Real-Time Measurement of Molecular Tension during Cell Adhesion

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Imaging and Diagnostics

Real-time measurement of molecular tension during cell adhesion and migration using multiplexed differential analysis of tension gauge tethers Myung Hyun Jo, Wayne Taylor Cottle, and Taekjip Ha ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01216 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 4, 2019

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ACS Biomaterials Science & Engineering

Real-time measurement of molecular tension during cell adhesion and migration using multiplexed differential analysis of tension gauge tethers Myung Hyun Jo1, W. Taylor Cottle1and Taekjip Ha1-4* 1Biophysics

and Biophysical Chemistry, 2Biomedical Engineering, 3Biophysics, Johns Hopkins

University, 4Howard Hughes Medical Institute, Baltimore, MD 21205, USA.

Corresponding Author [email protected]

KEYWORDS Cell traction force, Force transmission, Cell adhesion, Tension Gauge Tether, Integrin

ACS Paragon Plus Environment

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ABSTRACT

Cells must respond specifically and dynamically to mechanical cues from the extracellular environment and dysregulation of extracellular force sensing leads to a variety of diseases. Therefore, it is important to deconvolve the many inputs that transduce mechanical signals and understand how these signals are interpreted and responded to. DNA and peptide-based molecular force sensors have been previously developed to measure forces applied through single membrane receptors including integrins and Notch receptors. The tension gauge tether (TGT) exploits the physical rupture force of double-strand DNA to measure and modulate the force applied through single receptor-ligand bonds and can cover a wide range of tension (10-60 pN). By exploiting a fluorescent dye-quencher pair and collecting differential fluorescence signals over time, we characterized the quenched tension gauge tether (qTGT) system and developed image analysis protocol to measure molecular tension in quasi-real time. We show that this differential qTGT analysis method can simultaneously measure multiple levels of integrin-mediated molecular tension over a wide timescale during the onset of adhesion and cell migration.


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Introduction Cells rely on mechanical feedback and interaction with their micro-environments to regulate cell adhesion, migration, differentiation, and cell-cell communication1–4. Various cell membrane receptors interact through ligand binding to support mechanical interaction and signaling. The integrin superfamily is a prominent class of extracellular matrix (ECM)-receptors which recognize peptide sequences of ECM, establish physical linkages, and mediate signaling5. For example, many of these heterodimeric integrins (α5β1, αvβ1, and αvβ3) recognize the tripeptide Arg-Gly-Asp (RGD) sequence which is shared by several extracellular matrix proteins6,7. The ligand bound and mechanically activated integrins can trigger a variety of signal transduction cascades8–10. To efficiently conduct the complex signal transduction, integrins start to cluster into multi-protein structures such as nascent adhesions and adhesion complexes11,12. Some of these structures mature into larger macromolecular assemblies called focal adhesions (FAs) which are composed of various structural and signaling proteins13,14. FAs link intracellular actin bundles to the extracellular substrate and mediate regulatory effects15,16. Adhesive structure formation and disassembly are dynamic during adhesion and migration17; FA components undergo regulated turnover18. However, many details of functional interplay between components and the role of mechanical tension remain unclear partly due to the inability to measure dynamic mechanical forces in living cells at the molecular level. In particular, mechanobiology of adhesive structures is understudied during cell landing which can mimic the landing of a metastatic cell. To address this need, molecular tension sensors have recently been developed to exploit DNA19–22 or peptide23,24 structures where the length or structural stability is force dependent. The


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sensors are immobilized on a surface and conjugated with peptide or protein ligand to bind transmembrane receptors on the cell surface such as integrins and notch receptors. Fluorescent probes conjugated to the sensors report on conformation changes of the sensors induced by cellular forces. Among these sensors, the tension gauge tether (TGT) is suitable for studying strong molecular tension generated by adherent cells because it has a broader tension detection range (10-60 pN) than other assays by exploiting the stable double helix structure of DNA19. Rupture of the double helix DNA region of TGT is irreversible, so a fluorescence image of TGT rupture patterns provides a snapshot of the dynamic rupture pattern history. Though this tension map offers an accumulated summary of cell-generated force19,25, it is not ideal for realtime observation of fine structures due to photo-bleaching and negative contrast. To address this limitation, we characterized the previously demonstrated variation of TGT26,27 that we term here quenched tension gauge tether (qTGT) and used a differential image processing method, termed differential qTGT analysis, to determine only the rupture events that occurred between frames. In this way, we could reconstruct quasi-real-time tension maps free from photobleaching. By adjusting the image acquisition interval, we were able to minimize illumination for fluorescent imaging while still monitoring early adhesion without missing rupture events. We were also able to multiplex 12 pN and 54 pN qTGTs to measure both force outputs simultaneously in real time during adhesive structure formation in cell adhesion and migration28.


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Experimental Section 1. TGT and qTGT synthesis A typical TGT is a double-stranded DNA (dsDNA) conjugated with a ligand for a target receptor and a biotin for immobilization. BHQ2-conjugated DNA oligos were purchased from Biosearch Technologies and other oligos from Integrated DNA Technologies. The duplex region of TGT is 18 base pair of a previously reported sequence29. Ligand strand:

5- /BHQ-2/GGC CCG CAG CGA CCA CCC/Thiol C6 SS/ -3

Biotinylated strand (12-pN): 5'-/Biotin/TTT TTT GGG TGG TCG CTG CGG GCC/AmMO/-3' Biotinylated strand (54-pN): 5'-GGG TGG TCG CTG CGG GCC /iAmMC6T/TT TTT /Biotin/ 3' TGT and qTGT were assembled by hybridizing the ligand-conjugated DNA strand and biotinylated strand. To minimize background signal from the dye-labeled biotinylated qTGT strand, the ligand- and BHQ2- labeled strand were mixed at a 1.5:1 ratio for hybridization. 1.1. Ligand-conjugated strand RGD peptide ligand was conjugated to a thiol-modified DNA oligo using an amine-tosulfhydryl crosslinker. Cyclo (Arg-Gly-Asp-D-Phe-Lys) (PCI-3696-PI, Peptides International) was first reacted with sulfo-SMCC (22622, Thermo Scientific). The maleimide functionalized product was then conjugated to the deprotected thiol group on DNA oligo at pH 7.3 for both steps. The final products were purified by HPLC with a C18 reversed phase column. 1.2. Biotinylated strand


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NHS-PEG12-Biotin (21312, ThermoFisher scientific) was used to add a linker with a biotin for TGT strands as described in previously published protocol29. To minimize steric hindrance from the immobilization surface in the qTGT setup, we added a (dT)6 linker between the biotin and the duplex. NHS ester form of Cy3 or Atto647N was conjugated to the amine modified biotinylated strand for qTGT. The conjugated products were purified by HPLC with a C18 reversed phase column. 1.3. Double-stranded DNA rupture force estimation To estimate the DNA double helix rupture force, the model formulated by de Gennes was used with parameters which are obtained from magnetic tweezers experiment30,31.

𝑇𝑡𝑜𝑙 = 2 𝑓𝑐 [𝑥 ―1tanh

(𝑥2𝑙) + 1] ,

where fc is breaking force per base pair, l is the number of base pairs, and x is the spring constant factor of DNA. 2. Surface passivation and qTGT dish preparation The glass substrate was passivated by PEGylation to minimize non-specific binding of cell-secreted extracellular matrix proteins. Glass coverslips cleaned by piranha etching solution (a 3:1 mixture of sulfuric acid and hydrogen peroxide) were coated by Biotin-PEG (MW 5,000) and mPEG (1:19). A cell imaging chamber was 3D-printed with PLA filament and attached to the coverslip with epoxy. Neutravidin protein (0.4 mg/ml) was incubated in the chamber for 15 min and washed out thoroughly. A droplet of qTGT solution (3 µL, 1 µM) was placed on the coverslip (10 min) to locally immobilize qTGT. To correct the lateral drift in sub-pixel accuracy,


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gold nanoparticles (100 nm, biotin coated) or magnetic beads (1 µm, streptavidin coated) were immobilized sparsely. 3. Live cell imaging of cell adhesion HEK293FT and 3T3 cells were cultured in DMEM (10mM NaPyr, 10mM L-glutamate, 1x Antibiotic-Antimycotic (Gibco™), 10% fetal bovine serum (FBS)) and CHO-K1 cells were cultured in αMEM (1x Antibiotic-Antimycotic (Gibco™), 10% FBS). All cells were cultured in 5% CO2 at 37 °C. Cells were gently harvested by incubating with PBS based dissociation buffer or 0.05% trypsin for 2 min. The cells were plated on the qTGT immobilized chamber in serumdeprived cell media except for the migration study (10% FBS). Temperature and CO2 concentration in the imaging area were maintained at physiological conditions (37 °C and 5%). A Nikon Eclipse Ti microscope equipped with Nikon total internal reflection fluorescence (TIRF) microscopy module and perfect focus system driven by Elements software was used. Custom filter sets (Chroma and Thorlabs) were used for TIRF imaging as well as reflection interference contrast microscopy (RICM). All image was recorded using an electron-multiplying charge-coupled device (EMCCD; Andor iXon 888). 4. Differential qTGT analysis Time-lapsed images were obtained with a Nikon perfect focus system and the lateral drift was corrected by tracking immobilized fiducial makers. The drift trajectories were obtained using ImageJ Trackmate32 at the subpixel level. The images were registered using the trajectories, and pixel-wise intensity increase was collected frame by frame to reconstruct quasi-


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real-time tension maps. The pixel-wise intensity change was one-dimensional median-filtered in time (N=3) to reduce noise. The image processing was done with Matlab scripts.


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Result and Discussion TGT is composed of two complementary DNA strands. Typically, one strand has a ligand which binds a target receptor on the cell surface and the other strand is biotinylated for immobilization on the substrate. When a cellular receptor binds to the ligand and exerts mechanical force, TGT can endure up to a certain level of force which is called its tension tolerance (Figure 1A). The tension tolerance is dependent on the relative position of the ligand and biotin. When a mechanical force comparable to the tension tolerance ruptures the DNA duplex, the cell receptor is detached from the surface losing its mechanical tension. The ligandconjugated strand can be labeled with a fluorescent probe so that the loss of fluorescence signal from the surface is an indication of force exceeding the tension tolerance. Imaging fluorescently labeled TGTs, therefore, provides an accumulated map of the molecular forces exerted by the cell through the ligand-receptor bonds. Because the conventional TGT produces a negative contrast image, the image quality can suffer when TGT density is low or rupture events are not extensive26,33. Yet, to exploit the TGT assay for live cell imaging, the assay must detect a small number of rupture events occurring in a limited time interval. Using a quenched TGT strategy similar to what was reported by the Wang lab26,27, we aimed to achieve a positive contrast by moving the fluorescent probe to the biotinylated strand and adding a quencher to the ligand-conjugated strand (Figure 1B). In this quenched TGT assay (qTGT), BHQ2, a broad-spectrum quencher (560-670 nm), quenches the fluorescence signal. Upon tether rupture, the signal is dequenched, producing a positive contrast signal that recapitulates the pattern produced by cellular forces.


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To determine the quenching efficiency, Cy3-labeled DNA strands and the hybridized qTGTs were imaged at the single molecule level by using prism type TIRF microscopy. 1.5-fold excess BHQ2 quencher strand was added to hybridize with the Cy3-labeled strand. 40 pM DNA solution was incubated with the neutravidin-functionalized, passivated quartz substrate (Figure 1C). Hybridized DNAs were imaged with a long exposure time (1 sec) due to their low intensity. Both 12 pN and 54 pN qTGT showed more than 98 % quenching efficiency (Figure 1D) after excluding unhybridized molecules that showed strong single Cy3 fluorescence signals. Quenching efficiencies were also measured in solution using a fluorimeter, and the apparent quenching efficiencies were much lower, 80-90 %, likely due to unhybridized species or missing quenchers (Figure S1). The apparent quenching efficiencies of Atto647N-conjugated qTGTs were also measured in bulk solution and were similar to those of Cy3 qTGTs (Figure S1).


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Figure 1. Schematic of qTGTs and quenching efficiency quantification. (A) A Schematics of Tension Gauge Tether assay for cell adhesion study. (B) Fluorescent probe labeling strategies for Tension Gauge Tether assay to visualize the cumulated tension transmitted by cells. (C) Cy3-labeled DNA strand (white arrows) and the hybridized qTGT (yellow arrows) imaged at single-molecular level using prism type TIRF microscopy. 40 pM of each strand was immobilized and the number of spots was similar (n = 3; BHQ2 hybrid, 12 pN = 515 ± 9; Cy3 labeled strand, 12 pN = 541 ± 5; BHQ2 hybrid, 54 pN = 459 ± 45; Cy3 labeled strand, 54 pN = 551 ± 46). Scale bar, 10 μm. (D) Normalized intensity histograms for Cy3-labeled DNA strands and hybridized qTGT show quenching efficiencies.


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To demonstrate the positive signal contrast in a cellular context, we immobilized cyclic RGD peptide- and Cy3-conjugated 54 pN qTGT on a PEG-passivated and neutravidinfunctionalized glass coverslip, and plated CHO-K1 cells on the surface and incubated for 2 hours. Adhering CHO-K1 cell ruptured 54 pN qTGTs, showing streak patterns brighter than the background (Figure 2A), likely due to the migration of focal adhesions that are connected by actin stress fibers, as was shown in previous adhesion studies using TGT25. To compare the performance of TGT and qTGT side-by-side, we incubated 3T3 fibroblast cells for one hour on three different surfaces: TGT-54pN-Cy3, qTGT-54pN-Cy3 and qTGT-54pN-Atto647N. The fibroblast cells spread, leaving a skid-like rupture pattern. The intensity profiles show the contrast-to-noise ratio (CNR) where the signal contrast is normalized against the noise level of the intact regions without cell-induced rupture. Both qTGTs showed about 3-fold higher CNR than TGT (Figure 2B). Using qTGTs of different tension tolerance values makes it possible to monitor molecular tensions of different levels. We next tested low-tension tolerance qTGTs, qTGT-12pN-Cy3 and qTGT-12pN-Atto647N. As previously reported19, CHO-K1 cells did not stably adhere after 30 minutes incubation and caused extensive DNA tether rupture underneath (Figure 2C). The intensity profiles, normalized to the average signal of the intact qTGT region, show a qTGT rupture pattern that is much brighter than the background signal (35-fold for Cy3 and 15-fold for Atto647N).


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Figure 2. Positive contrast qTGT improves the molecular tension map for cell adhesion. Rupture patterns of qTGT during cell adhesion. (A) CHO-K1 cells were plated on qTGT54N-Cy3 for 2-hr to adhere with matured focal adhesion. Actin was imaged with SiR-Actin. Scale bar, 10 μm. (B) 3T3 fibroblast cells are plated on the TGT with different labeling schemes (TGT-54pN-Cy3, qTGT-54pN-Cy3, and qTGT-54pN-Atto647N; 1 μM) and incubated for 1 hr. Cell morphologies are shown in DIC and Cy3 or Atto647N fluorescence images are shown for ruptured TGT or qTGT. Intensity profiles are shown for each condition, the intensity is normalized to show contrast-noise-ratio. Scale bar, 10 μm. (C) CHO-K1 cells were plated on qTGT-12pN-Cy3 and qTGT-12pN-Atto647N (1 μM) for 30min. A small amount of 54 pN TGT (10 nM) was added to facilitate the cell adhesion. For background signal comparison, the edges of qTGT immobilized spots are shown. Intensity profiles across the qTGT immobilized region and the passivated glass substrate are shown.


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The intensity is normalized by the background signal of each qTGT. The boundaries are marked by dashed lines. Scale, 50 μm.

The improved qTGT rupture detection capability enhances the cellular force monitoring in real time. In Figure 3A, the first image shows the cumulated qTGT-12pN-Cy3 rupture during the adhesion of CHO-K1 cells for the first 51 minutes. Subsequent five images at 1 min intervals show additional Cy3 signal increases where individual integrin-ligand bonds experienced forces exceeding the tension tolerance. To identify cellular force between adjacent time points, newly emerging qTGT rupture events were determined by extracting the Cy3 signal increase between two sequential time-lapse images. The differential images thus acquired clearly showed new qTGT rupture signals even on areas with pre-existing rupture. From the bottom panel of Figure 3A, which shows the overlay of differential rupture images with the rupture signals accumulated for 5 minutes between t=51 minutes and t=56 minutes, we can deduce that the force-induced rupture propagated toward the cell center. We found that drift correction is critical for accurately acquiring the differential images (Figure S5). We used fiducial marker-based lateral drift correction to achieve sub-pixel accuracy in addition to the microscope-embedded auto-focus system as described in Experimental Section. In Figure 3A and 3C, the frame-to-frame Cy3 signal increase visualizes the mechanical tension of integrins at 1 min time resolution. The temporal resolution can be adjusted depending on the cellular behavior of interest by adjusting the interval of time-lapse images. For monitoring early cell adhesion, 0.5-1 min intervals were used (Figure 3 and 4), while 10 min intervals were sufficient to capture cell migration which is a slower process (Figure 5). Continuous imaging,


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which can cause excess photo-bleaching and phototoxicity, is not necessary because irreversible qTGT ruptures retain information on the molecular force exerted during the time interval but if necessary, the qTGT can withstand frequent imaging to further increase temporal resolution. Integrin clusters form large multi-protein structures called focal adhesion with adaptor proteins including talin, paxillin, and vinculin to develop mechanical links and transduce tension between intracellular actin bundles and the extracellular substrate. By applying our differential qTGT analysis, we could monitor the strong cellular tension (54 pN) exerted on the substrate with concurrent cellular morphological changes and focal adhesion formation during the entirety of early adhesion. 3T3 fibroblast cells stably transfected with GFP-paxillin were gently dissociated from culture and added onto a cyclic RGD conjugated qTGT-54pN-Cy3 surface to monitor the onset of adhesion at the moment of landing. The spreading behavior of a 3T3 cell was monitored at 1 min time intervals for 1 hr (Figure 3B and 3C). DIC images show cell morphology changes and reflection interference contrast microscopy (RICM) images show detailed cell membrane contacts near the glass substrate. The cell in Figure 3B spread out for 21 min to form a polarized morphology. RICM images show that spike-like filopodia probed the surface after cell landing, then the cell body started to spread out and made close contacts with the substrate. GFP-paxillin imaging showed that during early adhesion paxillin formed elongated structures near the edge of the cell which is typical of focal adhesions. Overlaid images of GFP-paxillin and the frame-to-frame Cy3 signal increase showed high spatial coincidence, suggesting that developing focal adhesions exerted forces exceeding 54 pN through single integrin receptors (Figure 3C and S2). Similarly, we observed the early adhesion of HEK cells on the 54 pN qTGT surface together with actin imaging using SiR-actin. The cell shape in DIC image and the actin distribution show that the


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HEK cell formed anchorage-dependent morphology while exerting strong forces exceeding 54 pN through single integrins near the anchors (Figure 3D).


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Figure 3. Differential qTGT analysis visualizes real-time tension map during early cell adhesion. (A) Workflow for differential qTGT analysis. Florescent signal (Cy3) increase between neighboring raw image frames was detected after drift correction to visualize the molecular force (12 pN) in real-time. CHO-K1 cells were plated on the qTGT immobilized surface (qTGT-12pN-Cy3 and qTGT-54pN-Atto647N, 0.5 μM each during tether deposition). Cy3 signal of 12 pN qTGT is shown here. (B-C) 3T3 fibroblast cell adhesion on qTGT-54pN-Cy3 (1 μM). DIC and RICM show the morphological change during the adhesion and GFPpaxillin images show the development of adhesive structures. Cy3 signal increase is shown to visualize 54 pN force in real-time. Time-lapse images were taken at 1 min interval. Every 4th frame was analyzed (B) to summarize the adhesion. (C) GFP-Paxillin (green) and realtime rupture (red) is overlaid. (D) HEK 293FT cell adhesion on qTGT-54pN-Cy3 (1 μM).


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The development of actin stained by SiR-Actin (red) and Cy3 increase (green) are shown in overlaid images. Scale bar, 10 μm. Next, in order to simultaneously monitor integrin tension of different magnitudes, we cultured 3T3 cells on a multiplexed qTGT surface. Using a surface with equal amounts of qTGT12pN-Cy3 and qTGT-54pN-Att647N (0.5 μM each during tether deposition), we could detect instantaneous rupture of weak (12 pN) and strong (54 pN) tethers together with the associated morphological changes of the cell during early adhesion (Figure 4A). Because differential qTGT analysis allowed for 30 second time resolution without continuous illumination we were able to identify small and transient adhesive structures (Figure 4B). When measuring 12 pN qTGT rupture during the onset of cell adhesion, we observed several puncta structures which have not been described in previous TGT studies25,34. 32 ± 6 circular spots were observed in each frame for the first 4 min with an average diameter of 0.5 ± 0.2 μm (Figure 4C and S4). The rapid appearance and small diameters suggest that these ruptures are caused by focal complexes which are small dynamic structures consisting of integrin, paxillin, and talin35. Adhesion is initiated by small nascent adhesions (~0.25 um)36, some of which quickly mature into circular shaped focal complexes (1-5 min), then continue to grow into elongated focal adhesions (Figure 4B). The puncta are short-lived, with an average lifetime of less than one 30 second frame (Figure 4B and 4D), suggesting that small integrin clusters can bind to the RGD ligands and generate short bursts of 12 pN force over small regions to probe the surface during early adhesion. The images obtained from the 54 pN qTGT showed primarily the elongated rupture pattern (Figures 4A & 4B). As shown in the overlaid images, 54 pN rupture was detected less


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frequently and colocalized with 12 pN signal (Manders overlap coefficient = 0.96; Figure S3), likely because both 12 pN and 54 pN qTGTs were ruptured together when the cell exerted strong forces onto the region. The extended and elongated structures, presumably FAs, mature over time as seen by the growing colocalized signal. This suggests that the formation of a mature FA structure facilitates the application of stronger forces.


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Figure 4. Multiplexed qTGT analysis can capture dynamic early cell adhesion. 3T3 cell adhesion carried out on multiplexed qTGT (qTGT-12pN-Cy3 and qTGT-54pNAtto647N, 0.5-μM each) 30 sec time intervals. (A) The overlays of DIC, 12 pN, and 54 pN molecular tension show adhesion and spreading. Scale bar, 10 μm. (B) Highlighted region shows dynamic punctate and elongated rupture patterns. Scale bar, 2 μm. (C) The number of 12 pN qTGT rupture puncta in early adhesion over time. (D) Lifetime of 12 pN qTGT rupture puncta.


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Finally, we observed cell locomotion following landing and spreading. The 3T3 cell shown in Figure 5 began migration 40 min after landing on a multiplexed qTGT surface. The cell migrated 20 μm over an hour period while forming blunt-ended protrusions as shown in DIC and RICM images. One major advantage of the qTGT system is that cell behaviors can be imaged over long time periods without excessive illumination. Additionally, the overlay images of RICM and GFP-paxillin show that paxillin is localized to the protrusions and molecular tension detected by 12-pN and 54-pN qTGT also localized to protrusions. The second advantage of our differential analysis is the ability to distinguish new ruptures along the path of cell migration. The overlay images in Figure 5 shows that fibroblast migration is a dynamic behavior that generates a wide range of force (12-54 pN) through integrin receptors, and differential qTGT analysis can visualize a broad range of forces during cell migration.


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Figure 5. Multiplexed qTGT analysis can visualize force exertion during cell migration. 3T3 cell migration on the multiplexed qTGT surface. Time-lapse images of cell morphology (DIC and RICM), the location of focal adhesion (GFP-paxillin), and molecular force (Cy3 increase for 12 pN and Att647N increase for 54 pN) at 10 min interval. Scale bar, 10 μm


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Conclusion We performed differential qTGT analysis using multiplexed qTGT force sensors in order to monitor two levels of molecular tension experienced through single integrin receptors in real time. The TGT is well suited for the study of the mechanically dynamic behavior of cells because it covers a broader tension range (10-60 pN) than other contemporary tension sensors which exploit DNA hairpin structures (4-18 pN)21 or elastic peptides (1-10 pN)23. In addition, the irreversibility of DNA duplex rupture allows us to monitor real-time molecular forces without requiring continuous imaging, making it possible to perform long-term imaging with minimal phototoxicity or photobleaching without missing rupture events. Using this analysis, we found that fibroblast cells transiently apply weak forces (> 12 pN) through single integrins during early adhesion which we speculate to be due to the dynamic focal complex formation. As some of these structures mature, cells are able to apply stronger forces exceeding 54 pN. These two force regimes have been studied individually using recently developed real-time molecular tension sensors21,27, but we showed that both regimes can be visualized simultaneously, offering insights into adhesive structure formation and integrinmediated mechano-transduction. Additionally, we demonstrated the potential use of this approach to study cell migration which requires long time live-cell measurements. Because this assay only requires drift correction and image processing, it can be easily applied to a variety of TGT-based cellular tension studies on various membrane receptors and cellular processes.


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ASSOCIATED CONTENT PDF for all figure. Figure S1. Quenching efficiency of qTGT in solution. Figure S2. Colocalization of focal adhesion and 54-pN qTGT rupture. Figure S3. Colocalization of 12-pN and 54-pN qTGT rupture. Figure S4. Focal complex induced molecular force in early adhesion. Figure S5. Drift correction is critical for differential qTGT analysis. Figure S6. Workflow of qTGT differential analysis.


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AUTHOR INFORMATION Corresponding Author [email protected] Author Contributions M.H.J. and T.H. conceived and designed the project. M.H.J. developed analysis tools, performed the analysis, and synthesized tension sensors. M.H.J. and T.C. carried out the experiments. M.H.J., T.H., and T.C. wrote the paper. T.H. supervised the project. Funding Sources This work was supported by U.S. NSF grant PHY 1430124 to T.H. T.H. is an investigator of the Howard Hughes Medical Institute. M.H.J. was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (2018R1A6A3A03012786). ACKNOWLEDGMENT We thank Dmitriy Bobrovnikov for help with sample purification, and Aditi Biswas for providing PEGylated coverslips, and thank Yang Liu for helpful comments in manuscript preparation. ABBREVIATIONS dsDNA, double-stranded DNA; ECM, extracellular matrix; EMCCD, electron-multiplying interference charge-coupled device; FA, focal adhesion; FC, focal complex; RGD, tripeptide Arg-Gly-Asp domain; RICM, reflection interference contrast microscopy; TGT, tension gauge tether; qTGT, quenched tension gauge tether; TIRF, total internal reflection fluorescence;


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For Table of Contents Use Only

Real-time measurement of molecular tension during cell adhesion and migration using multiplexed differential analysis of tension gauge tethers Myung Hyun Jo1, W. Taylor Cottle1and Taekjip Ha1-4* 1Biophysics

and Biophysical Chemistry, 2Biomedical Engineering, 3Biophysics, Johns Hopkins

University, 4Howard Hughes Medical Institute, Baltimore, MD 21205, USA.


31

Fig 1. Quenched Tension Gauge Tether Scheme B

dsDNA

Count

Count

1 Biotin dsDNA rupture 2 Neutravidin 3 4 Passivated and functionalized surface 5 B6 Quencher Dye 7 Dye 8 Signal loss Signal increase 9 C10 11 Cy3 strand +BHQ2 strand 12 13 14 15 0 128 255 16 D17 500 300 12 pN 400 18250 300 19200 150 200 20100 100 50 21 0 0 0.6 0.8 1.0 1.2 1.4 0.00 0.02 0.04 0.06 22 23200 600 54 pN 500 24150 400 25100 300 Environment 26 50 ACS Paragon Plus 200 27 28 29 30 31 32 33 34 35 36

100

0 0.6

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Integrins

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1.0

1.2

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0 0.00

0.02

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qTGT 12pN Figure 3C (right)

600

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cRGD

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Tension > Ttol ACS Biomaterials Science & Engineering Page 32 of 36 Cell

quenched 400

not quenched "underestimated"

200

qTGT 12pN Figure 3C (right)

600

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quenched 400

not quenched "underestimated"

200 0

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Figure 1. Schematic of quenched tension gauge tether assay. (A) A Schematics of Tension Gauge Tether assay for cell adhesion study. (B) Fluorescent probe labeling strategies for Tension Gauge Tether assay to visualize the cumulated tension exerted by cells. (C) Fluorescence spectrums show the quenching efficiencies for various combinations of organic dye s and labeling positions. The organic dye labeled DNA strands and the hybridized with a quencher labeled DNA (100-nM) were scanned. (D) Cy3-lab eled DNA strand and the hybridized are imaged at single-molecular level by using prism type TIRF microscopy. 1.5X quencher labeled strand was a dded to hybridize the Cy3-labeled stand (C-D). Scale bar, 10-μm. Note Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies)

Fig 2. Quenched Tension Gauge Tether improves the tension map (BHQ2 can be used for Cy3 and Atto647N) qTGT-54pN-Cy3

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B

DIC

TGT rupture (54pN)

line 1 ACS Biomaterials Science & Engineering

line 2

line 2

0

CNR

CNR

TGT Cy3

DIC

0

SI Rupture pattern in the lower density (10-50nM) for 3T3? More data

161112 qTGT_12pN_RGDfK_BHQ2_Cy3 (54pN added) 60X1.5 30 min incubation of CHO-K1

50

Intensity (A.U.)

A

45 40 35 30

CNR

Normalized

CNR

CNR

qTGT Cy3 qTGT Atto647N

-5

CNR

Normalized

TGT rupture (54pN) Actin

Subplot order changed 1 -10 -10 2 0 5 10 15 20 25 0 5 10 15 20 25 line 1 9/30 Distance (m) Distance (m) 3 30 Distance (m) line 2 20 4 20 5 10 10 6 line 1 0 0 7 0 5 10 15 20 25 0 5 10 15 20 25 Distance (m) Distance (m) 8 30 25 line 2 25 20 9 20 15 15 10 10 10 5 5 11 0 0 12 0 5 10 15 20 25 0 5 10 15 20 25 line 1 Distance (m) Distance (m) 13 C14 qTGT-12pN-Cy3 qTGT-12pN-Atto647N 15 20 40 16 15 30 17 10 20 18 passivated passivated 5 10 19 ACS Paragon Plus Environment 0 0 20 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 21 Distance (m) Distance (m) 22 23 Figure 2. Positive contrast qTGT improves the molecular tension map for cell adhesion. 24 Rupture patterns of qTGT during cell adhesion. (A) CHO-K1 cells were plated on qTGT-54N-Cy3 for 2-hr to adhere with matured focal adhesion. Actin was imaged with SiR-Actin. Scale bar, 10 μm. (B) 3T3 fibrobla 25 st cells are plated on the TGT with different labeling schemes (TGT-54pN-Cy3, qTGT-54pN-Cy3, and qTGT-54pN-Atto647N; 1 μM) and incubated for 1 hr. Cell morphologies are shown in DIC and Cy3 or Atto647N 26 fluorescence images are shown for ruptured TGT or qTGT. Intensity profiles are shown for each condition, the intensity is normalized to show contrast-noise-ratio. Scale bar, 10 μm. (C) CHO-K1 cells were plated on 27 qTGT-12pN-Cy3 and qTGT-12pN-Atto647N (1 μM) for 30-min. Small amount of 54 pN TGT (10 nM) was added to facilitate the cell adhesion. For background signal comparison, the edges of qTGT immobilized spot s are 28 shown. Intensity profiles across the qTGT immobilized region and passivated glass substrate are shown. The intensity is normalized by the background signal of each qTGT. The boundaries are marked by dashe d 29 lines. Scale, 50 μm. 30 31 32 33 34 35 36 -5

25 20 15 10

5 0

0

5 10 15 20 25 30 35 40 45 50

Fig 3. Time-lapse reconstitution of molecular force B

A

1 min

5


DIC

DIC

1. Obtain time-lapse images with fiducial markers

9

13

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Page 34 of 36

RICM

2. Correct the drift by

Cy3 increase GFP-Paxillin (54 pN) Cy3 increase (54 pN) GFP-Paxillin

Cy3 increase (54 pN)

Sir-Actin

DIC

Cy3 increase

Y

Cy3

tracking the markers 1 2 3 1 2 3 X 4 2 3 5 3. Collect the fluorescence signal (F) 6 increase frame by frame Fincrease = Fn – Fn-1 7 D8 C 1 min 2 3 4 5 6 0 min 5 10 15 25 30 SI? 9 Schemetics? 10 7 8 9 10 11 12 40 45 50 55 60 11 35 12 13 13 14 15 16 17 18 0 min 5 10 15 25 30 1 2 3 14 – = 15 2 1 2 ACS Paragon Plus Environment 19 20 21 22 23 24 40 45 50 55 60 16 35 17 18 19 4. Collect the fluorescence signal increase frame by frame using Matlab script. 20 Fig 3. Differential analysis of qTGT for real-time tension map visualization during cell adhesion. 21 (A) Cy3 signal increase between neighboring raw Cy3 image frames are detected to visualize the molecular force (12-pN) in real-time. Arrows indicate the new qTGT ruptures. CHO-K1 cell is plated on the qTGT immobilized 22 surface (qTGT-12pN-Cy3 and qTGT-54pN-Atto647N, 0.5-μM each). Cy3 signal of 12-pN qTGT is shown here. (B-C) 3T3 fibroblast cell adhesion on qTGT-54pN-Cy3 (1-μM). DIC and RICM shows the morphological chang 23 e during the adhesion and GFP-paxillin images show the development of adhesive structures. Cy3 signal increase is shown to visualize 54-pN force in real-time. Time-lapse images were taken at 1-min interval. Every 4th frame 24 is analyzed and shown in (B) to summarize the adhesion. (C) GFP-Paxillin (green) and real-time rupture (red) is overlaid. (D) HEK 293FT cell adhesion on qTGT-54pN-Cy3 (1-μM). The development of actin stained by SiR-A 25 ctin (red) and Cy3 increase (green) are shown in overlaid images. Scale, 10-μm. 26 27 #To be improved: 28 29 30 31 32 33 34 35 36

Fig 4. simultaneous imaging of 12-pN and 54-pN force. G:\ExpData_Cell\2017\170825_qTGTmix_3T3\90x_qTGTmix_3T3_003_located\ana by a4 A

DIC Cy3 increase (12 pN) Atto647N increase (54 pN)

Page 35 of 36


B

400*13/90 : 0.8 = 10 : x, x = 0.1385 400*13/90 : 0.9 = 10 : x, x = 0.157

1

02min

2-10fr X 0.5min

0.5

1

1.5

2

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Count

Number

B 60 X 120 pixel 3 120*13/90 : 1.2 = 2 : x, x = 0.138 4 C 5 60 50 36 3.5 4 4.5 5 5.5 40 7 30 20 8 10 9 0 1 2 3 4 10 Time (min) 6 6.5 7 7.5 8 8.5 11 D 200 9 9.5 10 10.5 11 11.5 12 150 cRGD 12 pN 13 100 14 ACS Paragon Plus Environment Cy3 50 15 B biotin 0 1.5 2 2.5 3 3.5 4 30 60 90 16min Lifetime (sec) BHQ2 17 18 Atto677N 19 54 pN 20 21 22 4. Multiplexed qTGT analysis for cell adhesion. Figure 23 cell adhesion procedure on multiplexed qTGT (qTGT-12pN-Cy3 and qTGT-54pN-Atto647N, 0.5-μM each). Time-lapse images were taken at 30 sec interval. (A) The overlay of DIC, 12-pN, and 54-pN molecular tension sho 3T3 w24 the adhesion and spreading. Scale bar, 10 μm. (B) Highlighted region shows dynamic punctate and elongated rupture patterns. Scale bar, 2 μm. (C) The number of 12 pN qTGT rupture puncta in early adhesion over time. (D) Li fetime 25 of 12 pN qTGT rupture puncta. 26 27 28 29 30 31 32 33 34 35 36

Fig 5. Molecular force measurement during cell migration on Multiplexed qTGT

DIC

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3T3 fibroblast locomotion after its landing and spreading can be monitored in longer observation. The 3T 3 cell shown in Figure 5 started to migrate 40-min after its landing, it moved about 20-μm for 1-hr on mult iplexed qTGT surface forming blunt-ended protrusions. It is known that fibroblast cell can quickly switch b etween a lamellipodia-driven migration mode and lobopodia-mediated locomotion depending on the envir onmental conditions and that the lobopodia still assemble focal adhesions (Michael Sixt 2012, Petrie 201 2), the 3T3 fibroblast migration on qTGT looks like lobopodial. The overlay images of RICM and GFP-pa xillin show that paxilllins are localized to the protrusions. Molecular tension detected by 12-pN and 54-pN qTGT was also localized to the protrusions. This observation shows that fibroblast migration is a dynamic behavior generating strong molecular force (> 54-pN) on integrin receptors, and qTGT analysis is a powe rful tool to visualized the wide range of force with a long observation time.

12pN+54pN

54pN

12pN

GFP-paxillin

RICM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

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Fibroblasts migrate on two-dimensional (2D) surfaces by forming lamellipodia—ac tin-rich extensions at the leading edge of the cell that have been well characterize d. In this issue, Petrie et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.20120 1124) show that in some 3D environments, including tissue explants, fibroblasts p roject different structures, termed lobopodia, at the leading edge. Lobopodia still a ssemble focal adhesions; however, similar to membrane blebs, they are driven by actomyosin contraction and do not accumulate active Rac, Cdc42, and phosphatid ylinositol 3-kinases.

Fig 5. Multiplexed qTGT analysis for cell migration 3T3 cell migration on multiplexed qTGT surface. Time-lapse images of cell morphology (DIC and RICM), location of focal adhesion (GFP-paxillin), and molecular force (Cy3 increase for 12-pN and Att647N increase for 54-pN) at 10-min interval. Scale, 10-μm

Real-Time Measurement of Molecular Tension during Cell Adhesion

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