Biomaterials Advances 150 (2023) 213431

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Biomaterials Advances
journal homepage: www.journals.elsevier.com/materials-science-and-engineering-c

DNA tension assays reveal that force-dependent integrin activation

regulates neurite outgrowth in primary cortical neurons
Ying-Chi Chen a, Ying Li a, b, Ching-Cher Sanders Yan a, Chao-Ping Hsu a, d, Pei-Lin Cheng c, *,
Hsiung-Lin Tu a, d, *
a
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
b
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan
c
Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
d
Genome and Systems Biology Degree Program, Academia Sinica and National Taiwan University, Taipei 10617, Taiwan

A R T I C L E I N F O A B S T R A C T

Keywords: Biomechanical inputs are ubiquitously present in biological systems and are known to regulate various cell
Tension force probes functions. In particular, neural cell development is sensitive to mechanical regulation, as these cells reside in one
Neurite initiation of the softest microenvironments in the body. To fully characterize and comprehend how mechanical force
Mechanotransduction
modulates early neuronal processes, we prepared substrates functionalized with DNA probes displaying integrin
Single cell mechanobiology
Neurons
ligands, including cRGD and laminin, to quantify integrin-mediated molecular tension during neurite initiation
in primary cortical neurons. Our live-cell imaging analysis reveals that integrin-mediated tension force is highly
dynamic and distributed across the cell body, with the overall tension signal gradually increasing during neurite
outgrowth. Notably, we detected a consistent level of mechanical force (amplitude = 4.7–12 piconewtons, pN)
for cell integrin-ligand interactions. Further quantifications reveal that neurons exhibit faster cell spreading and
neurite outgrowth upon interacting with ligands functionalized with 4.7 pN relative to 12 pN probes. These
findings indicate that the magnitude of integrin-mediated mechanical feedback regulates neuronal activity
during early neuritogenesis. Additionally, we observed that mechanical tension is correlated with calcium
signaling, since inhibiting calcium influx substantially reduced mechanical tension. Thus, our findings support
that the magnitude of integrin-mediated mechanical feedback regulates neuronal activity during early neurito­
genesis and that mechanical force is an essential element complementing well-known biochemical regulatory
mechanisms orchestrating the integrin activation machinery and controlled neurite outgrowth in cortical
neurons.

1. Introduction cellular processes and development [4]. Mechanical forces also

Mechanical signaling is emerging as a critical intracellular
messenger that can regulate diverse cellular functions, including adhe­
sion, migration, and proliferation [1], and its dysregulation has sub­
stantial implications for normal activity and pathological conditions [2].
These biomechanical stimuli are converted into intracellular signals by
mechanotransduction pathways such as the integrin signaling cascade.
Cells interact with the extracellular matrix (ECM) via an integrin-
mediated machinery known as focal adhesions (FAs), which are dy­
namic molecular complexes that mediate bidirectional signaling re­
actions upon ECM activation and environmental stimulation [3–5]. A
recent study showed that coordinated FA regulation is central to various
orchestrate interactions among neural cells and play a key role during
brain development [6]. For instance, somatosensory adult neurons are
responsive to mechanical manipulations including stretching, vibration,
and touch, and the growth cones of developing neurons depend on
mechanical inputs from the environment for their maturation [7,8].
Various tools and techniques have been developed to measure the
mechanical interactions between neural cells and the extracellular
environment, including traction force microscopy (TFM) [9,10], elastic
substrates [11], micropost arrays [12], and atomic force microscopy
(AFM) [13,14]. More recently, live-cell-imaging-based strategies—such
as single-molecule force spectroscopy (SMFS) and molecular tension
fluorescence microscopy (MTFM)—as well as tension gauge tethers, are

* Corresponding authors at: Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
E-mail addresses: plcheng@imb.sinica.edu.tw (P.-L. Cheng), hltu@gate.sinica.edu.tw (H.-L. Tu).

https://doi.org/10.1016/j.bioadv.2023.213431
Received 9 November 2022; Received in revised form 12 April 2023; Accepted 13 April 2023
Available online 18 April 2023
2772-9508/© 2023 Published by Elsevier B.V.
Y.-C. Chen et al.
also being deployed to characterize the mechanical behaviors of cells at
single-cell and single-molecule resolutions [15–20]. Notably, these tools
can be used under nearly physiological conditions, enabling precise
mapping of tension signals arising from well-defined and specific in­
teractions between cell receptors and ligands [16,21]. Indeed, many
such techniques have been applied to reveal key insights into various
biological processes. For instance, using MTFM, one study showed that
platelet aggregation during vascular injury is regulated by mechanical
force, ultimately leading to hemostasis at the physiological level [22].
Likewise, Liu et al. employed MTFM to show that activation of T cells is
also tightly modulated by a well-defined molecular force of 12–19 pN
that is mediated by T-cell receptor ligation [23].
To study how biomechanical inputs modulate neurodevelopmental
processes, we and others have also reported that early neuritogenesis of
hippocampal and cortical neurons is highly sensitive to their biome­
chanical microenvironments [24–28]. In particular, we observed
elevated neurite outgrowth for neurons grown on substrate exhibiting
soft-tissue-equivalent softness (elastic moduli = 0.1 kPa), and upon
interacting with ligands displaying an “optimal” level of surface vis­
cosity [24,25]. These results highlight that developing neurons are
sensitive to the biomechanical properties of their surroundings and may
respond via differentially activated proteins, such as paxillin and cal­
pain, to evoke distinct cellular responses [24,25]. Despite these studies,
the tension characteristics and effects of different mechanical stimula­
tions on neurons remain incompletely characterized. Understanding
such processes could, in principle, serve as a pivotal step for further
elucidating how neuron-ECM interactions are mechanically regulated
and provide insights aiding biomaterial or scaffold designs for neuro­
developmental and neurodegenerative applications [29,30].
In this study, we present direct visualizations and quantitative ana­
lyses of the sub-cellular distribution of integrin-mediated molecular
forces during neurite initiation in cortical neurons by using MTFM. More
specifically, to investigate tension force characteristics and the func­
tional outcomes driving neuritogenesis upon receiving different levels of
mechanical stimuli, we prepared MTFM substrates functionalized with
either 4.7 pN or 12 pN tension probes containing integrin ligands (cyclic
RGD, cRGD, or laminin). Probes lacking an integrin ligand were used as
a control. Through live-cell imaging experiments and quantitative ana­
lyses, we show that cortical neurons exhibit a surprisingly consistent
level of integrin-mediated tension force of 4.7–12 pN during early in
vitro neuritogenesis. Moreover, we reveal that neurons spread and
extend their neurites faster when engaged with 4.7 pN tension probes
relative to 12 pN ones, indicating that integrin-generated tension can
exert positive feedback to control subsequent neurite outgrowth.
Furthermore, at a subcellular level, we observed that increased me­
chanical signals are correlated with the site of neurite outgrowth and
that calcium flux is essential for mechanically-coupled integrin activa­
tion. Together, our data evidence how biomechanical modulation, in
addition to well-documented biochemical regulation, plays a central
role in controlling the early neurite outgrowth of primary cortical
neurons.
2. Materials and methods
2.1. Materials and reagents
Cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG-PEG)] (cRGD; MW = 894) was
purchased from Peptides International (Louisville, KY, USA). All oligo­
nucleotide sequences were synthesized and desalted by Integrated DNA
Technologies (Coralville, Iowa, USA), unless otherwise noted. Azide-
NHS, Bis-NHS-PEG5, triethylamine acetate (TEAA), neurobasal me­
dium and streptavidin were acquired from Thermo Fisher Scientific
(Rockford, IL, USA). BAPTA-AM, triethylamine (99 %, TEA), dime­
thylformamide (99 %, DMF), acetonitrile (>99 %, ACN), trifluoroacetic
acid (99.5 %, TFA), dimethyl sulfoxide (99 %, DMSO), ascorbic acid
(>99.0 %), and sodium bicarbonate (99 %) were purchased from Sigma-
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Biomaterials Advances 150 (2023) 213431
Aldrich (St. Louis, MO, USA) and used without further purification.
Copper(II)-Tris[(1-benzyl-4-triazolyl)methyl]amine (Cu-TBTA com­
plex) was obtained from Lumiprobe Corporation (USA and Worldwide).
Phosphate buffered saline (PBS) and trypsin were purchased from
Caisson (Smithfield, VA, USA). Gem21 NeuroPlex was purchased from
GEMINI Bio-products (West Sacramento, CA, USA). Hoechst 33342,
calcein AM, ethidium homodimer-1 (EthD-1), Fluo-4 AM (F14201), anti-
phospho-FAK, AlexaFluor-488 goat anti-rabbit secondary antibody
(35552), and paraformaldehyde were purchased from Invitrogen
(Carlsbad, CA, USA). Biotinylated laminin was purchased from Cyto­
skeleton Inc. (CO, USA).
2.2. Synthesis of the c[RGDfK(PEG-PEG)] ligand strand with Cy3
The cRGD peptide was conjugated with the fluorescently-labeled
DNA strand by click-chemistry reaction according to a previously pub­
lished protocol (Fig. S2) [31]. In brief, the primary amine of the cRGD
peptide was activated with an azide group in DMF for 12 h. Product I
(Fig. S2A) was purified by reverse-phase HPLC (C18; flow rate: 1 mL/
min; solvent A: 0.1 M TFA, solvent B: 100 % ACN; initial conditions: 10
% solvent B with a gradient of 1.5 % solvent A per minute). An elec­
trospray ionization time-of-flight (ESI-TOF) mass spectrometer (Waters,
LCT, Manchester, U.K.) was then used to confirm the mass of product I
(Fig. S2B and D). In the following step, product I was coupled to the Cy3-
labeled DNA strand by click reaction. The reaction was conducted in 50
% DMSO containing a mixture of hexynyl-ligand strand-Cy3, product I,
ascorbic acid, and Cu-TBTA complex for 12 h. Product II was separated
from unlabeled oligonucleotides by reverse-phase HPLC (C18; flow rate:
1 mL/min; solvent A: 0.1 M TEAA, solvent B: 100 % ACN; initial con­
ditions: 10 % solvent B with a gradient 1.0 % solvent A per minute)
(Fig. S2C) after purification and confirmed by matrix-assisted laser
desorption/ionization (MALDI) TOF/TOF (Bruker, New ultra­
fleXtremeTM, Bremen, Germany) (Fig. S2D).
2.3. Tension probe surface functionalization
Tension probe-functionalized surfaces were prepared as previously
described [23]. In brief, #1 glass coverslips (24 × 60 mm, Marienfeld)
were first cleaned with Micro-90 detergent (Cole-Parmer) to remove oil
residues. Then, the glass coverslips were sonicated in isopropyl alcohol
(IPA) and deionized (DI) water for 10 min each. Dried glass coverslips
were placed in an oven at 70 ◦ C for 30 min. The glass coverslips were
bonded with a homemade polydimethylsiloxane (PDMS) chamber by
means of oxygen plasma treatment followed by ultraviolet light sterili­
zation overnight. In Fig. S1, we summarize the fabrication procedure for
the functionalized surfaces. The glass coverslips were functionalized
with 1 % 3-(aminopropyl)triethoxysilane (APTES; in ethanol, v/v %) for
1 h, and then washed three times with absolute ethanol. Then, the
coverslips were dried in an oven at 70 ◦ C for 10 min. After cooling, the
chambers were reacted with 1 mg/mL Bis-NHS-PEG5 in DMSO and
incubated for 16 h at room temperature. Subsequently, the coverslips
were washed three times with ethanol and dried at 70 ◦ C for 10 min. The
chambers were then incubated for 1 h at room temperature with 100 μL
of 10 nM pre-assembled DNA tension probes, which contained either the
biotin-based or cRGD-based probes (Fig. S1). The surface functionalized
with cRGD-conjugated probes was rinsed thoroughly before conducting
cell-imaging experiments. Meanwhile, for the surfaces conjugated with
the biotin-based probe, the laminin ligand was further functionalized by
means of streptavidin-biotin interaction. Specifically, the biotin-
conjugated surface was incubated with 50 nM streptavidin for 45 min,
followed by incubation with 0.1 μg/mL biotinylated laminin for an
additional 45 min. Finally, the laminin-probe functionalized surface was
thoroughly washed with PBS three times before undergoing subsequent
experiments.
Y.-C. Chen et al. Biomaterials Advances 150 (2023) 213431

Fig. 1. Mechanical forces regulate cell spreading, integrin tension force generation, and focal adhesion establishment and maturation. (A) An illustration of the
contact zone between a cell and the tension probe surface. The inset at right shows the interaction of integrins with DNA probes in the presence of ligands. (B)
Representative fluorescence images for substrates functionalized with different probe sets (shown in C). The lower schematic shows the configurations of the closed
and open tension probes. Scale bar is 100 μm. (C) Determination of the quenching efficiency (QE) of DNA hairpins on the surfaces. Fluorescence signal was analyzed
by TIRF to calculate the QE. Each column represents the mean ± SEM from at least three independent experiments.

2.4. Hairpin hybridization and measurement of quenching efficiency strands (10-fold molar excess) and DNA handles.

All DNA oligonucleotides were allowed to fold into their secondary

hairpin structure in DI water at a concentration of 10 nM in a 1.5-mL
Eppendorf ThermoMixer C. All oligonucleotides were first denatured
at 95 ◦ C for 5 min, before undergoing a renaturation step when the
temperature was allowed to return to room temperature at a rate of
2.5 ◦ C/min.
To determine the quenching efficiency of the DNA-based tension
probes, we recorded the fluorescence intensity of the closed hairpin on
glass substrates under experimental conditions (37 ◦ C, cell media;
Fig. 1B and C). To mimic the DNA probes unfolded upon interaction with
the receptor, the tension probes were hybridized to their complementary
2.5. Cell culture, immunostaining and BAPTA-AM treatment
Cortical neurons were prepared from embryonic day (E) E17.5 rat
embryos as previously described [25,32]. After cell harvesting, cortical
neurons were cultured in neurobasal medium supplemented with
Gem21 NeuroPlex in a 37 ◦ C incubator with 5 % CO2 and high humidity
until the time of experiments.
Rat cortical neurons were plated and allowed to spread on the ligand-
tethered tension probe-conjugated surface for 5 or 10 h at 37 ◦ C with 5 %
CO2. The cell density used in this study is typically ~6–8 neurons/100 ×
100 μm2. Then, the cells were fixed for 12 min with 4 %

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Y.-C. Chen et al.
paraformaldehyde in 1× PBS at 37 ◦ C in an incubator, before being
washed three times with PBS. The fixed cells were permeabilized in 0.3
% Triton X-100 for 12 min and blocked with 3 % BSA overnight at 4 ◦ C.
The cells were then incubated overnight at 4 ◦ C with primary anti-
phospho-FAK. Unbound primary antibodies were removed by washing
three times with PBS at room temperature in a shaker with mild agita­
tion (65 rpm), and the cells were then incubated overnight at 4 ◦ C with
AlexaFluor-488-labeled goat anti-rabbit secondary antibody (1:1000).
For BAPTA-AM-treated cortical neurons, cells were treated with BAPTA-
AM (10 μM) and Fluo-4 AM (2 μM) for 30 min, followed by careful buffer
exchange of the culture medium.
2.6. Microscopy
Cells were imaged with total internal reflection fluorescence (TIRF)
and reflective interference contrast microscopy (RICM) on a Nikon
Eclipse Ti2 microscope with a Nikon Perfect Focus System (PFS) driven
by the Nikon Elements software package. The microscope was equipped
with an oil immersion Apo TIRF 60× NA 1.49 objective and a cooled
electron-multiplying charge-coupled device camera (EMCCD; iXon3;
Andor Technology). The sample was illuminated with a Sola epifluor­
escence light source (Lumencor) for RICM and a 561 nm laser light
source for TIRF, respectively. All live-cell experiments were performed
at 37 ◦ C with 5 % CO2 in a heated micro-incubator (Tokai Hit Co.,
Shizuoka-ken, Japan), whereas fixed cell experiments were conducted at
room temperature.
2.7. Image processing
We summarize how we obtained the image, average intensity, and
distribution probability of pair-binding tension force in Fig. S3. The
images were analyzed using Fiji (ImageJ), Nikon Elements software, and
Python language. Maximum intensity projection images were obtained
by selecting Max intensity as the projection type using Fiji. For all im­
ages, signal values were subtracted from an adaptive threshold and then
normalized to the control group in order to represent the full dynamic
range. Images further analyzed by custom-written Python code were
subtracted from background fluorescence to determine morphological
parameters including the area of the cell footprint (RICM area), the
activity of tension probes, and the integrated tension. To define the cell
boundary in tension calculations, the cell footprint area from the RICM
image was established according to the adaptive threshold obtained
using Otsu’s method [33,34]. The integrated tension was then deter­
mined by calculating the total tension probe fluorescence intensity for
the open probes within the cell boundary and subtracting the back­
ground signal measured from an off-cell (or non-cellular) region. The
background fluorescence was obtained from the quenched DNA tension
probes not experiencing any cellular tension.
Each step in the flowchart shown in Fig. S3 can be described as fol­
lows: (i) collect raw fluorescence images of neurons cultured on the
surfaces functionalized with tension probes; (ii) for images taken at
different times, perform a linear stack alignment using the scale-
invariant feature transform (SIFT) algorithm to correct for displace­
ments among different images [35,36]; (iii) obtain cell contours in the
RICM images by applying the threshold derived from Otsu’s method to
differentiate regions inside and outside of each cell, which fills gaps
within cells; (iv) conduct cell alignment on the multi-stock images; (v)
calculate tension signal within cell boundaries; (vi) subtract background
noise (after setting a threshold) obtained from an off-cell region to
obtain the actual tension signal; and, finally, (vii) process and convert
the tension signal to mean intensity and a probability distribution.
2.8. Statistical analysis
All experimental values are expressed as mean ± SEM (standard
error of the mean) for n ≥ 3 independent experiments.
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Biomaterials Advances 150 (2023) 213431
3. Results and discussion
3.1. Fabrication and characterization of DNA molecular tension probes
In this study, we functionalized substrates hosting DNA probes
conjugated to an integrin ligand, i.e., either cRGD or laminin, as a ten­
sion force-sensing approach (Fig. S1) [37]. This approach, also known as
an MTFM platform, was previously reported by K. Salaita et al., which
can be applied to studying the mechanotransduction properties of
various cell types [16,31,38] This assay enables mapping of mechanical
responses during various cellular processes at the piconewton (pN)
range and at subcellular resolution [16]. Specifically, the MTFM plat­
form comprises three single strands of DNA: (1) a reporting strand
containing a ligand and a fluorophore; (2) a hairpin strand containing a
stem-loop, the GC content of which can be adjusted to account for
different unzipping forces; and (3) a quenching strand containing a
quencher [23,31]. Upon a cell receptor binding the ligand and pulling it
away from the completely hybridized DNA probe, the fluorophore on
the reporting strand is “de-quenched” and thus emits fluorescence.
Previous studies have reported that integrin signaling affects the
formation of FAs and regulates the mechanical responses of neurons
upon interacting with the ECM [39]. The integrin signaling pathway is
activated by specific ligands, including laminin, cRGD, and others
[24,40]. Furthermore, the connection between integrin signaling and
mechanotransduction is mainly mediated by dynamic assembly and
disassembly of the actin cytoskeleton network. Rat cortical neurons
express integrin receptors during early development, but they have not
been well characterized in terms of integrin-related mechanical re­
sponses at the subcellular level [24]. Therefore, we implemented MTFM
to monitor the tension characteristics of primary cortical neurons upon
their initial interaction (i.e., the first ~10 h) with integrin ligands. Our
experimental setup is depicted in Fig. 1A. After plating neurons on the
DNA tension probe substrates, dynamic cell integrin-ligand interactions
were measured by monitoring the fluorescence of folded or unfolded
DNA probes using live-cell TIRF microscopy. Under our experimental
conditions, Cy3 fluorescence is quenched in the absence of ligand
binding due to the close proximity of the quencher (BHQ2) on the
quenching strand. Upon cell integrin-ligand binding, the quencher is
pulled away from the fluorophore due to unzipping of the hairpin
strand, resulting in strong fluorescence signal detectable by TIRF
imaging.
First, we tested if our assay could distinguish between the folded and
unfolded DNA hairpins based on their relative fluorescence intensities.
Substrates decorated with varying ratios of the reporter versus
quenching strands were prepared to mimic the different levels of applied
tension forces. A substrate functionalized with force probes without the
quencher acted as a positive control to reflect the fully unzipped state
(“no-quencher strand” in Fig. 1C). By comparing fluorescence signal
from these surfaces to the control substrate, we determined the
quenching efficiency (QE) for each sample. Our results reveal an
increasing trend of QE from 5 % to 92 % as the ratio of the quenching
strand increased (Fig. 1B and C), consistent with findings from previous
studies [23,31]. More importantly, this analysis confirmed that QE is
dose-dependent, as both 10 nM and 20 nM of quencher elicited an
equally high QE of ~92 %. Furthermore, to demonstrate that tension
probes can emit fluorescence properly upon external stretching of the
hairpin strand, we prepared an additional set of tension probes in which
we added an extra DNA strand complementary to the hairpin strand
(Fig. 1B and C) [23]. This configuration allowed the formation of DNA
pairs with a well-separated fluorophore (Cy3) and quencher (BHQ2)
pair, resulting in high-level fluorescence comparable to that of the
unquenched state (Fig. 1B and C). Taken together, our results demon­
strate that our DNA-based tension probes were successfully functional­
ized on the surfaces and that their relative fluorescence intensities can
be used to report the folded and unfolded states of tension probes,
thereby revealing associated tension signals.
Y.-C. Chen et al. Biomaterials Advances 150 (2023) 213431

Fig. 2. Real-time measurement of molecular tension forces for neurons on substrates functionalized with tension probes. (A) Maximum intensity projection-based
RICM images (top row) and tension force signals (middle row) of neurons on the 4.7 pN probe surfaces for different time intervals. The yellow outlines in the RICM
images represent the cell outline for the previous time period. (B) Tension forces of cortical neurons were mapped on cRGD-functionalized tension probe surfaces
with different force probes by TIRF microscopy during the initial 10 h (0− 10 h). (C) A comparison of the relative Cy3 mean signal intensity and (D) a plot showing the
probability distribution of accumulated activated time of neurons on the cRGD tethered-tension probe surfaces for 4.7 pN (red line), 12 pN (blue line) and no-ligand
(black line) groups. Note the probability is defined as the number of events displaying a particular accumulated time divided by the total number of events for a single
cell (measured using a region of interest of 3 × 3 pixels; 0.65 μm2; n = 11, 15 and 8 for the 4.7 pN, 12 pN and no-cRGD substrates, respectively).

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Fig. 3. Time-lapse images showing that the amplitude of tension force modulates the spreading area of neurons. (A) RICM images of cortical neurons on the 4.7 and
12 pN substrates functionalized with or without cRGD at a few selected time-points. The dotted outline in the RICM images represents the cell outline from the
previous time period. Plots displaying (B) the normalized spreading area and (C) integrated tension force signal of neurons on the 4.7- and 12-pN tension surfaces
functionalized with or without cRGD as a function of time. Scale bars: 5 μm (n = 11, 15 and 8 for the 4.7 pN, 12 pN and no-cRGD substrates, respectively).

3.2. Visualization of integrin-mediated tension in cortical neurons
DNA tension substrates have been used to study receptor-ligand in­
teractions in various cell types [22,23], revealing many insights into
these biological processes. However, to our knowledge, their application
to neuronal cells has not been reported previously. We harvested em­
bryonic rat cortical neurons and used them to examine tension charac­
teristics during their initial contact with the substrate via integrin
signaling activation. Compared to other cellular tension platforms,
MTFM provides improved sub-cellular resolution and specificity. After
seeding neurons on substrates pre-functionalized with the cRGD-
tethered DNA probes, time-lapse TIRF microscopy at a 20-sec imaging
frequency showed that, following initial contact, neurons gradually
spread out and started to project neurites after being cultured on these
substrates for 10 h (Fig. 2A and Movie S1). Interestingly, we also
observed a significant level of Cy3 signal underneath these neurons.
These signals, which reflected an F1/2 (force required to unzip 50 % of
the probes) of 4.7 pN [23,31], were spatially localized and exhibited
dynamic on-and-off tension signals. Using a maximum intensity pro­
jection to visualize our time-lapse images, we noted that these tension
signals were generated solely underneath the cell body and that there
was a sustained level of tension signal during this timeframe (Fig. 2B).
Areas without neurons did not show any noticeable fluorescence. Such
dynamic on-and-off tension forces for integrin signaling in neurons is
interesting, as most previous studies using DNA tension probes on other
cell types, such as T cells and cancer cells, have revealed more gradual
and wave-like tension signals that propagated towards the cell periphery
[16,22,23,31].
Next, we wondered if the measured tension exhibits a specific force
range. Accordingly, we prepared a cRGD-tethered tension substrate with
a hairpin strand of 77 % GC content, which has been characterized
previously as exhibiting a tension value (F1/2) of 12 pN [23,31].
Intriguingly, we observed that when neurons engaged with these 12-pN
tension probes, there was a significantly reduced level of fluorescence
relative to that determined for the 4.7-pN substrate (Fig. 2B, C and
Movie S2). This outcome indicates that most cRGD-integrin interactions
cannot activate molecular tensions >12 pN, i.e., the cRGD-integrin-
mediated tension for primary cortical neurons is between 4.7 and 12
pN. Furthermore, taking advantage of the unique assay set-up, we
analyzed the overall durations of time-lapse tension signals (Fig. S3).
The results showed that recorded tension signals displayed substantially
longer half-lives upon interacting with the 4.7 pN tension probe

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Fig. 4. Live-cell measurements showing that elevated tension force signals correlate with neurite outgrowth. (A) Merged tension force (red) and RICM (gray) images
of a neuron displaying neurite outgrowth on the 4.7-pN tension substrate at two selected time-points, i.e., I: ~4.5 h and II: ~7 h. (B) A heatmap image of measured
tension force signals for the neuron shown in (A). (C) Recorded time traces of tension force intensity from the neuron shown in (A) over a 6-h period (4–10 h).
compared to the 12 pN one (Fig. 2D). Importantly, to further verify that
the Cy3 fluorescence was specific to the integrin-mediated interaction,
we prepared a probe substrate lacking the integrin ligand (cRGD). A
similar analysis revealed that neurons cultured on this latter substrate
failed to generate any noticeable fluorescence signals, thus confirming
that the observed tension signal was specific to the cRGD-integrin
interaction (Fig. 2B bottom panel and Movie S3).
Laminin is another ligand for the integrin receptor and it is also
commonly used to study neurons in in vitro settings, therefore we pre­
pared DNA probes functionalized with laminin as an alternative integrin
activator to assess their tension characteristics. Our data show that
neurons mostly engaged laminin with a force of >4.7 pN, i.e., similar to
the cRGD-integrin interaction, and only rarely did we measure a mo­
lecular force of >12 pN (Figs. 2 and S4). These results indicate that
although laminin and cRGD are both integrin ligands, they seem to
stimulate differential integrin signaling activation both biochemically
and mechanically. Taken together, these findings demonstrate that the
measured tension signal is highly dynamic and specific to different
integrin ligands and that the corresponding molecular tensions occur in
a characteristic force range of between 4.7 and 12 pN, which is depen­
dent upon the specific interaction between the cellular receptor and
ligands.
3.3. The tension signal is not attributable to endocytosis and can be
blocked by inhibiting actin polymerization
To confirm if the observed tension signals are due to specific
neuronal integrin and ligand binding rather than endocytosis of tension
probes (specifically unzipped reporting strands) inside the cells, we
performed confocal live-cell imaging of neurons after culturing them on
the MTFM platform for 17 h. Importantly, if the reporting strands are
indeed endocytosed (presumably as a result of mechanically-induced
melting and unzipping of tension probes), then the fluorophores
would be internalized and emit signal from the intracellular space (i.e.,
cytosol). Careful examination of our confocal data revealed no detect­
able fluorescence signal within the cytosol, confirming that the signals
we recorded were generated by ligand-receptor binding at the cell-
substrate interface (Fig. S5). The correlation between observed tension
signals and integrin activation was further validated by means of
immunofluorescence staining, which showed colocalization of tension
signals and phosphorylated focal adhesion kinase (pFAK), the presence
of which directly supports activated integrin signaling (Fig. S6).
Notably, actin plays a crucial role in neurite growth [8,41,42], and may
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Biomaterials Advances 150 (2023) 213431
govern mechanical regulation for many cell types. Therefore, we
wondered if the observed tension signals could be altered upon blocking
actin activity. To answer this question, we used latrunculin B to inhibit
actin polymerization in neurons while monitoring the tension signals in
real-time. As shown in Fig. S7, tension signals of cortical neurons were
rapidly and completely eliminated within ~30 s of adding latrunculin B
(Movie S4). Together, these results confirm that tension forces are pri­
marily exerted from cortical neurons through the integrin receptor.
Moreover, our findings demonstrate that DNA tension probes can be
deployed successfully to report the integrin-specific and cytoskeletal-
mediated molecular forces of neurons.
The relationship between axonal growth and mechanical force in
terms of neuronal mechanotransduction has been explored substantially
using TFM and it has been linked to molecular events, including cell
adhesion, neurite outgrowth, and growth cone guidance [43–45].
Various studies have also shown that cytoskeletal dynamics involve
force generation and growth cone dynamics [45,46]. Typically, the re­
ported force values from these studies range from pN to hundreds of nN,
depending on the types and parts of the neurons being examined, as well
as the characterization methods [47,48]. As a complementary platform,
we used MTFM in the current study to identify relatively well-defined
molecular forces ranging from 4.7 to 12 pN, which were generated
upon neurons interacting with tension probes in cortical neurons.
Notably, this range agrees well with the tension produced when actin
filaments polymerize in vitro (~5–10 pN per filament) [45,49], further
supporting the notion that the observed tension force is mediated by
integrin signaling.
3.4. Distinct integrin signaling-mediated forces mediate cell spread and
neurite outgrowth
Although our analyses revealed that the majority of integrin acti­
vation in cortical neurons triggers molecular forces of between 4.7 and
12 pN, we wondered if such tension forces may be correlated with
particular neuronal activities. Therefore, we characterized how the dy­
namics of neuron spread and integrin tension are impacted by the me­
chanical force generated through DNA tension probes exhibiting
different unzipping forces. Neurons were seeded on the tension probe
substrates and monitored simultaneously by RICM and TIRF imaging. As
illustrated in Fig. 3A and B, we observed substantially increased
spreading area for neurons interacting with the 4.7-pN cRGD-probe.
Importantly, this outcome contrasted sharply with results for neurons
that interacted with the 12-pN cRGD tension probes or when they were
Y.-C. Chen et al.
Fig. 5. Integrin-mediated tension force signals depend on neuronal calcium
influx. (A) RICM, tension force and Fluo-4 images of neurons on the 4.7 pN
cRGD-functionalized tension substrates without (top) or with (middle) BAPTA-
AM treatment, as well as on the no-cRGD tension surface (bottom). (B) Quan­
titative analyses of the integrated tension force signals from the three groups
shown in (A) (n = 12, 3 and 5 for the 4.7 pN, 4.7 pN/BAPTA-AM and no-cRGD
substrates, respectively).
seeded on a tension substrate lacking the integrin ligand (cRGD).
Notably, in both these latter cases, the neurons displayed a substantially
lower degree of spread within the same time period (Fig. 3A and B).
Moreover, our results revealed that tension signals obtained from neu­
rons on the 4.7-pN substrate increased more rapidly than for neurons on
the substrates without ligand or with the 12-pN cRGD probe (Fig. 3C).
Together, these findings indicate that upon integrin binding to ligands,
neurons primarily trigger a tension force >4.7 pN and that this tension
force is crucial to efficiently mediate the downstream signaling cascades
that promote cell spread. When ligands engaged with the 12 pN probe,
most integrin receptors failed to generate sufficient force to unzip the
DNA hairpin, resulting in lower fluorescence signal and significantly
reduced cell spread. Given that the biochemical input was the same
(integrin-ligand binding) for neurons on both the 4.7- and 12-pN tension
probe substrates, our findings highlight that mechanotransduction may
modulate signaling outcomes that are orthogonal or inaccessible via
biochemical means.
8
Biomaterials Advances 150 (2023) 213431
We further explored the possibility that mechanical force may acti­
vate a feedback mechanism to modulate neurite outgrowth in cortical
neurons. Notably, we observed that increased tension force signal
correlated well with neurite extension in cortical neurons (Fig. 4). Our
data also revealed that integrin-mediated tension force was elevated on
the side where neurite initiation occurred. Together, these findings
support the notion that integrin-mediated signaling not only exerts a
well-defined tension force (in the range of 4.7 to 12 pN), but also implies
that mechanotransduction is essential to stimulate efficient cellular
processes such as neuritogenesis by cortical neurons.
3.5. Neuronal tension force is correlated with calcium influx
Next, we explored the interplay between mechanical tension and
calcium signaling, as previous studies have shown that local calcium
pulses induce cell adhesion and trigger neuronal outgrowth (Fig. 1A)
[50–52]. Ca2+ is a highly versatile intracellular messenger that regulates
various cellular events, including neurite growth [53]. Two different
Ca2+-dependent processes have been shown to modulate growth cone
motility; moderate Ca2+ levels facilitate motility and extension, whereas
higher Ca2+ levels inhibit growth cone motility and arrest growth [54].
If a mechanical response can induce optimal Ca2+ influx, it is feasible
that it can facilitate neurite elongation and growth cone movements
[50,55]. Again using the MTFM platform, we observed that mechanical
tension forces could be enhanced by Ca2+ influx (Fig. 5A). Specifically,
we used Fluo-4 AM, a chemically fluorescent Ca2+ probe, to determine
Ca2+ influx in neurons on the tension probe substrates. Time-lapse im­
ages showed that Ca2+ influx into neurons on the cRGD-probe surface
was sensitive to mechanical force, presumably via mechanosensitive
channels. Indeed, previous studies have indicated that such mechano­
sensitive channels are critical regulators of Ca2+ influx in response to
mechanical force [56–58]. In contrast and as an important control, both
Ca2+ influx and tension force were reduced in neurons on the tension
substrates lacking the integrin ligand (cRGD). To investigate if Ca2+ is
required for the mechanical response promoting neurite outgrowth, we
used BAPTA-AM to chelate both intracellular and extracellular Ca2+ of
neurons plated on tension substrates for 15 h. These BAPTA-AM-treated
neurons displayed notably reduced tension force signals (Fig. 5A).
Moreover, we detected higher tension force and fluorescence signal for
neurons on the 4.7-pN tension substrate compared to the 12-pN sub­
strate (Figs. S8 and 5B), supporting that Ca2+ is a major factor partici­
pating in the mechanical regulation of neurite outgrowth in cortical
neurons.
The action of neurite growth involves mechanical forces that regu­
late integrin activation, which modulates integrin clustering and the
transmission of mechanical forces. Integrin clusters serve as a scaffold
for the recruitment and activation of proteins such as paxillin, integrin-
linked kinase (ILK), and FAK to promote FA assembly during neurite
initiation [24], neuronal differentiation [59], and axon outgrowth [60].
The tensile forces applied by actomyosin stress fibers on FA can stabilize
the entire structure and trigger various biochemical events [61].
Consequently, the exertion of mechanical forces can also trigger Ca2+
flux into the cytoplasm, which facilitates binding of myosin motors to
the cytoskeleton [62]. Clearly, these scenarios observed in this study
highlight the positive feedback between mechanical regulation and Ca2+
influx that underlies integrin signaling.
4. Conclusions
In this study, we systematically characterized DNA tension assays
that report different force ranges and successfully applied them to study
integrin-mediated neurite growth in cortical neurons. Our results reveal
that when neurons ligated with the 4.7 pN probes, the integrin-mediated
tension exhibits a longer half-life than when it interacts with 12 pN
probes. Functionally, our results also show that the amplitude of me­
chanical activation modulates the growth dynamics of neurites from
Y.-C. Chen et al.
primary neurons. Neurons on the 4.7 pN probe substrate displayed faster
cell spread and neurite outgrowth than those interacting with 12 pN
probes. Moreover, our live-cell TIRF imaging uncovered that the
maximum force and activity of the ligand-integrin interaction during
neurite outgrowth are primarily concentrated near the neurite elonga­
tion region and that they dynamically change over time. Biochemically,
Ca2+ activity is essential for mechanically-coupled integrin activation.
Our results imply that in addition to well-characterized biochemical
regulation, mechanical modulation arising from cell-microenvironment
interactions also actively regulate neuronal morphogenesis. We also
highlight that the recorded tension force characteristics for cortical
neurons observed in this study differ substantially from those observed
for other cell types, with wave-like force propagation patterns often
being observed for these latter [16,22,23,31], implying a bi-phasic
mechanism for force generation (gated at ~5 pN) by neuronal and
non-neuronal cells. Looking forward, it will be interesting to study
molecular tension characteristics for neurons interacting within soft-
tissue-type microenvironments, as well as to examine whether and
how mechanosensitive proteins are differentially activated upon their
binding to different receptors or with tension probes of varying force
magnitudes.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioadv.2023.213431.
CRediT authorship contribution statement
Ying-Chi Chen: Methodology, Investigation, Validation, Formal
analysis, Data curation, Writing – original draft. Ying Li: Methodology,
Investigation, Formal analysis. Ching-Cher Sanders Yan: Methodology,
Visualization. Chao-Ping Hsu: Methodology, Supervision. Pei-Lin
Cheng: Supervision, Resources, Project administration, Funding acqui­
sition, Writing – review & editing. Hsiung-Lin Tu: Conceptualization,
Supervision, Project administration, Funding acquisition, Writing –
original draft, Writing – review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
Data availability
Data will be made available on request.
Acknowledgments
We appreciate the financial support from the Academia Sinica
iMATE program (AS-iMATE-108-21) and GCS program (AS-GCS-112-
L02), and the Ministry of Science and Technology in Taiwan (MOST 110-
2113-M-001-019-MY2). The authors thank the IMB Imaging Core Fa­
cilities (Institute of Molecular Biology, Academia Sinica) for technical
and equipment support. The authors also thank Prof. W.-T. Chiu (Na­
tional Cheng Kung University, Taiwan) for his constructive comments on
this work.
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