond lifetime under force may amplify the downstream effects of cognate ligand binding events relative to the shorter bond lifetime (under force) of non-cognate ligands, through the classic kinetic proof reading model.Catch bonds thus complement the kinetic proofreading model and offer a potent means for mechanical proofreading.
T cells are mechnaosensitive, transducing TCR forces into biochemical signals, such as calcium flux 15 .Additionally, the TCR transmits piconewton forces to the pMHC, and these forces are important to T cell antigen recognition 20 .2D affinity measurement of the TCR-pMHC bond demonstrated that agonist pMHC actually has a shorter bond lifetime than weak agonist pMHC 8 .Conversely, biomembrane force probe measurements revealed that the TCR-pMHC interaction exhibits catch bond behavior, but only for agonist antigen 24  (Fig  3A).The catch bond behavior of the TCR-pMHC bond was also confirmed by optical tweezer measurements 25 .Sibner and colleagues recently interrogated TCRs that bind pMHC but are not activated.When agonist peptides to the formerly non-responsive TCRs were generated, catch bonds that correlated with CD 45 exclusion from the T cell-antigen presenting cell contact area were identified in the new, activating interactions 44 .This finding further emphasizes the importance of catch bonds, and thus mechanical proofreading, in T cell antigen discrimination.The accumulation of TCR-pMHC bond lifetimes may link TCR forces with the serial-engagement model of T cell activation 38 , allowing the catch bond behavior of many TCR-pMHC interactions to cumulatively trigger sustained calcium signalling during T cell activation 24,45 .The increase in bond lifetime for agonist pMHC could serve as an extraordinarily specific indicator that a T cell has found agonist antigen, specificity purchased at the cost of mechanical work.
Catch bonds are not limited to the TCR-pMHC interaction.A catch bond has been demonstrated in the bond between lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) 41 .Combined with the observation that dendritic cells (DC) immobilize ICAM-1 on their surface in response to inflammatory signals 46 , these results raise the possibility that that the LFA-1-ICAM-1 catch bond may help T cells recognize stimulated DCs.
Finally, catch bonds likely mediate mechanical proofreading in non-immune biological systems.For example, the platelet glycoprotein GPIbα exhibits catch bond behavior when interacting with Von Willebrand factor 42 .Bacterial adhesion under flow is regulated by a catch bond between FimH and mannose 47 .Leukocyte adhesion against the flow of blood depends on a catch bond 35 .It is likely that catch bonds are a biologically general mechanism for mechanical proofreading, ensuring that biological processes occur only at specific, desirable interfaces via extremely specific mechanically stable receptor-ligand bonds.
Mode 2: Stress Test-Most receptor-ligand interactions are not catch bonds; however, slip bonds in groups of mechanically strained receptors can also facilitate mechanical proofreading.Receptor clustering is crucial to many biological signaling pathways and may also provide a mechanical advantage to force-bearing receptors.When force-bearing receptors cluster together, the stress (defined as force per unit area) applied at a cell-cell junction may be reduced because the force is distributed over many bonds.Receptor clustering therefore offers an opportunity for a mechanical proofreading "stress test," which selects for cellular structures composed of many force-bearing proteins.Collectively, clustered proteins may withstand forces that would rupture any single bond.Note that for the stress test mechanism to function, the cellular force-generating machinery must be connected to groups of receptors (e.g. one actin stress fiber transmitting force to many integrins).
Consider a cluster of N receptors withstanding a total force F at a cell-cell junction.Force balance dictates that each receptor is bearing a force of approximately F/N.Clustering leads to an N fold decrease in the force experienced per ligand-receptor pair.Based on the Bell model 32 , the reduction in force on each receptor-ligand pair will lead to a significant enhancement in the bond lifetime, increasing the probability that the cellular structure will survive long enough to initiate biochemical signaling.To achieve the desired specificity and sensitivity, biological systems can alter both F and N. Increasing F produces shortened bond lifetime, decreasing sensitivity.Conversely, increasing N reduces the per-receptor force, facilitating signalling under force, potentially decreasing specificity.High F and high N may achieve both high sensitivity and specificity by enabling only highly multivalent interations to initiate biochemical signalling.
B cell receptor (BCR) antigen internalization is an example of a mechanical proofreading stress test.BCR signaling is intricately related to the B cell cytoskeleton 48 .Clusters of BCRs are thought to use mechanical force to internalize antigen.Natkanski et al. demonstrated that B cells internalize plasma membrane sheet bound antigen in a myosin IIa dependent manner, and that clathrin-coated structures seem to be associated with antigen internalization 40 .Interestingly, BCR microclusters were observed to resist contractile forces for 20-30 seconds; however, AFM measurements demonstrated that single BCR-antigen bonds do not endure long enough for the observed membrane invagination to occur 40 .The authors hypothesize that forces exerted by B cells shorten the BCR-antigen bond lifetime, ensuring that only multivalent, high-affinity antigens which collectively withstand the cellular force are internalized (Fig 3B) 40 .These findings are supported by the observation that B cells exert measurable traction forces through the B cell receptor, and that the magnitude of traction force scales with the number of clustered BCRs involved in force transmission 49 .Additionally, DNA-based molecular tension sensor measurements demonstrated that the BCR utilizes molecular forces to extract antigen from follicular dendritic cells, and that stiff follicular dendritic cells produce stronger BCR forces and more stringent antigen affinity discrimination 50 .Collectively, this evidence points towards a mechanical stress test facilitating BCR selection for high-affinity antigen.
A mechanical stress test may also be important to TCR signalling.The TCR is known to exert pN forces on the TCR-pMHC bond 20 , but the importance of nanoclusters in TCR force transmission is not known.Super-resolution imaging has suggested that TCRs form nanoclusters, and that clustered TCRs are more likley to be phosphorylated and to participate in downstream signalling 51 .TCRs are known to cluster at the surface of microvilli on the T cell surface 52 and TCR-bearing microvilli are selectively stabilized at the T cell-antigen presenting cell interface 53 .Collectively, this evidence raises the possibility that TCR nanoclusters, and potentially microvilli, may facilitate a mechanical stress test.
Mechanical stress within other supramolecular complexes may also be important.For example, the focal adhesion has been proposed to behave like a molecular clutch, with mechanical unfolding of adaptor proteins serving to recruit more integrins to the adhesion site to share the applied load 54 .Thus focal adhesions may sense and respond to substrate stiffness via a mechancial proofreading stress test, where specific combinations of force and adhesion site density produce focal adhesion growth and maturation.
Mode 3: Strain test-Another mode of mechanical proofreading is a "strain test."Strain is defined as the change in length of an object relative to its original length.Analogously, many receptors undergo conformational changes due to ligand or allosteric interactions.Some conformational shifts are facilitated by pN scale forces 55,56 .Conformational shifts may expose cryptic binding sites, leading to protein recruitment or to phosphorylation of a previously inaccessible site.Force-reconfigurable proteins therefore offer a third potential mode of mechanical proofreading.In the mechanical proofreading strain test, a receptorligand bond must withstand a threshold force to produce a conformational shift before eliciting biochemical signaling.Note that for the strain test to function, a large energy threshold must prevent the receptor from spontaneously experiencing the conformational shift in the absence of a large input of mechanical work by a cell.
Integrins are well-studied examples of the mechanical proofreading strain test.They exhibit profound conformational shifts, existing in a low-affinity, bent conformation at rest, but able to adopt a high affinity extended state 57 .Integrins may be an ultrasensitive molecular switch, able to extend in response to pN forces 55 .
In the context of lymphocytes, the integrin α L β 2 , also known as LFA-1, binds to ICAM, and is important to immune cell-cell adhesion and to T cell function 58 .LFA-1 undergoes outside-in activation in response to surface bound, but not soluble ICAM 59 , and molecular forces increase the rate of LFA-1 extension and slow the rate of LFA-1 bending (Fig 3C) 56 .It is believed that actin forces transmitted through LFA-1 to immobilized ICAM-1 induce extension of LFA-1 and that actin is critical to the formation and maintenance of the immunological synapse 60 .Additionally, recent interference photoactivation localization microscopy (iPALM) measurements, which resolve the location of fluorophores along the microscope optical axis with ~10 nm resolution, have measured the extension of LFA-1 at a T cell-surface interface 61 .Outside-in activation of LFA-1 has functional consequences; for example, severing ICAM's cytoskeletal anchorage prevented natural killer cells from forming junctions with target cells and disrupted granule polarization 62 .Additionally, when DCs are induced to mature via treatment with lipopolysaccharide, they utilize their actin cytoskeleton to restrict ICAM-1 surface mobility.T cells sense the change in ICAM-1 mobility, producing more extended LFA-1 on the T cells surface 46 .Collectively, these findings demonstrate that LFA-1 exhibits force-sensitive outside-in activation, positioning LFA-1 as a potential mechanical proofreading strain sensor.
The T cell receptor (TCR) may also utilize a mechanical strain test during T cell antigen recognition.Optical tweezer measurements demonstrated a force dependent extension in the FG loop of the TCR 25 .The FG loop extends 8-15nm under the influence of pN forces.Deleting the FG loop of the TCR removed the force-dependent extension behavior and reduced the ability of T cells to respond to antigen as assessed by IL-2 production 25 .The FG loop extension of the TCR therefore presents another example of a potential mechanical proofreading strain test.Because TCR forces are between 12 and 19 pN 20 , and because the FG loop extension is known 25 , the height of the mechanical proofreading energy barrier that TCR forces must surmount has been calculated as ~37 k B T (assuming 10nm displacement and 15pN forces), or almost twice the energy of one ATP hydrolysis.This energy of discrimination could explain the remarkable specificity of T cell antigen recognition 27 .The mechanical proofreading strain test is therefore capable of extreme specificity.
The strain test mode of mechanical proofreading is not limited to the immune system.For example, platelets action must be tightly regulated to prevent erroneous clot formation.The platelet integrin α IIb β 3 is anisotropcially mechanosensitive, requiring lateral forces to undergo outside in activation to enable platelet spreading and activation on a surface 63,64 which may explain why platelets ignore soluble fibrinogen in the blood, only binding to fibrinogen attached to other activated platelets.Additionally, the platelet glycoprotein complex GPIb-IX-V, has a mechanosensitive domain that extends several nanometers under force 65 , making it another likely candidate for a strain test for mechanical proofreading.

Combinations of mechanical proofreading modes
The modes of mechanical proofreading may also work together to produce increased specificity.For example, the GP1b-IX-V:von-Willebrand factor bond is a catch bond 42 .The prolonged bond lifetime under force may facilitate the opening of the mechanosensitive domain of GP1b-IX-V 66 .Similarly, the TCR exhibits catch bond behavior, an extension of its FG loop, and receptor clustering 24,25,51 .Various mechanisms invoked to explain the extreme sensitivity and specificity of the TCR-pMHC bond involve each of the proposed modes of mechanical proofreading 2,11 .The molecular clutch model of the focal adhesion 54,67 argues that talin unfolding under force (a strain test) regulates integrin recruitment to the focal adhesion (regulating stress), thus these two models may work cooperatively in focal adhesions.Elucidating which mechanisms are critical to regulating cellular decision-making will require careful experimentation to isolate the effects of each of the modes of mechanical proofreading.

Conclusion
Here, we have focused on applications of mechanical proofreading within the immune system, but it is likely a biologically general mechanism.Because mechanical forces generally disrupt receptor-ligand interactions, as described by the Bell model 32 , careful examination of force-exerting systems is required to determine what functional benefit is derived by expending cellular energy on force generation.Mechanical forces may be vital to biological differentiation between soluble and a surface bound ligand.Soluble ligands are not capable of resisting mechanical force.Indeed, it is conceivable that from a mechanistic point of view, mechanical forces are the principle difference between cell-cell interfaces and receptors interacting with soluble ligands.
Mechanical proofreading likely has great physiological relevance.Zhu and colleagues found that during thymic selection, negative selection ligands produced cooperative trimolecular catch bonds (TCR-pMHC-CD8) while positive selection ligands formed slip bonds 68 .This result strongly suggests that mechanical proofreading, by means of catch bonds, assists in the elimination of strongly self-reactive thymocytes during negative selection.Additionally, Huse and colleagues recently found that the exertion of force by T cells is spatially and temporally linked to the release of cytotoxin release and target cell killing 28 .Thus mechanical proofreading is also implicated in the killing of target cells by cytotoxic T cells.Finally, T cells have been demonstrated to respond to increased stiffness of a substrate presenting activating antibodies to CD3 and CD28 with increased IL-2 secretion 69 .Importantly, human primary immune cells, including macrophages and dendritic cells, modulate their stiffness in response to inflammatory signals 70 .Collectively, the ability of antigen presenting cells to modulate their stiffness in response to inflammatory signals combined with the stiffness sensitivity of T cells suggests that mechanical proofreading may play a role in enabling T cells to respond appropriately to complex environmental signals.
Additionally, mechanical proofreading has the potential to resolve longstanding debates in immunology.For example, mechanical proofreading may provide insight into how adaptive immune responses are initiated.TCR binding affinity for pMHC does not always correlate with T cell activity [7][8][9] .The ability of a pMHC to bind to the TCR does not guarantee that the TCR will activate 71 .Garcia and colleagues recently interrogated TCRs that bind pMHC but are not activated.When agonist peptides were produced to the formerly non-responsive TCRs, these new agonist pMHC-TCR bonds exhibited catch bond behavior 44 .This finding further emphasizes the importance of mechanical forces, catch bonds, and mechanical proofreading in T cell antigen discrimination.
Finally, mechanical proofreading may have utility for cell-based immunotherapies.Kam and colleagues have recently demonstrated the use of polydimethylsiloxane microbeads coated in activating antibodies to CD3 and CD28 to enhance the ex vivo activation and proliferation of both CD4 + and CD8 + T cells for immunotherapy 72 .Intriguingly, T cells expansion was found to be enhanced in soft beads, implicating a role for mechanical forces in ex vivo T cell expansion.It is also possible that some variety of mechanical proofreading may operate in chimeric antigen receptor (CAR) T cell recognition of antigen.Chen and colleagues recently rewired CAR-T cells to recognize soluble antigen 73 .However, CAR signaling in these cells required ligand-mediated CAR dimerization, which the authors believe implicates a role for mechanotransduction in CAR signaling.Interestingly, the chimeric antigen receptor is comprised of an antibody-derived fragment for ligand binding.Antibodies exhibit slip bond character under force 74 .Mechanotransduction during CAR signaling likely proceeds via a different mechanism than the catch bonds that influence TCR-pMHC interactions.
Experiments designed to test the models outlined here may be critical in determining the mechanical origins of cellular decision-making.For example, deletion of mechanically sensitive domains in the TCR reduced IL-2 production 25 .Likewise, the mecahnosensitivity of platelets has been partially explianed via a mechanosensitive domain discovered in the platelet glycoprotein GpIbα 65 .Deletion of a portion the mechanosensitive domain produces constitutive GpIbα signalling 75 .These experiments may be facilitated by the recent development of powerful molecular tools capable of measuring both the magnitude and orientation of molecular forces 18,20,21,64,76 .We anticipate that understanding the precise role of molecular forces in receptor-ligand interactions will provide fundamental insight into the exquisite specificiy of cellular decision-making.CD8 + T cells exhibit more potent activation when the pMHC-TCR complex is more mechanically stable.(E) Optical tweezers, formed by trapping a bead near the focus of a tightly focused laser, enable precise control over the movement of the bead and are useful for exerting pN forces on receptor-ligand interactions.(F) Optical tweezer manipulation of pMHC-presenting beads enabled the application of pN forces to TCR-pMHC complexes.TCR-pMHC bond lifetime increases for agonist pMHC (catch bond behavior) but decreases for non-agonist pMHC (slip bond behavior).The TCR FG loop is thought to elongate under cellular force and to be responsible for catch bond behavior.Stabilizing the FG loop with the H57Fab frag further enhances bond lifetime.Panels A-D are adapted from with permission from [1] while panels E and F are adapted from [23] licensed under Creative Commons (CC BY 4.0).Abbreviations: NSET is near surface energy transfer, BF is brightfield, RICM is reflection interference contrast microscopy which displays cell-surface contact area, TGT is tension gauge tether, T tol is the tension tolerance of a TGT, the force at which the TGT ruptures within 2 seconds bBSA is biotinylated bovine serum albumin, SA is streptravidin *** Indicates p < 0.001 and **** indicates p < 0.0001.(A) Molecular forces may skew the energy landscape of a biological process.Under most conditions, pN molecular forces destabilize receptor-ligand complexes, shifting thermodynamic equilibrium.The plot depicts an idealized free energy diagram for a twostate receptor-ligand interaction in the presence and absence of pN force.G 0 is the standard free energy of the interaction pathway in the absence of force, Δx is the distance to the transition state of receptor-ligand unbinding, and ‡ indicates the transition state on the free energy diagram.(B) Molecular forces also generally reduce bond lifetime (increasing k off under force).This force-dependent reduction in bond lifetime is known as a slip bond and is well-described mathematically by the Bell model.Select bonds exhibit increased bond lifetime under force, a phenomenon known as a catch bond.The plot depicts the force-bond lifetime relationship for idealized catch and slip bonds.k off 0 is the zero-force off rate, k B is the Boltzmann constant, T is the temperature.(C) Molecular forces may also alter the energy landscape of a protein, facilitating conformational shifts, exposing cryptic binding sites for accessory proteins (dotted line).Accessory proteins may produce secondary messengers, amplifying the signal produced by force-induced conformational shifts.(a) In rare cases, forces borne by receptor-ligand interaction reduce the off rate of that interaction, a behavior known as a catch bond.The TCR-pMHC bond is known to exhibit catch bond behavior.The increased bond lifetime under force may facilitate downstream signaling, acting as a form of mechanical proofreading.(b) Mechanical proofreading may occur via a stress test if single bonds are insufficient to withstand the applied cellular force.
Only when the force is spread across many bonds will the entire force-bearing structure survive to produce biological signaling.This mode of mechanical proofreading may occur in BCR-antigen internalization.(c) Mechanical proofreading may occur via a strain test.Piconewton receptor forces act to produce a conformational shift in a receptor only if the strength of the receptor-ligand bond is sufficient.A potential example of the mechanical proofreading strain test is the LFA-1:ICAM bond.LFA-1 is known to extend in response to interactions with ICAM-1 and to modulate T cell functions.

Mode 1 :
Alteration of Bond Lifetime: Receptor-ligand interactions at cell-cell or cellextracellular matrix junctions frequently experience force.These pN forces alter receptorligand bond kinetics (Fig 2B, Fig 3A).Most frequently, receptor-ligand forces produce reduced bond lifetime (slip bonds)

Figure 1 :
Figure 1: Piconewton molecular forces are important to TCR antigen specificity.(A)Molecular tension probes are comprised of a fluorophore-quencher pair flanking a forceextensible domain.Piconewton molecular forces extend the probe, separating the fluorophore-quencher pair.DNA-based molecular tension probes, highlighted here, produce up to a 100x increase in fluorescence under the influence of cellular forces.(B) Using DNA molecular tension probes, OT-1 CD8 + T cells were found to exert defined(12-19pN) forces through the TCR.(C) Tension gauge tethers rupture irreversibly when receptor forces reach a threshold, capping the maximum tension a cell can exert through a receptor.(D) OT-1 CD8 + T cells exhibit more potent activation when the pMHC-TCR complex is more mechanically stable.(E) Optical tweezers, formed by trapping a bead near the focus of a tightly focused laser, enable precise control over the movement of the bead and are useful for exerting pN forces on receptor-ligand interactions.(F) Optical tweezer manipulation of pMHC-presenting beads enabled the application of pN forces to TCR-pMHC complexes.TCR-pMHC bond lifetime increases for agonist pMHC (catch bond behavior) but decreases for non-agonist pMHC (slip bond behavior).The TCR FG loop is thought to elongate under cellular force and to be responsible for catch bond behavior.Stabilizing the FG loop with the H57Fab frag further enhances bond lifetime.Panels A-D are adapted from with permission from[1] while panels E and F are adapted from[23] licensed under Creative Commons (CC BY 4.0).Abbreviations: NSET is near surface energy transfer, BF is brightfield, RICM is reflection interference contrast microscopy which displays cell-surface contact area, TGT is tension gauge tether, T tol is the tension tolerance of a TGT, the force at which the TGT ruptures within 2 seconds bBSA is biotinylated bovine serum albumin, SA is streptravidin *** Indicates p < 0.001 and **** indicates p < 0.0001.

Figure 2 :
Figure 2: Effects of pN forces on receptor-ligand interactions.