Label-free single-molecule quantification of rapamycin-induced FKBP

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Label-free single-molecule quantification of rapamycin-induced FKBPFRB dimerization for direct control of cellular mechanotransduction Yinan Wang, Samuel Barnett, Shimin Le, Zhenhuan Guo, Xueying Zhong, Pakorn Kanchanawong, and Jie Yan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b03364 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Label-free single-molecule quantification of rapamycin-induced FKBP-FRB dimerization for direct control of cellular mechanotransduction Yinan Wang1,4, Samuel F. H. Barnett2,4, Shimin Le1, Zhenhuan Guo2, Xueying Zhong2, Pakorn Kanchanawongb,2,3, Jie Yana,1,2 1Department

of Physics, National University of Singapore, Singapore 117546. Institute, National University of Singapore, Singapore 117411. 3Department of Biomedical Engineering, National University of Singapore, Singapore 117583. 4These authors contributed equally. aEmail: [email protected] bEmail: [email protected] 2Mechanobiology

Abstract Chemically induced dimerization (CID) has been applied to study numerous biological processes and has important pharmacological applications. However, the complex multi-step interactions under various physical constraints involved in CID imposes a great challenge for the quantification of the interactions. Furthermore, the mechanical stability of the ternary complexes has not been characterized, hence their potential application in mechanotransduction studies remains unclear. Here, we report a single-molecule detector which can accurately quantify almost all key interactions involved in CID and the mechanical stability of the ternary complex, in a label-free manner. Its application is demonstrated using rapamycin-induced heterodimerization of FRB and FKBP as an example. We revealed the sufficient mechanical stability of the FKBP/rapamycin/FRB ternary complex, and demonstrated its utility in the precise switching of talin-mediated force transmission in integrin-based cell adhesions. Keywords: CID, rapamycin, talin, mechanotransduction, magnetic tweezers

Introduction CID refers to the use of small molecules to induce dimerization of two proteins. It has been applied to modulate numerous biological processes through precise spatiotemporal control of protein-protein interactions1-3, such as gene expression4, 5, protein translocation6, 7, and signal transduction8, 9. In addition, CID also has important pharmacological applications10, 11, for which it has been suggested that small molecules that can induce dimerization between two proteins could be a generic approach to modulate the activity of the target proteins with physiological functions12, 13. In many of these applications, one or both of the reacting proteins are often tethered to membrane7, 8 or to chromosomes4, 5. As such, the formation and the stability of the ternary complex may be influenced by various physical constraints, such as membrane or chromosome tethering and, importantly, mechanical force9. A CID reaction involves multiple-step ternary interactions of two complementary adapter domains A1 and A2, and a dimerizer D. Hereafter, we use ‘’/’’ to indicate a complex, and ‘’-‘’ to indicate an interaction. A CID reaction to form the final A1/D/A2 ternary complex includes the following reversible sub-step binary interactions: A1-D, A2-D, A1/D-A2, and A2/D-A1 (Fig. 1A). To fully understand a CID reaction, each sub-step binary interaction must be quantified, however, this process is challenging.

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Figure 1. CID interactions. (A) Intermediate/final complexes of CID in solution. The association and dissociation rates of each sub-step binary interactions are indicated by 𝑘𝑜𝑛 and 𝑘𝑜𝑓𝑓 (B) Five configuration states of the CID detector in rapamycin solution under force, which can be categorized in to a looped state- (A1/D/A2) and four unlooped states - (A1, A2), (A1/D, A2), (A1, A2/D) and (A1/D, A2/D). In the unlooped states, rapamycin can interact with the A1 and A2 domains in the detector with certain kinetic rates. In addition, when one of the domains is bound with rapamycin, a transition between the unlooped and looped states can occur with certain force-dependent rates 𝑟𝑙𝑜𝑜𝑝(𝐹) and 𝑟𝑢𝑛𝑙𝑜𝑜𝑝(𝐹), respectively. The current biochemical quantifications of CID systems suffer from various limitations such as sensitivity and specificity13-15. As a result, very few CID systems have been fully quantified. In addition, the mechanical stability of the final A1/D/A2 ternary complexes has not been quantified for any of the existing CID systems, raising a question concerning how effectively these CID systems can be used to modulate cellular processes where the proteins are under force. The CID systems have begun to be applied in mechanobiological studies where proteins could be subject to appreciable forces9, 16. For example, in an earlier study, the rapamycinmediated CID system was successfully used as a synthetic receptor-ligand system to substitute the force-bearing native ligand-receptor interaction of the transmembrane Notch receptor9. Therefore, a rigorous characterization of the mechanical properties of these dimerizers becomes highly important but has long been technically challenging for existing techniques. This crucial information is needed to implement the CID system in various mechanotransduction processes that involve different level of forces. To address this, we developed a highly sensitive label-free single-molecule assay that can quantify the lifetime of the ternary complex under force as well as the kinetics of each reversible sub-step binary interactions with high sensitivity and specificity. This assay is based on mechanical manipulation of a single-molecule detector which is a chimeric protein construct consisting of three components: an adapter domain A1, a complementary adapter domain A2, and a 196 a.a. intrinsically disordered peptide with the majority (182. a.a) derived from the FH1 domain of formin mDia117, 18 between them (Supp. Methods, detector sequence). The long linker used in the construct acts as an amplifier based on the large extension increase step when


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the two domains are separated under force. This stepwise extension change is a readout of the experiment to indicate the state of the detector.

Figure 2. Rapamycin detector force responses. (A) Top panel shows the schematic of the rapamycin detector domain map. Bottom panel shows the schematic of the detector under force and predominant interactions. The association and dissociation rates between rapamycin and ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 FKBP in the detector are indicated by 𝑘𝐹𝐾𝐵𝑃 and 𝑘𝐹𝐾𝐵𝑃 , respectively. 𝑜𝑛 𝑜𝑓𝑓 𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 ― 𝐹𝑅𝐵 The looping and unlooping rates are indicated by 𝑟𝑙𝑜𝑜𝑝 (𝐹) and 𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 ― 𝐹𝑅𝐵 (𝐹), respectively. (B) Top-panel shows 𝐻𝑏𝑒𝑎𝑑 recorded from four 𝑟𝑢𝑛𝑙𝑜𝑜𝑝 consecutive force-increase scans in 2.5 μM rapamycin. The bottom panel shows the histogram of the unlooping/unfolding forces recorded from 59 force cycles from 7 independent tethers. The detector is placed under different forces within the physiological range. Upon induction of the dimerizer D, it can exist in one of the following five configuration states (A1, A2), (A1/D, A2), (A1, A2/D), (A1/D, A2/D) and (A1/D/A2) (Fig. 1B). (A1/D/A2) can form through a transition from the (A1/D, A2) or from (A1, A2/D). As (A1/D/A2) involves looping of the long flexible linker against force, such transition can only occur at sufficiently low forces. The A1/D/A2 complex can also be opened up by applying a sufficiently high force, causing a transition to (A1/D, A2) or (A1, A2/D). Hereafter, the (A1/D/A2) state of the detector is referred to as the looped state, while the other states are collectively referred to as the unlooped state. Most of the CID systems developed are based on rapamycin and its derivatives, which induces dimerization between a 12-kDa FK506 binding protein (hereafter referred to as FKBP) and the FRB domain ( ~93 a.a.) of the mTOR kinase14. Here we use the rapamycin CID system as an example to demonstrate how to use this single-molecule assay to quantify the affinity and kinetics of the key reversible sub-step interactions involved rapamycin (D) induced dimerization of FKBP (A1) and FRB (A2), as well as to quantify the mechanical stability of the FKBP/rapamycin/FRB (A1/D/A2) complex. We showed that the mechanical stability of the FKBP/rapamycin/FRB complex is sufficiently high such that it can be used to control focal adhesion maturation in a temporally precise fashion, allowing the dissection of how distinct talin domains contribute to talin-mediated mechanotransduction process.

Results Single-molecule detector of rapamycin-induced FKBP and FRB dimerization


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The top panel of Fig. 2A shows the single-molecule rapamycin CID detector. Two repeats of titin I27 domains are added to each side of the detector serving as a spacer and as a specific control. The detector is specifically tethered between a coverslip through its C-terminus and a super-paramagnetic bead through a 572-bp DNA handle at its N-terminus. The looped and the unlooped states have different extensions which can be detected using magnetic tweezers at a nanometer spatial resolution and millisecond temporal resolution19-23 (Fig. 2A, bottom panel). We recorded the bead height (𝐻𝑏𝑒𝑎𝑑) at different forces. Fig. 2B shows representative data of 𝐻𝑏𝑒𝑎𝑑 during force-increase scans at a loading rate of 2.0 ± 0.4 pN/s in 2.5 𝜇M rapamycin. Three sequential steps were observed in each force scan, corresponding to unlooping of the detector, unfolding of FRB and unfolding of rapamycin bound FKBP. The step at forces of ~35 pN was confirmed to be from the unfolding of the rapamycin-bound FKBP (Fig. S6). The other two steps observed at similar forces of ~10 pN can be explained by unlooping followed by unfolding of the FRB domain by analyzing the number of released residues from the extension changes for each step (Fig. S8). Specifically, the first step with a large step size of ~ 49 nm is contributed by the unlooping of the detector, and the second step with a step size of ~ 22 nm is contributed by the unfolding of the FRB domain (Supp. Info. 1). Around 2 pN, near-balanced stepwise fluctuation between the looped and unlooped states was observed, with a step size of ∆𝑧 ~ 20 nm (Fig. S7). Therefore, below 1.5 pN and above 2.5 pN, the detector is predominantly in the looped and unlooped states, respectively, with ~100% probability (Fig. S7). Trans-binding of rapamycin to tethered FKBP It has been known that rapamycin and its derivatives bind FKBP with a much stronger affinity than binding to FRB14. Therefore, at low rapamycin concentrations, its binding to FKBP predominates (Fig. S10). We quantified the FKBP-rapamycin interaction between the detector FKBP and the solution rapamycin at a concentration 𝑐 = 1 nM. Cycles of sequential force jump among 1.0 ± 0.2 pN (looping force, 5 sec), 2.0 ± 0.4 pN (detecting force, 4 sec) and 4.0 ± 0.8 pN (binding force, 30 sec) were implemented. The binding force ensures the unlooped state, allowing binding and unbinding by rapamycin. The looping force allows immediate formation ( < 1 sec) of FKBP/rapamycin/FRB ternary complex when FKBP is bound with rapamycin. The detecting force was used to detect the state of the detector. At this force, the looped state is ~20 nm shorter than the unlooped state, which can be unambiguously distinguished (Fig. S7). The cycles were repeated for a few thousands' times from 13 independent tethers. Left panel of Fig. 3A shows 𝐻𝑏𝑒𝑎𝑑 in six representative consecutive cycles obtained from a tether. 𝐻𝑏𝑒𝑎𝑑 at the detecting force in the first three cycles are ~20 nm higher than those in the last three cycles, indicating that the detector FKBP was unbound by rapamycin in the first three cycles and bound by rapamycin in the last three cycles (Fig. 3A, right panel). In addition, in the last three cycles, after jumping from the detecting force to the binding force, delays of a few seconds before unlooping was observed (black arrows in Fig. 3A), indicating that the FKBP/rapamycin/FRB ternary complex can withstand ~4 pN forces for several seconds. This example shows that whether the FKBP in the detector is bound with a rapamycin can be determined based on whether the detector can form the looped state at the looping force of ~1 pN for 5 seconds, which was detected based on the bead height after a subsequent jump to detecting force of ~2 pN. Using this approach, we measured the time taken from loopable (rapamycin bound) state to unloopable (rapamycin unbound) state (𝜏𝑏𝑜𝑢𝑛𝑑), and the time taken


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from unloopable state to loopable state (𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑) (details can be found in Supp. Methods, under "𝜏𝑏𝑜𝑢𝑛𝑑 and 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 measurement").

Figure 3. Trans-binding of rapamycin to detector-FKBP. (A) The left panel shows a representative time trace of 𝐻𝑏𝑒𝑎𝑑 during six consecutive cycles of sequential force jumping among ~1 pN (black), ~2 pN (purple), and ~4 pN (dark red). The state of the detector at ~2 pN is indicated with dashed horizontal lines. Black arrows indicate delayed unlooping after jumping from ~2 pN to ~4 pN. The right panel shows the zoomed-in time trace of 𝐻𝑏𝑒𝑎𝑑 in cycle 3 and cycle 4. As indicated by the dashed horizontal lines, the 𝐻𝑏𝑒𝑎𝑑 at ~2 pN (purple) in cycle 3 is ~20 nm higher than that in the cycle 4. (B-C) The normalized histogram of the lifetimes of the unbound (B) and bound (C) states, which are fitted with a single-exponential 𝐹𝐾𝐵𝑃 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝐹𝐾𝐵𝑃 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 𝜏𝑏𝑜𝑢𝑛𝑑 decay functions 𝐴𝑜𝑛𝑒 ―𝑐𝑘𝑜𝑛 and 𝐴𝑜𝑓𝑓𝑒 ― 𝑘𝑜𝑓𝑓 . Fitting to the normalized histograms of 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 (Fig. 3B) and 𝜏𝑏𝑜𝑢𝑛𝑑 (Fig. 3C), we obtained ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 the association rate 𝑘𝐹𝐾𝐵𝑃 = 2.6 ± 0.3 × 106 M ―1s ―1 and dissociation rate 𝑜𝑛 ―3 ―1 𝐹𝐾𝐵𝑃 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 of the FKBP-rapamycin interaction, which = 1.6 ± 0.1 × 10 s 𝑘𝑜𝑓𝑓 ―𝐷 𝐴1 ― 𝐷 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 correspond to the 𝑘𝐴1 and in Fig. 1. The dissociation constant is 𝐾𝐹𝐾𝐵𝑃 = 𝑘 on off 𝑑 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝑘𝐹𝐾𝐵𝑃 𝑜𝑓𝑓 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝑘𝐹𝐾𝐵𝑃 𝑜𝑛

= 0.6 ± 0.1 nM, in good agreement with ~0.2 nM estimated in previous

measurements using surface plasmon resonance (SPR) method14. The standard errors are estimated based on bootstrap analysis (Supp. Info. 6).


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Figure 4. Trans-binding of FKBP/rapamycin to the detector-FRB. (A) Schematic of the experiment design and predominant interactions. (B) A representative time trace of 𝐻𝑏𝑒𝑎𝑑 during five consecutive cycles of force jump among ~1 pN (black, 5 s), ~2 pN (purple, 4 s), and ~4 pN (dark red, 10 s). (C) The normalized histogram of the lifetimes of the unbound state 𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 ― 𝐹𝑅𝐵 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 fitted with 𝐴𝑜𝑛𝑒 ―𝑐𝑘𝑜𝑛 . (D) The normalized histogram of the lifetimes of the ― 𝐹𝑅𝐵 𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 ― 𝐹𝑅𝐵 ― 𝑘𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝜏𝑏𝑜𝑢𝑛𝑑 𝜏𝑏𝑜𝑢𝑛𝑑 bound state fitted with 𝐴𝑜𝑓𝑓,1𝑒 𝑜𝑓𝑓,1 . + 𝐴𝑜𝑓𝑓,2𝑒 ― 𝑘𝑜𝑓𝑓,2 Trans-binding of FKBP/rapamycin to tethered FRB The detector can also quantify the FKBP/rapamycin-FRB interaction, corresponding to the A1/D-A2 interaction in Fig. 1A. 100 nM rapamycin and 1 nM FKBP were used in this assay. ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 Since the rapamycin concentration is > 100 times of 𝐾𝐹𝐾𝐵𝑃 and < 0.01 times of 𝑑 𝐹𝑅𝐵 ― 𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 , this condition ensures ~100% of FKBP bound with rapamycin and ~0 𝐾𝑑 probability of the detector FRB bound with rapamycin (Supp. Info. 5 for more details). If the detector FRB is bound with FKBP/rapamycin, looping is inhibited until the bound FKBP/rapamycin dissociates (Fig. 4A). Fig. 4B shows five representative consecutive force-jump cycles. The data show that 𝐻𝑏𝑒𝑎𝑑 at the detecting force (~2 pN) in the first two cycles are ~20 nm higher than that in the last three cycles, indicating that the detector-FRB was bound by free FKBP/rapamycin in the first two cycles (therefore the detector is unloopable), while it was unbound in the last three cycles (therefore the detector is loopable). Repeating the cycles for > 1500 times from 7 independent tethers, we obtained the lifetimes of the loopable and unloopable states, which correspond to the lifetimes of the detector-FRB unbound and bound with free FKBP/rapamycin complex, respectively.


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Fitting the normalized histogram of 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 with a single-exponential decay function, the association rate of solution FKBP/rapamycin to the detector FRB is determined to be ― 𝐹𝑅𝐵 = 9.6 ± 1.2 × 106 M ―1s ―1. Interestingly, the normalized histogram of 𝑘𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝑜𝑛 𝜏𝑏𝑜𝑢𝑛𝑑 cannot be well fitted with a single-exponential function. Fitting the data using doubleexponential function instead, two apparent dissociation rates are estimated: ― 𝐹𝑅𝐵 ― 𝐹𝑅𝐵 ∈ (3.2, 8.2) × 10 ―2 s ―1 and 𝑘𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 = 3.5 ± 1.1 × 𝑘𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝑜𝑓𝑓,1 𝑜𝑓𝑓,2 ―3 ―1 (Fig. 4C-D). The faster dissociating species (species ‘’1’’) is predominant, 10 s contributing to more than 85% of the observed dissociation. Based on it, the dissociation ― 𝐹𝑅𝐵 𝑘𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 𝑜𝑓𝑓,1

― 𝐹𝑅𝐵 constant can be estimated to be 𝐾𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 = 𝑘𝐹𝐾𝐵𝑃/𝑟𝑎𝑝𝑎𝑚𝑦𝑐𝑖𝑛 ― 𝐹𝑅𝐵 ∈ (3.3 8.5) nM, in 𝑑 𝑜𝑛

reasonable agreement with ~12 nM measured in previous SPR measurement14. Please refer to Supp. Info. 6 for error estimations.

Figure 5. Mechanical stability for FKBP/rapamycin/FRB. (A) Representative time trace of 𝐻𝑏𝑒𝑎𝑑 during five consecutive cycles of force-jump between ~1 pN and ~5 pN. (B) The lifetime (mean ± s.e.) of the ternary complex at different forces fitted with Bell's model. The mechanical stability of FKBP/rapamycin/FRB ternary complex In order to explore the applications of the rapamycin CID system to modulate mechanotransduction of cells, we quantified the lifetime of the ternary complex at different forces in 10 nM rapamycin. The lifetime was measured by force jumping from 1.0 ± 0.2 pN where the detector exists in the looped state, to a set of higher forces to obtain the lifetime of the looped state at each force. Fig. 5A shows 𝐻𝑏𝑒𝑎𝑑 in five representative consecutive force cycles between 1.0 ± 0.2 pN and 5.0 ± 1.0 pN. In each cycle, after jumping to ~5 pN, the ternary complex can withstand a lifetime of a few seconds before unlooping. More than 400 cycles of force jumping from 9 independent tethers were obtained, from which the average of the lifetime of the ternary complex was recorded. Fig. 5B shows the average lifetime of the 𝐹𝛿 𝑇

―𝑘

ternary complex at different forces. Fitting with Bell's model24 𝜏(𝐹) = 𝜏0𝑒 𝐵 determines a transition distance of 𝛿~1.3 nm and predicts a zero-force lifetime of 𝜏0~38 s. Implementation of rapamycin-based CID for the direct control of force transmission in focal adhesions As Fig. 5B shows that the ternary complex can withstand at least a few pN forces over a few seconds, this raised an interesting possibility for intracellular mechanobiological application of the CID since recent measurements using a Forster Resonance Energy Transfer (FRET) tension


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gauge revealed that key mechanotransduction molecules such as vinculin or talin experience tension of a few to several pN magnitude25-29, which can be explained by a force-buffering function of rod-like proteins such as talin that contain a tandem of protein domains25, 30. We hypothesize that the rapamycin-based CID could thus be exploited as a molecular switch to control the connectivity of force-transmission pathways, and allow direct and moleculespecific interrogation of mechanosensing and mechanotransduction. For the integrin-based focal adhesions (FAs), earlier studies using super-resolution microscopy revealed the nanoscale organization of proteins into a supramolecular assembly, with talin serving as the “backbone” of FAs, connecting the integrin receptor with the actin cytoskeleton31-33. As such, we sought to test whether talin could be engineered with the CID into a switchable backbone to permit precise control of FA formation (Fig. 6A-B). Cell spreading and adhesion on ECM-coated substrate typically undergoes distinct phases of morphological changes (P1: non-contractile spreading, P2: contractile spreading, and P3: polarization)34. While integrin activation is sufficient to support the P1 phase, talin is indispensable for the P2 and P3 phases35. Talin is a highly elongated protein (2541 a.a.) that consists of a linear tandem of globular domains, linked together by flexible linkers32, 36-38. The N-terminal FERM domain serves as a key integrin-activating and integrin-binding site, while the C-terminal rod region contains a series of -helical bundles, termed R1-R13, which encompass at least two distinct actin-binding sites (ABS2 and ABS3), as well as numerous binding sites to partner proteins such as vinculin39 (Fig. 6A). To implement a CID switchable talin system, we generated expression vectors consisting of talin-1 with the FRB and FKBP modules inserted at various positions. At the truncation sites, the N-terminal fragments of talin-1 are coupled with the FRB module and termed talinN#-FRB, where # corresponds to the R# domain adjacent to the FRB module. Likewise, the C-terminal fragments are termed FKBPtalinC#. Upon rapamycin induction, it is expected to physically connect talin-N#-FRB and FKBP-talinC#, restoring a direct physical linkage (Fig. 6A-B), furnishing a complete FA “backbone” and permitting mechanical force propagation from the actin cytoskeleton to the integrin ECM receptors. These constructs were tagged with fluorescent proteins (FPs, either mApple or mEmerald40, 41) for visualization, and expressed in Mouse Kidney fibroblast lacking both isoforms of talin42.


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Figure 6. Temporal control of talin-mediated force-transmission supramolecular linkages. (A) Schematic diagram of talin-1 highlighting major integrin and actin binding regions (not all protein-protein interactions sites are depicted). (B) Schematic diagram of CID switchable talin constructs. The truncated talin constructs respectively fused with FRB and FKBP are expected to connect upon rapamycin binding, completing a direct force-transmission link. (C) The changes in cell morphology and FA profiles after the addition of 2.5 μM rapamycin. Inset corresponds to time-lapse montage in (D). See also Mov. S1 (Green: FKBP-talinC11mEmerald fragment; Magenta: mApple-talinN10-FRB fragment). Scale bar: 10 μm. (D) Magnified view of inset in (C). Asterisk (yellow) denotes rapid advancement of lamellipodia upon rapamycin addition. Arrowhead (red) denotes an FA that forms within 30 s of rapamycin addition. Top row (merge channels) with talin-FRB in magenta and talin-FKBP fragment in green. Middle and bottoms rows: inverted contrast view of talinN10-FRB and FKBP-talinC11 channels, respectively. Scale bar: 2 μm. (E) Mean FRAP recovery curves of FAs before (blue) and after (orange) rapamycin induction. Bar graphs showed the quantification of the recovery half-time and mobile fraction (n=50 for each condition, based on two pooled experiments). (F) Traction force (false color) maps before and after rapamycin induction and the talinN10 fluorescence channel. Color bar: traction stress in Pa. (G) Quantification of total traction forces


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generated by cells in the presence and absence of rapamycin (n=11 for each condition). Scale bar: 20 µm. Error bars: s.d. (* = P < 0.05; ** = P < 0.005; *** = P < 0.0005). As shown in Fig. 6C-D and Fig. S11E-F, the talin-null cells expressing talinN10-FRB and FKBP-talinC11 were imaged by live-cell Total Internal Reflection Fluorescence (TIRF) microscopy before and after rapamycin induction (conc. 1 µM). Prior to rapamycin induction, the cells generated minimal tension on the substrate as measured by traction force microscopy (TFM) (Fig. 6F-G). Although lamellipodia formed, due to the ability of talinN10-FRB to activate integrin and support P1 spreading (Fig. S11C), mature FAs (as probed by immunostaining for endogenous vinculin) were absent, indicating that the P1 to P2 transition is disrupted. Consistently, cells treated with Mn2+ which activates integrin, or individually expressing mApple-talinN10-FRB are unable to form mature FAs, while cells expressing FKBP-talinC11-mEmerald form lamellipodia only upon Mn2+ treatment, but not FAs (Fig. S11A,C,D). Interestingly, upon rapamycin induction, we observed a rapid formation of FAs within 30s in cells that co-express talinN10-FRB and FKBP- talinC11 (Fig. 6C-D, S11F). As shown in the montage (Fig. 6C-D) and Mov. S1, the cells undergo polarization and initiated directed cell migration with the characteristic FA maturation and FA turnover, similar to typical fibroblast migration43. In conjunction, TFM measurement (Fig. 6F-G) revealed a significant increase in the traction force magnitude from 20.4 nN ± 17 to 47 ± 16 nN, thus indicating that force-transmission across talin is activated upon the FRB/rapamycin/FKBP ternary complex formation. Additionally, we made use of Fluorescence Recovery After Photobleaching (FRAP) to assess the dynamics of the CID-switchable talin (Fig. 6E). Both before and after rapamycin induction, we observed a t1/2 of ~40 s. On the other hand, the mobile fraction decreased significantly upon rapamycin induction to ~60%, indicating that the ternary complex formation may stabilize talin in FAs, as expected. The observed t1/2 is comparable in magnitude to the previously measured lifetimes of talin44, thus likely reflecting the binding dynamics of the talin N-terminal head domain. While the FKBP/rapamycin/FRB complex has an average lifetime of 30-50 seconds measured in vitro experiments14, the ternary complex formed in cells has earlier been shown to be largely irreversible45. Consistent with this, we also observed that FAs remained intact and the cells were spreading despite five cycles of wash-out and with more than 90 minutes of monitoring (Fig. S12C). The apparently slower dissociation of the FKBP/rapamycin/FRB complex in cells may be related to the crowded environment and potential interactions with other macromolecules in cytosol. These results altogether demonstrated that CID-based switchable talin enable precise control of FA maturation and support cell migration.


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Figure 7. Modular design of CID-talin to probe adhesion formation and maturation. (A) Schematic of switchable talin constructs with the FRB/FKBP inserted between R10 and R11. (B) Rapamycin-induced cell spreading and FA formation in conjunction with ROCK inhibition. Cells were pre-incubated with 5 µM Y-27632 for 45 minutes prior to imaging. (C) Schematics of extra-long talin and shortened talin as generated by the FRB/FKBP dimerizers. (D) Montages of rapamycin-induced cell spreading and FA formation using the switchable talin constructs from (C). (E) Schematic of switchable talin constructs based on (A) but with mutations in ABS3 to inhibit F-actin binding. (F) Montages from live-cell movies before and after rapamycin-induction using constructs in (E). (G) Normalised fluorescence intensity of talin-C11-ABS3mut-mEmerald in talin-N10 clusters channel from (F) (n=43 from 8 separate cells, error bars: s.d.). (H) Model of chemically induced dimerization for the control of in vivo mechanotransduction. Scale bars: 20 µm. Insets in B,D, and F corresponds to zoomed-in views in Fig. S13. Since FA maturation generally requires actomyosin contractility43, we also performed rapamycin induction in conjunction with Rho Kinase inhibition (by treatment with Y-27632). As shown in Fig. 7A-B and Fig. S13, while the cells still exhibited a rapid spreading we


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observed a significant attenuation of FA maturation, suggesting that rapamycin-induced FA maturation appears to depend on both the direct physical connection through talin and actomyosin contractility. Consistently, using immunofluorescence detection of endogenous FA proteins, we found that, similar to the control (cells rescued by wild-type talin), rapamycininduced FAs prominently contain vinculin, a marker of mature FAs known to be recruited to talin by a contractility-dependent mechanism (Fig. S11B)25, 46, 47. To demonstrate the versatility of the CID approach, we also generated FRB and FKBP constructs with different insertion sites and used them to create an extra-long talin rod (talin-N12 + talin-C1) that almost doubled the length of talin rod or the deletion (talin-N3 + talin-C11) that removed a significant portion of the rod domain. As shown in Fig. 7C-D, these constructs are able to support FA maturation to different extent, with the extra-long talin-N12/talin-C1 supporting larger FAs whereas the deletion talin-N3/talin-C1 supporting smaller FAs that formed around the cell periphery, similar to earlier observation in a truncation experiment37. Taken together, these data thus suggest that CID-based switchable talin can be engineered in a highly modular fashion to recapitulate major aspects of mature FAs. While talin is well-documented to play important roles both in terms of biochemical signalling39 and as the biophysical ‘molecular clutch’48, a key question has remained regarding the contribution of the biochemical factors (signalling by specific protein domains) versus the biophysical factors (physical connectivity and force transmission) in talin-mediated mechanotransduction functions. Here, we demonstrate how the CID-mediated control of force transmission may permit a more direct dissection of such processes. In particular, talin contains two major actin binding sites (ABS) in the rod domains, whose contribution to talin-mediated mechanotransduction is still not fully understood. Recent studies identified ABS2 (R4-R8 domain) as the primary load-bearing ABS in FAs, while the role of ABS3 (R13 domain) has been less clear26, 49. Based on a previous study showing that talin with ABS3 truncation (residue 1-2197) was unable to localize to FAs50, we hypothesize that ABS3 may be required for the initial phase of interaction between talin and F-actin. To test this, we perform rapamycin-induction of FA maturation using mApple-talinN10-FRB and FKBP-talinC11mEmerald with the mutations (K2443D, V2444D, and K2445D, denoted ‘DDD’, for short) that impair ABS3 F-actin binding51. As shown in Fig. 7E-G and Mov. S2, upon rapamycin induction, the mEmerald fluorescent intensity exhibited a clear step increase indicative of the recruitment of the FKBP-talinC11-(DDD)-mEmerald by CID. However, FA formation is significantly impaired, with no clear P2 and P3 phases of cell spreading (compared to Fig. 6CD). In contrast, in a situation where ABS2 is absent (Fig. 7D), FA growth was able to proceed, although full maturation was significantly attenuated. These results thus supported the previously proposed notion that ABS3 could be necessary as a ‘trigger’ for the initial engagement of talin with F-actin, but once initiated, ABS2 may predominate as the main forcebearing link in FAs37.

Discussions In summary, we have developed a generic label-free single-molecule assay that is capable of quantifying crucial sub-step interactions involved in CID under various physical constraints. We have demonstrated its applications by quantifying the crucial sub-step interactions involved in rapamycin-induced heterodimerization between FRB and FKBP, and the mechanical stability of the FKBP/rapamycin/FRB complex. The novelties, other applications, advantages, and limitations of this method compared with traditional bulk assays are provided in Supp. Info. 3. As demonstrated in the rapamycin CID system, it is possible to quantify the kinetics and affinity of the sub-step interactions involved in a CID system using a single-molecule


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detector that consists of a pair of complementary domains, A1 and A2, linked by a long flexible linker, in a fluorescence label-free manner. A high-performance CID system is expected to operate over a broad concentration range, with ―𝐷 ―𝐷 a high stability of the final ternary complex. Denoting 𝐾𝐴1 and 𝐾𝐴2 the binding affinity 𝑑 𝑑 of the dimerizer to the two adapter domains and assuming that the A1-D interaction has a higher ―𝐷 affinity than that of the A2-D interaction, the operational concentration range is roughly 𝐾𝐴1 𝑑 ―𝐷 . Thus, a high-performance CID system requires highly differential binding < 𝑐 < 𝐾𝐴2 𝑑 ―𝐷 affinity of the dimerizer D to the two adapter domains, i.e., 𝐾𝐴2 is at least one order of 𝑑 𝐴1 ― 𝐷 magnitude greater than 𝐾𝑑 . The rapamycin CID system meets all these requirements, with ―𝐷 an ultra-high A1-D affinity with a sub nM 𝐾𝐴1 measured in previous SPR experiment14 and 𝑑 ―𝐷 in this study, and a much lower A2-D affinity with an above 10 μM 𝐾𝐴2 , and a high A1/D𝑑 𝐴1/𝐷 ― 𝐴2 A2 affinity with a nM range of 𝐾𝑑 . In addition, these crucial sub-step interactions have fast association rate ( > 106 M ―1s ―1. These characteristics provide the rapamycin CID system a wide operational concentration range about 1 nM ― 10 μM, within which highly stable ternary complex can form rapidly. In the quantification of the sub-step interactions of a CID system using the single-molecule detector method, the degeneracy of the states in the unlooped conformation of the detector ―𝐷 ―𝐷 needs to be considered. However, for a high-performance CID system (𝐾𝐴1 ), ≪ 𝐾𝐴2 𝑑 𝑑 such degeneracy does not affect the measurement. One can choose a dimerizer concentration ―𝐷 to quantify the A1-D interaction, at which the lower-affinity domain (A2) is 𝑐 ~ 𝐾𝐴1 𝑑 predominantly un-associated with D. In contrast, to quantify the A1/D-A2 interaction, one can ―𝐷 ―𝐷 choose a dimerizer concentration of 𝐾𝐴1 , at which the A1 domain in the ≪ 𝑐 ≪ 𝐾𝐴2 𝑑 𝑑 detector and in solution are saturated bound with D, while the A2 domain is predominantly unassociated with D making it available to interact with the solution A1/D complexes. As such, the quantification method developed in this study can be generally applied to any highperformance CID system. Although not demonstrated, the approach described in the manuscript can be used to quickly determine whether a CID system is high-performance. This can be done by increasing the ―𝐷 dimerizer concentration till looping occurs at a concentration around 𝐾𝐴1 . For a high𝑑 performance CID system, keeping increasing the concentration will lead to a plateau of the ―𝐷 looping probability spanning a concentration at least 10 times of 𝐾𝐴1 (Fig. S10, black curve). 𝑑 In addition, the lifetime of the A1/D/A2 ternary complex is a strong indicator of the strength of the interaction between A1/D and A2. Once a CID system is determined to be highperformance, further detailed quantification of the kinetics and affinity of key sub-step interactions can be done using the same procedure as demonstrated in the example of rapamycin CID system in the manuscript. Our results show that for FKBP-rapamycin interaction, the lifetimes of the unbound and bound states both follow single exponential decay profiles, suggesting that this interaction can be understood based on two-state transition with defined transition rates. In contrast, for the interaction between FKBP/rapamycin and FRB, while the lifetime of the unbound state follows a single exponential decay profile, a double-exponential function is needed to fit the lifetime data of the bound state that contains a major ( > 85%) and a minor ( < 15%) conformation species. The two conformation species dissociate with different rates, resulting in the observed double-exponential decay profile. Since majority of the lifetime data ( > 85%) of the bound state can still be fitted with a single-exponential decay function, it is reasonable to estimate the


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dissociation rate based on single-exponential decay fitting to the major fraction of the lifetime data. The presence of such two bound conformations won’t affect the association rate measurement. The measured association rate (~107 M ―1s ―1) is close to the diffusion limited association rate; therefore, the measured association rate mainly reflects the rate of diffusion of FKBP/rapamycin to the binding site (FRB). While the assumption of the existence of two conformations of the FKBP/rapamycin/FRB complex can explain the measured dissociation kinetics, to prove it is beyond the scope of this research. Importantly, we determined that the FKBP/rapamycin/FRB complex can withstand pN range of forces for seconds to tens of seconds, which allows us to use it to modulate talin-mediated mechanotransduction in FAs. To our knowledge, this has been the first time that the mechanical stability of a CID system is quantified, and it has also been the first time to achieve temporal control of the connectivity of integrin/talin/actin force-transmission supramolecular linkage using a CID system. Our results revealed that within 30s, FA formation can be clearly observed, and that cells are able to undergo FA maturation, turnover, and polarized migration. This example thus further established talin as the main force-transmission linkage, which, once connected, enable cells to respond to the local environment within minutes upon the linkage assembly. We note that the utility of the FKBP/rapamycin/FRB system as a general molecular switch is further supported by a recent study by our collaborators showing that the rapamycin-induced FKBP-FRB dimerization can effectively control the connectivity of KANK-mediated supramolecular linkages to control FA disassembly 16. Thus, this principle should be highly generalizable to a wide range of mechanotransduction processes (Supp. Info. 8). Furthermore, the single-molecule CID detector also has important potential pharmacological applications. Rapamycin is a FDA approved drug11 and a number of rapamycin derivatives have been developed to achieve more favourable pharmacokinetic profiles52, which can be quantified using the rapamycin detector. Other drugs based on CID can be similarly quantified by substituting FRB and FKBP with corresponding protein domains in the detector. In addition to the CID systems which use a small molecule to induce dimerization, the lightinduced dimerization (LID) systems have been utilized to modulate various cellular processes through using certain-wavelength light to control the dimerization between photosensitive protein domains53-56. In some recent studies55, 56, the LID systems have also been applied in mechanobiological studies where the proteins are under force, implying that the LID systems likely confer certain level of mechanical stability that can be harnessed to modulate many mechanotransduction processes. In principle, the chimeric single-molecule detector described in this work can be used to quantify the mechanical stability of any LID system by substituting the FKBP and FRB to corresponding interacting domains.

Methods Protein expression and purification (single-molecule experiments). Three stretchable proteins were prepared for the single-molecule manipulation experiments: 1. FRB-FH1-FKBP detector (the amino acid sequence of FRB-FH1-FKBP detector can be found in Supp. Methods); 2. stretchable FRB domain; 3. stretchable FKBP. All plasmid constructs for them were synthesized by IDT gBlocks and cloned into an expression vector (pET151). These plasmid constructs were co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in Luria-Bertani (LB) media. They were purified through his-tag and then


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eluted by TEV cleavage. In addition to the stretchable FKBP construct, the in-trans FKBP protein was purchased from Abcam (Recombinant human FKBP12 protein, ab167985). Single-molecule manipulation. An in house-made back-scattered vertical magnetic tweezers was used in the single-molecule manipulation experiments with a spatial resolution of ~1 nm and temporal resolution of ~200 Hz23, 57. In the experiments, the protein sample is tethered on one end to the coverslip through SpyTag/SpyCatcher system, while the other end is linked to a superparamagnetic bead through a 572-bp double strand DNA linker (detailed protocols can be found in Supp. Methods, under “Tethering methods and coverslip preparation”). This system is performed in a laminar flow channel linked to a syringe pump, which allows mild and stable solution exchange. The extension change of the protein sample is measured by the height change of the superparamagnetic beads, which is attached to the surface through the protein of interest. Details of force calibration and force control can be found in Supp. Methods. Cell culture. Mouse Kidney fibroblast with talin-1 and talin-2 deletion was kindly provided by Dr. Carsten Grashoff (University of Muenster, Germany) and has been described previously26. Cells were cultured in a 5% CO2, 37oC humidified atmosphere. The culture media is DMEM Media (Life Technologies), supplemented with 10% FBS, sodium pyruvate, and Pennicillin/Streptomycin. For transfection, cells were diluted to a density of 6 ×10^6 cells/ml and mixed with 0.5-2.0 μg of expression vectors per each electroporation reaction. Transfection was performed by electroporation (Neon, Life Technologies) per manufacturer's protocol. For imaging, transfected cells were sparsely plated at a density of 7000/cm2 on fibronectin-coated coverslips or glass-bottom dishes (Iwaki, Japan). Cells were tested monthly for mycoplasma contamination. Generation of switchable talin constructs. All fluorescent proteins (FP) fusion constructs were based upon either N1- or C1- Clontech-style vectors with pCMV promoters. For pmApple-talinN10-FRB, the construct contains the mApple fluorescent protein at the Nterminus, conjugated with mouse talin-1 (residue:1-1973) and FRB sequence. For pEGFP-N1FKBP-talinC1-mEmerald, the construct contains FKBP at the N-terminus, conjugated with mouse talin-1 (residue: 1974-2541), followed by mEmerald fluorescent protein at the Cterminus. More details on other constructs can be found in the Table 1 below. Expression vectors were generated by gene synthesis (Epoch Life Sciences, USA), verified by sequencing, and amplified by QIAgen Miniprep or Maxiprep kits per manufacturer's protocol. In Text talin-N10 talin-C11 talin-C1 talin-N3 talin-N12 talinFL talin-C11ABS3mut

Description mApple-talin(1-1973)-FRB FKBP12-talin(1974-2541)-mEmerald FKBP12-talin(434-2541)-mEmerald mApple-talin(1-911)-FRB mApple-talin(1-2299)-FRB Full length talin (1-2541) FKBP12-talin(1974-2541)(K2443D, V2444D, and K2445D)-mEmerald

Table 1. Nomenclature for switchable talin constructs. Live-cell Total Internal Reflection Fluorescence (TIRF) Microscopy. TIRF imaging of live cells were performed using a Nikon Eclipse Ti inverted microscope (Nikon Instruments), equipped with a motorized TIRF illuminator, with a polarization-maintaining optical fibercoupled laser combiner (100 mW 405 nm, 60 mW 488nm, 50 mW 561 nm, and 100 mW 642nm


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solid-state lasers, Omicron Laserage), a light emitting diode-based epifluorescence excitation source (SOLA, Lumencor), an ORCA-flash 4.0 sCMOS camera (Hamamatsu), a 60X N.A 1.49 Apo TIRF objective lens (Nikon Instruments), and an Okolab stage-top chamber with CO2 and temperature control (Okolab, Italy). Cells were cultured on Iwaki glass-bottom dish and pre-warmed rapamycin at the indicated concentration were manually added to the specimen. Live-cell FRAP Imaging. FRAP imaging of live cells was performed on an Olympus IX81 inverted microscope (Olympus) equipped with the iLas2 Targeted Illumination System (Cairn Research), fiber coupled laser lines (40 mW 405 nm, 200 mW 488 nm, 130 mW 561 nm and 40 mW 640 nm), 100X N.A. 1.49 UApoN TIRF objective lens (Olympus), Evolve512 EMCCD (Photometrics). A prebleach series consisting of 10 frames was aquired prior to targeted photobleaching of focal adhesions. The recovery was then monitored at 5 second intervals for 5 minutes. All analysis was performed with custom MATLAB scripts. Data was normalized and bleach corrected using a single term exponential function. Mobile fractions and half-life of each adhesions was characterized using the following expression. 𝑦 = 𝐴(1 ― 𝑒 ―𝑏𝑥) Traction Force Microscopy. A 4.1 kPa gel was prepared on squeaky clean coverslips as described in58. Prior to polymerization 200 nm 660/680 fluosphere beads (Life Technologies) were added to the solution to an appropriate density for TFM imaging. The polyacrylamide surface was activated with 1.5 mg/ml Sulpho-SANPAH (ThermoFisher) under 360 nm light for 90 seconds and coated with 1 mg/ml fibronectin for 1 hour. Transfected cells were then seeded on the gels and allowed to spread overnight. Imaging was performed on an inverted Nikon Ti Eclipse microscope (Nikon Instruments) equipped with an X1 spinning disk confocal unit, 60X N.A. 1.4 TIRF objective (Nikon) and Prime 95B sCMOS camera (Photometrics). The cell was monitored using 488 and 561 nm lasers whereas the displacement of the beads was imaged with a 647 nm laser. Bead displacement and traction forces were analyzed with MATLAB software developed previously59.

Supporting Information Supplementary methods provide more details of force calibration and force control, 𝜏𝑏𝑜𝑢𝑛𝑑 and 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 measurement, protocol of tethering methods and coverslip preparation in singlemolecule experiments. Supplementary information includes more details of data analysis of single molecule manipulation experiments, comparison between this assay using a singlemolecule detector with traditional bulk assays (Supp. Info. 1-5), error estimations (Supp. Info. 6), more details of rapamycin induced changes in cell morphology and FA profiles in living cell (Supp. Info. 7), and discussion on the significance of controlling the connectivity of mechanosensing force-transmission supramolecular linkages (Supp. Info. 8). Figures describing the representative time trace of 𝐻𝑏𝑒𝑎𝑑 in 1 nM rapamycin to measure the 𝜏𝑏𝑜𝑢𝑛𝑑 and 𝜏𝑢𝑛𝑏𝑜𝑢𝑛𝑑 for FKBP-rapamycin interaction (Fig. S1); three different tethering methods used in the single-molecule experiments (Fig. S2); the mechanical response of the rapamycin detector in the absence and presence of rapamycin (Fig. S3-9); probabilities of the four unlooped states versus the concentration of rapamycin (Fig. S10); immunofluorescence microscopy image of focal adhesion components and the actin cytoskeleton in Tln1-/-Tln2-/- cells (Fig. S11); colocalization of talinN10 with focal adhesion components and the actin cytoskeleton and the stability of rapamycin-induced focal adhesions to washout (Fig. S12); magnified views of rapamycin-induced focal adhesion from Fig. 7 (Fig. S13). Movies describing the rapid induction of FA maturation and cell migration by rapamycin (Mov. S1); essential ABS3 for creation of new focal adhesions (Mov. S2). Tables listing the fitted mean values and standard errors of binding rates (Table S1).


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Author Contributions J.Y. and P.K. conceived and supervised the research. Y.W. performed the single-molecule manipulation experiments and analyzed the data, S.B. and Z. G. performed the cell experiments, S. L. designed and expressed the single-molecule rapamycin detector, Y. W., J.Y., P.K., and S.B. wrote the manuscript.

Acknowledgement The authors thank the protein expression facility and science communication core of the Mechanobiology Institute. The research is funded by the National Research Foundation (NRF), Prime Minister's Office, Singapore under its NRF Investigatorship Programme (NRF) Investigatorship Award No. NRF-NRFI2016-03, to J.Y.), Singapore Ministry of Education Academic Research Fund Tier 3 (MOE2016-T3-1-002 to J.Y., in part), Human Frontier Science Program RGP00001/2016 grant (to J.Y., in part), the National Research Foundation, Prime Minister's Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme (to J.Y. and P.K.), the Ministry of Education Academic Research Fund Tier 2 (MOE-T2-1-124 and MOE-T2-1-045, to P.K.). S.B. is supported by the National Research Foundation Quantum Engineering Programme, QEP-P-7).

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Label-free single-molecule quantification of rapamycin-induced FKBP

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