A General Approach for Generating Fluorescent Probes to Visualize Piconewton Forces at the Cell Surface

Article abstract of DOI:10.1021/jacs.5b11602

Mechanical forces between cells and their extracellular matrix (ECM) are mediated by dozens of different receptors. These biophysical interactions play fundamental roles in processes ranging from cellular development to tumor progression. However, mapping the spatial and temporal dynamics of tension among various receptor-ligand pairs remains a significant challenge. To address this issue, we have developed a synthetic strategy to generate modular tension probes combining the native chemical ligation (NCL) reaction with solid phase peptide synthesis (SPPS). In principle, this approach accommodates virtually any peptide or expressed protein amenable to NCL. We generated a small library of tension probes displaying different ligands, flexible linkers, and fluorescent reporters, enabling the mapping of integrin and cadherin tension, and demonstrating the first example of long-term (～3 days) molecular tension imaging. This approach provides a toolset to better understand mechanotransduction events fundamental to cell biology.

Full text of DOI:10.1021/jacs.5b11602

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI
Communication
A General Approach for Generating Fluorescent Probes
to Visualize Piconewton Forces at the Cell Surface
Yuan Chang, zheng Liu, Yun Zhang, Kornelia Galior, Jeffery Yang, and Khalid Salaita
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11602 • Publication Date (Web): 12 Feb 2016
Downloaded from http://pubs.acs.org on February 13, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
A General Approach for Generating Fluorescent Probes to
Visualize Piconewton Forces at the Cell Surface
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Yuan Chang, Zheng Liu, Yun Zhang, Kornelia Galior, Jeffery Yang and Khalid Salaita*
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia, United States
Supporting Information Placeholder
within 24 hrs.11 Ligand exchange is further exasperated in
ABSTRACT: Mechanical forces between cells and their ex-
biological media containing ~100 ꢀM thiol bearing mole-
cules.12 Alternatively, Blakely and colleagues used amine-
thiol heterobifunctional linkers to immobilize DNA tension
probes.13 However, this crosslinking chemistry limits the
choice of ligands to molecules lacking lysine and cysteine.
Therefore, new bio-orthogonal approaches for covalent im-
mobilization of molecular tension probes are needed.
tracellular matrix (ECM) are mediated by hundreds of differ-
ent receptors. These biophysical interactions play fundamen-
tal roles in processes ranging from cellular development to
tumor progression. However, mapping the spatial and tem-
poral dynamics of tension among various receptor-ligand
pairs remains a significant challenge. To address this issue,
we have developed a synthetic strategy to generate modular
tension probes combining the native chemical ligation (NCL)
reaction with solid phase peptide synthesis (SPPS). In princi-
ple, this approach accommodates virtually any peptide or
expressed protein amenable to NCL. We generated a small
library of tension probes displaying different ligands, flexible
linkers, and fluorescent reporters, thus mapping integrin and
cadherin tension, and demonstrating the first example of
long-term (~3 days) molecular tension imaging. This ap-
proach provides a toolset to better understand mecha-
notransduction events fundamental to cell biology.
Scheme 1. Synthetic scheme for generating ligand-general mo-
lecular tension fluorescence microscopy (MTFM) probes.
Cells sense and transduce physical signals from their ex-
ternal environment using hundreds of different cell surface
receptors. Transmembrane adhesion receptors, such as in-
tegrins and cadherins, mediate cell-cell and cell-extracellular
matrix (ECM) adhesions, and transmit the mechanical forces
responsible for giving tissue their intrinsic architecture.1 Be-
cause integrin2–4and cadherin5 receptor function is highly
dependent on mechanics, there is a pressing need to develop
specific molecular probes to map receptor forces throughout
the chemo-mechanical coupling cycles controlling cell fate.
We overcome this challenge by developing a new class of
stable and modular MTFM probes that is generated using
solid phase peptide synthesis (SPPS) along with the NCL
reaction (Scheme 1). This approach allows one to site-
specifically incorporate a ligand and a pair of chromophores,
and is compatible with virtually any peptide of interest. SPPS
was used to generate a “spring” composed of a C-terminal
alkyne-modified amino acid followed by lysine, discrete
PEG24/PEG48, and terminated with an N-terminal cysteine, 1
(Scheme 1). The lysine was coupled to an NHS-ester modified
tetramethylrhodamine (TAMRA) fluorophore. The peptide
conjugate was then cleaved and purified using reverse phase
HPLC (RP-HPLC) and verified using MALDI-TOF (Figure S1).
We also synthesized a small library of α-thioester modified
peptide ligands selected based on their specificity toward the
integrin and cadherin receptors (Figure S2-S3, SI Material
and Methods). These peptide ligands recapitulate many of
the cell responses in cell-cell and cell-ECM adhesions.14,15
These ligands included: 1) cyclic RGDfK (cRGDfK) and linear
We recently developed molecular tension-based fluores-
cence microscopy (MTFM) to visualize receptor forces.6–10 In
MTFM, an extendable polymer (eg. polyethylene glycol (PEG)
or DNA) is flanked by a fluorescent donor at one terminus
and a quencher or acceptor at the second terminus and the
construct is immobilized on a surface. Receptor-mediated
forces extend the polymer “spring”, which de-quenches the
fluorophore allowing the transduction of a mechanical signal
into an optical readout. Recently, we found that biotin-
streptavidin-immobilized MTFM probes were dissociated
due to integrin receptor forces.8 This was unexpected be-
cause this association is described as the strongest non-
covalent bond in nature. Given that integrins exert sufficient
tension to dissociate the biotin-streptavidin bond, we next
developed nanoparticle-based MTFM probes that employ
thiol-gold binding for immobilization.7,10 Nonetheless, thio-
lated ligands are known to dissociate from the Au surface
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 11
GRGDS peptides derived from the fibronectin-III repeat 10
brush-like conformations.24,25 In contrast, the 1.3 nm length
1
2
3
4
5
6
7
8
(FN-III10), which primarily bind the α5β1 and αvβ3
integrins,16 2) the synergy site PHSRN derived from FN-III9,
which is reported to increase cell adhesion when combined
with the RGD sequence, 3) the PHSRN(SG)4RGDS peptide,
which includes a spacer between the PHSRN and GRGDS
peptides and better mimics the distance between the two
binding domains in FN17 and 4) the SHAVSS and
LRAHAVDING peptides, which bind the E-cadherin and N-
cadherin receptors, respectively.15,18 Standard SPPS protocols
were used to generate these α-thioester linear peptides, ex-
cept for the cRGDfK sequence for which we used a protocol
adapted from Xiao et al.19 Standard NCL conditions were
used to couple the peptide to 1 (Figure S4). We subsequently
took advantage of the free thiol group inherent to the NCL
reaction to site-specifically couple maleimide-modified dyes
that take part in FRET with the TAMRA fluorophore, 2. Typi-
cally, we employed Alexa 488 due to its appropriate Forster
radius R0 = 5.9 nm with TAMRA (Supplementary Note 1).
The molecular probes were then covalently immobilized
onto an azide-modified glass slide using standard click reac-
tion conditions. Fluorescence microscopy and FRET analysis
showed uniform surface coverage of the tension probes and
high quenching efficiency (Figure S5-7). Withholding Cu(II)
or using surfaces that lacked the azide did not lead to signifi-
cant binding of the probe, thus confirming the specificity of
the click reaction. The surface density of tension probes
ranged from 9000 to 11000 probes/ꢀm2, as determined by
quantitative fluorescence imaging (Figure S8).20
of the SBS was insufficient to crowd the tension probes (Fig-
ure 1d). Therefore, SBS provides superior passivation against
biofouling and concurrently improves probe sensitivity by
reducing background signal. SBS passivation was used in all
subsequent cell studies. This result represents the first
demonstration of using SBS for cell culture, outperforming
PEG polymers, the most widely used reagent for passivation
against biofouling.
To test the cRGDfK tension probe, NIH 3T3 cells were
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To maximize the interaction of the cell receptors with ten-
sion probes, we passivated the surface against non-specific
binding of cell-generated ECM and serum proteins. Typically,
PEG polymers are used for passivation21. However, we found
that employing PEG polymers for passivation increased
background signal and diminished sensitivity (Figure 1). To
solve this problem, we tested the zwitterionic silane (3-
(dimethyl-(3-(trimethoxysilyl)propyl)ammonio) propane-1-
sulfonate), (SBS) (Figure S5),22 and compared its efficiency of
passivation against that of PEG polymers (Mw = 2000, 5000
Da).23 SBS provided enhanced passivation against cell adhe-
sion in comparison to PEG polymers (Figure 1a-1b). Given the
importance of the molecular extension of the tension probes,
we next aimed to estimate the mean inter-fluorophore dis-
tance when the surface was PEG passivated rather than SBS
passivated. This was achieved by measuring the FRET effi-
ciency using a TAMRA-PEG24-fluorescein probe and report-
ing the acceptor-normalized donor intensity (Figure S9). The
donor-acceptor distance was greater for the PEG2000 and
PEG5000 surfaces in comparison to the SBS passivated sur-
face (Figure 1c). The mean inter-fluorophore distance for SBS
surfaces was approximately 2.6 nm, which is in agreement
with the predicted 2.4 nm distance based on the Flory model.
The TAMRA-fluorescein distance increases by ~1 nm when
the surface is passivated using PEG5000, which leads to a
~15% decrease in quenching efficiency. These results indicate
that the PEG polymer passivation (~2000 Da and ~5000 Da)
leads to molecular crowding of the tension probe, thus plac-
ing it in a more extended conformation compared to the
samples prepared with SBS passivation. The extension of the
tension probe upon passivation with PEG polymers is con-
sistent with the literature showing that polymers with fixed
grafting density and increasing molecular weight tend to
increase crowding and the transition of polymers to more
Figure 1. Role of passivation in MTFM probe conformation. (a) Repre-
sentative brightfield images of NIH 3T3 cells plated for 2.5 hrs on SBS,
PEG2000, bare glass, and cRGDfK substrates. Scale bar = 100 μm. (b)
Plot showing average cell density on cRGDfK, bare glass, PEG2000,
PEG5000, and SBS substrates as a function of time (24 hrs). Error bars
represent the standard error of the mean (SEM) of n = 3 substrates
where a total of 10 regions of interest were averaged from these sam-
ples. (c) Bar graph showing the average extension (left y-axis) and
acceptor intensity (right y-axis) of MTFM probes as a function of pas-
sivation. Error bars represent the SEM of n=3 substrates, where a total
of 10 regions were imaged. (d) Model showing the influence of pas-
sivation molecule on crowding and extension of tension probes.
plated onto TAMRA-QSY9 sensor modified surfaces for 1 hr
to allow the cells to adhere. We used epifluorescence micros-
copy to image the live cell tension response and reflection
interference contrast microscopy (RICM) to monitor cell-
substrate binding. A dynamic increase in fluorescence in the
tension channel (TAMRA) was observed in regions associat-
ed with the cell-binding pattern in RICM (Figure 2a and
Movie S1). Tension signal colocalized with GFP-tagged β3
integrin, confirming that tension signal is integrin mediated
(Figure S10, 11). The spatial distribution of tension was rela-
tively dynamic, changing on a time scale of tens of minutes
(Figure S12a). We used the worm-like chain (WLC) model
and the measured quenching efficiency to estimate the aver-
age tension per integrin ligand (Figure 2a and Supplementary
Note 2-3). Tension signal dissipated upon treating cells with
latruculin B (latB), an actin polymerization inhibitor, indicat-
ing that the signal can be reduced by inhibiting the cellular
cytoskeleton (Figure S12b and Movie S2). Taken together, the
dynamic fluctuations in tension signal, live cell dual channel
integrin/tension imaging, along with the latB experiment
show that the reversible fluorescence response is due to me-
ACS Paragon Plus Environment

Page 3 of 11
Journal of the American Chemical Society
chanical tension exerted by integrin receptors engaged to the
3b). Note that the magnitude of integrin tension was corre-
1
2
3
4
5
6
7
8
MTFM sensor. Given the predicted force-fluorescence curve
for the PEG24 probes, we expect that the dynamic range of
the probes is limited to ~15 pN; the stiffness of the probes
increases drastically as the polymer approaches its contour
length (Figure S13). The PEG48-based tension probes per-
formed similarly to the PEG24 probes, confirming the modu-
larity of the synthetic approach. The calculated dynamic
range of the PEG48 probes is limited to ~5 pN, which suggests
that these are more appropriate for signaling pathways that
lated with the average size of FAs. As cells fully spread, FAs
became slightly smaller in area, and this was accompanied by
a decrease in integrin tension. This represents the longest
imaging window for mapping receptor-ligand tension using a
molecular probe. Minimal change was observed in the fluo-
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Representative RICM, fluorescence, overlay of fluorescence
and RICM, and quantified heat map of tension for cells cultured on the
cRGDfK (TAMRA-QSY9) and SHAVSS (TAMRA-Alexa 488) tension
probe surfaces. Top row shows an NIH 3T3 cell cultured on the cRGDfK
tension probe for 1 hr, while the bottom row shows an MDCK cell cul-
tured on the SHAVSS peptide tension probe for 3 hrs. Raw fluorescence
data were converted to a force map. Scale bar = 10 μm.
Figure 3. Long-term live cell mechano-imaging using TAMRA-Alexa
488 sensor. (a) RICM and fluorescence images showing the cell-
substrate contact zone along with a map of integrin tension at the
indicated time points that spanned from 20 min to 64 hrs. Note the
changes in cell morphology and force pattern. Scale bar = 10 ꢀm. (b)
Plot showing the average tension within FAs (left y-axis), and average
FA area (right y-axis) as a function of time over a period of 64 hrs. The
error bars represent the SEM from n=3-4 cells, where 10-30 FAs were
employ a lower magnitude of tension such as the Notch
pathway.4,26
rescence background of both the donor and the acceptor
during the 64-hour imaging window (Figure S15).
To study the tension generated by E-cadherin receptors,
we plated endothelial cells (MDCK) on the SHAVSS peptide
tension probes. In contrast to the FA tension patterns ob-
served for the cRGDfK peptide sensor, we observed punctate
tension across the cell-substrate junction. The intensity of
tension signal was significantly lower for the SHAVSS pep-
tide compared to the cRGDfK peptide. The SHAVSS tension
signal was abolished upon treating cells with latB, showing
that the signal is generated by the cellular cytoskeleton (Fig-
ure S14). Immunostaining for the E-cadherin extracellular
domain EC4 displayed puncta at the basal cell surface re-
sembling the signal associated with E-cadherin tension in
our assays (Figure S14). We also found that 3T3 fibroblasts
did not adhere to the SHAVSS surface, confirming that E-
cadherin expression is necessary for cell adhesion. Im-
portantly, tension sensors specific to the N-cadherin ligand,
LRAHAVDING, did not yield detectable signal when rat dor-
sal root ganglion (DRG) neurons were cultured onto sub-
strates (data not shown). Taken together these results indi-
cate that E-cadherin-binding ligands experience lower values
of tension than that of integrin ligands, which may reflect the
mechanics of cadherin signaling or binding affinity and re-
ceptor density differences among these cell types.
The peptide PHSRN is found in the FN-III9, adjacent to
the 10th domain that contains the RGD peptide.27 PHSRN has
been identified as a synergy ligand that enhances the spread-
ing of cells on the RGD peptide.27–30 We asked whether
PHSRN carries a mechanical load much like the RGD peptide
that supports adhesion. Tension probes with the PHSRN,
cRGDfK, PHSRN(SG)4RGDS and linear GRGDS peptides were
immobilized on glass slides. Cells attached and spread ineffi-
ciently on PHSRN substrates (Figure 4a). In contrast, cells
plated onto surfaces comprised a binary mixture of PHSRN
and GRGDS/cRGDfK probes (1:1) spread efficiently. This is in
agreement with recent reports showing that PHSRN enhanc-
es cell spreading with RGD but is inactive when presented
exclusively.31,32 The tension signals for cells cultured on
PHSRN(SG)4RGDS, GRGDS and cRGDfK were similar and
greater than that of substrates with the binary mixture of
GRGDS/cRGDfK and PHSRN (Figure 4b). Note that the sam-
ples modified with the binary mixture of RGD ligands and
PHSRN display the same total density of MTFM probes but
50% of the RGD ligand density compared to the single com-
ponent surfaces. Although the affinity of integrins for
cRGDfK is greater than that of linear GRGDS, the signals
were similar for both ligands, which is in agreement with
results obtained using DNA-based tension probes.9 We were
not able to detect tension signals on the substrates present-
ing PHSRN exclusively. These data indicate that mechanical
tension is not transmitted through the PHSRN synergy lig-
and, rather its role is most likely in enhancing integrin-ligand
affinity. This conclusion clarifies a long-standing question
regarding the mechanical role of the PHSRN ligand in cell
adhesion. We expect that MTFM probes generated using this
modular approach will help elucidate the role of various
To test the capability of the tension probes in long-term
imaging, we incubated NIH 3T3 fibroblasts on cRGDfK-
Alexa488-TAMRA tension probes. Cells displayed tension
patterns at the cell periphery similar to that of the FA mark-
ers (Figure 3). After 20 min of incubation, the tension signal
became more elongated as cells polarized (Figure 3a). We
followed the cRGDfK tension pattern for a period of 64 hrs.
During this time, the average tension signal within FAs in-
creased initially and then decreased as cells spread (Figure
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 11
ECM components in mediating mechanotransduction pro-
Weizmann Institute, for rat embryonic fibroblast cells trans-
1
2
3
cesses. A general caveat of this approach is that the dynamic
range of the sensor is limited to ~15 pN; thus, while we are
fected with the β3-integrin-GFP and David Lynn, Depart-
ment of Chemistry, Emory University, for solid phase peptide
synthesizer.
4
5
6
7
8
REFERENCES
(1)
(2)
Hynes, R. O. Cell 2002, 110 (6), 673.
Tan, J. L.; Tien, J.; Pirone, D. M.; Gray, D. S.; Bhadriraju, K.;
Chen, C. S. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (4), 1484.
Legant, W. R.; Choi, C. K.; Miller, J. S.; Shao, L.; Gao, L.;
Betzig, E.; Chen, C. S. Proc. Natl. Acad. Sci. U. S. A. 2013, 110
(3), 881.
9
(3)
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(4)
(5)
Wang, X.; Ha, T. Science 2013, 340 (6135), 991.
Borghi, N.; Sorokina, M.; Shcherbakova, O. G.; Weis, W. I.;
Pruitt, B. L.; Nelson, W. J.; Dunn, A. R. Proc. Natl. Acad. Sci.
U. S. A. 2012, 109 (31), 12568.
Stabley, D. R.; Jurchenko, C.; Marshall, S. S.; Salaita, K. S. Nat.
Methods 2012, 9 (1), 64.
Liu, Y.; Yehl, K.; Narui, Y.; Salaita, K. J. Am. Chem. Soc. 2013,
135 (14), 5320.
Jurchenko, C.; Chang, Y.; Narui, Y.; Zhang, Y.; Salaita, K. S.
Biophys. J. 2014, 106 (7), 1436.
Zhang, Y.; Ge, C.; Zhu, C.; Salaita, K. Nat. Commun. 2014, 5,
5167.
Liu, Y.; Medda, R.; Liu, Z.; Galior, K.; Yehl, K.; Spatz, J. P.;
Cavalcanti-Adam, E. A.; Salaita, K. Nano Lett. 2014, 14 (10),
5539.
Bhatt, N.; Huang, P. J. J.; Dave, N.; Liu, J. Langmuir 2011, 27,
6132.
Mansoor, M. A.; Svardal, A. M.; Ueland, P. M. Anal. Biochem.
1992, 200 (2), 218.
Blakely, B. L.; Dumelin, C. E.; Trappmann, B.; McGregor, L.
M.; Choi, C. K.; Anthony, P. C.; Duesterberg, V. K.; Baker, B.
M.; Block, S. M.; Liu, D. R.; Chen, C. S. Nat. Methods 2014, 11
(12), 1229.
Rajagopalan, P.; Marganski, W. a; Brown, X. Q.; Wong, J. Y.
Biophys. J. 2004, 87 (4), 2818.
Ung, P.; Winkler; Winkler, D. A. J. Med. Chem. 2011, 54 (5), 1.
Pfaff, M.; Tangemann, K.; Müller, B.; Gurrath, M.; Müller, G.;
Kessler, H.; Timpl, R.; Engel, J. J. Biol. Chem. 1994, 269 (32),
20233.
Craig, J. a.; Rexeisen, E. L.; Mardilovich, A.; Shroff, K.;
Kokkoli, E. Langmuir 2008, 24 (18), 10282.
Noë, V.; Willems, J.; Vandekerckhove, J.; Roy, F. Van;
Bruyneel, E.; Mareel, M. J. Cell Sci. 1999, 112, 127.
Xiao, J.; Chen, R.; Pawlicki, M. A.; Tolbert, T. J. J. Am. Chem.
Soc. 2009, 131 (38), 13616.
Galush, W. J.; Nye, J. a; Groves, J. T. Biophys. J. 2008, 95 (5),
2512.
Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26 (1),
365.
Estephan, Z. G.; Jaber, J. a; Schlenoff, J. B. Langmuir 2010, 26
(22), 16884.
Jain, a; Liu, R.; Xiang, Y. K.; Ha, T. Nat. Protoc. 2012, 7 (3),
445.
(6)
(7)
(8)
(9)
(10)
Figure 4. The role of RGD and PHSRN peptides in mediating integrin
tension. a) Representative RICM and fluorescence tension images of
3T3 fibroblasts cultured onto PHSRN(SG)4RGDS, GRGDS, cRGDfK, 1:1
GRGDS: PHSRN, 1:1 cRGDfK: PHSRN, and PHSRN MTFM probes
(TAMRA-Alexa 488). Scale bar = 10 μm and contrasts are set identical-
ly. b) Bar graph showing the average tension normalized to the back-
ground for cells cultured onto the above substrates (a). Data obtained
in triplicate from n=8 cells in each category for a total of 40 cells,
where 10-30 FAs were analyzed from each cell. Note that the average
tension for the PHSRN probe was ~2% below the background signal
likely due to optical effects from cell adhesion.
(11)
(12)
(13)
able to detect differences in the ensemble average tension
signal, receptor forces that are >>15 pN are not distinguisha-
ble from lower magnitude signals. Therefore, the lack of sta-
tistical difference in tension signal between the GRGDS,
cRGDfK, and PHSRN(SG)4RGDS probes may be due to probe
sensitivity rather than the lack of biophysical difference.
(14)
(15)
(16)
In summary, we demonstrated that the integration of
SPPS and NCL for MTFM synthesis provides a general and
modular approach overcoming stability issues and providing
improved sensitivity over previous strategies for tension
probe design. This probe can be used to image receptor-
ligand forces for peptides amendable to the NCL reaction,
which are generally smaller peptides and proteins. There are
also some limitations in the choice of dyes that emit in the
near-infrared, as these are not generally compatible with
SPPS.
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
ASSOCIATED CONTENT
Supporting Information
Supporting Information and movies. This material is availa-
ble free of charge via the Internet at http://pubs.acs.org.
Backmann, N.; Kappeler, N.; Braun, T.; Huber, F.; Lang, H.-P.;
Gerber, C.; Lim, R. Y. H. Beilstein J. Nanotechnol. 2010, 1, 3.
De Gennes, P. G. Macromolecules 1980, 13 (19), 1069.
Kopan, R.; Ilagan, M. X. G. Cell 2009, 137 (2), 216.
Aota, S.; Nomizu, M.; Yamada, K. M. J. Biol. Chem. 1994, 269
(40), 24756.
Redick, S. D. J. Cell Biol. 2000, 149 (2), 521.
Petrie, T. a; Capadona, J. R.; Reyes, C. D.; García, A. J.
Biomaterials 2006, 27 (31), 5459.
Li, F.; Redick, S. D.; Erickson, H. P.; Moy, V. T. Biophys. J.
2003, 84 (2), 1252.
Feng, Y.; Mrksich, M. Biochemistry 2004, 43 (50), 15811.
Eisenberg, J. L.; Piper, J. L.; Mrksich, M. Langmuir 2009, 25
(24), 13942.
(25)
(26)
(27)
AUTHOR INFORMATION
Corresponding Author
k.salaita@emory.edu
(28)
(29)
Notes
(30)
The authors declare no competing financial interests.
(31)
(32)
ACKNOWLEDGMENT
K.S. is grateful for support from the NIH (R01-GM097399),
the Alfred P. Sloan Research Fellowship. We also thank Dr.
Benjamin Geiger, Department of Molecular Cell Biology at
ACS Paragon Plus Environment

Page 5 of 11
Journal of the American Chemical Society
Insert Table of Contents artwork here
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 11
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthetic scheme for generating ligand-general molecular tension fluorescence microscopy
(MTFM) probes.
87x67mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 7 of 11
Journal of the American Chemical Society
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Role of passivation in MTFM probe conformation. (a) Representative brightfield images of NIH 3T3
cells plated for 2.5 hrs on SBS, PEG2000, bare glass, and cRGDfK substrates. Scale bar = 100 µm. (b) Plot
showing average cell density on cRGDfK, bare glass, PEG2000, PEG5000, and SBS substrates as a function
of time (24 hrs). Error bars represent the standard error of the mean (SEM) of n = 3 substrates where a
total of 10 regions of interest were averaged from these samples. (c) Bar graph showing the average
extension (left y-axis) and acceptor intensity (right y-axis) of MTFM probes as a function of passivation.
Error bars represent the SEM of n=3 substrates, where a total of 10 regions were imaged. (d) Model
showing the influence of passivation molecule on crowding and extension of tension probes.
81x76mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 11
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Representative RICM, fluorescence, overlay of fluorescence and RICM, and quantified heat map of
tension for cells cultured on the cRGDfK (TAMRA-QSY9) and SHAVSS (TAMRA-Alexa 488) ten-sion probe
surfaces. Top row shows an NIH 3T3 cell cultured on the cRGDfK tension probe for 1 hr, while the bottom
row shows an MDCK cell cultured on the SHAVSS peptide tension probe for 3 hrs. Raw fluorescence data
were converted to a force map. Scale bar = 10 µm.
87x43mm (150 x 150 DPI)
ACS Paragon Plus Environment

Page 9 of 11
Journal of the American Chemical Society
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Long-term live cell mechano-imaging using TAMRA-Alexa 488 sensor. (a) RICM and fluorescence
images showing the cell-substrate contact zone along with a map of integrin tension at the indicated time
points that spanned from 20 min to 64 hrs. Note the changes in cell morphology and force pattern. Scale bar
= 10 µm. (b) Plot showing the average tension within FAs (left y-axis), and aver-age FA area (right y-axis)
as a function of time over a period of 64 hrs. The error bars represent the SEM from n=3-4 cells, where 10-
30 FAs were analyzed from each cell.
84x58mm (150 x 150 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 11
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. The role of RGD and PHSRN peptides in mediating integrin tension. a) Representative RICM and
fluorescence tension images of 3T3 fibroblasts cultured onto PHSRN(SG)4RGDS, GRGDS, cRGDfK, 1:1
GRGDS: PHSRN, 1:1 cRGDfK: PHSRN, and PHSRN MTFM probes (TAMRA-Alexa 488). Scale bar = 10 µm and
contrasts are set identically. b) Bar graph showing the average tension normalized to the background for
cells cultured onto the above substrates (a). Data obtained in triplicate from n=8 cells in each category for a
total of 40 cells, where 10-30 FAs were analyzed from each cell. Note that the average tension for the
PHSRN probe was ~2% below the background signal likely due to optical effects from cell adhesion.
88x59mm (299 x 299 DPI)
ACS Paragon Plus Environment

Page 11 of 11
Journal of the American Chemical Society
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
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
TOC
93x35mm (300 x 300 DPI)
ACS Paragon Plus Environment