Article

Neuronal paxillin and drebrin mediate BDNF-induced

force transduction and growth cone turning in a soft-
tissue-like environment
Graphical abstract Authors
Chen Chen, Chien-Hsin Chu, Ying Chu, ...,
Bi-Chang Chen, Hsiung-Lin Tu,
Pei-Lin Cheng

Correspondence
plcheng@imb.sinica.edu.tw

In brief
Chen et al. report that growth cones of
0.1 kPa neurons exhibit a highly
coordinated T-zone activity. Neurotro-
phic-factor-directed growth cone move-
ment on soft substrates depends on
drebrin phosphorylation and paxillin-
drebrin association, but not on Src activ-
ity, a mechanism distinct from the one
observed on conventional glass
coverslips.

Highlights
d Substrate stiffness changes molecular architectures of
growth cone subdomains

d T-zone factor drebrin modulates growth cone movement on

soft-tissue-like substrates

d Drebrin facilitates BDNF-elicited pressing force and

attraction on soft substrates

Chen et al., 2022, Cell Reports 40, 111188

August 16, 2022 ª 2022 The Author(s).
https://doi.org/10.1016/j.celrep.2022.111188 ll
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Article
Neuronal paxillin and drebrin mediate
BDNF-induced force transduction and growth
cone turning in a soft-tissue-like environment
Chen Chen,1,4 Chien-Hsin Chu,1,4 Ying Chu,1,4 Ting-Ya Chang,1 Sheng-Wen Chen,1 Shu-Yang Liang,1 Yun-Chi Tsai,2
Bi-Chang Chen,2 Hsiung-Lin Tu,3 and Pei-Lin Cheng1,5,*
1Institute of Molecular Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan
2Research Center for Applied Sciences, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan
3Institute of Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan
4These authors contributed equally
5Lead contact

*Correspondence: plcheng@imb.sinica.edu.tw
https://doi.org/10.1016/j.celrep.2022.111188

SUMMARY

Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms un-
derlying chemotropism-directed axonal growth cone movement in extremely soft environments remain un-
clear. Here, we show that drebrin, a growth cone T-zone protein, modulates growth cone turning in response
to brain-derived neurotrophic factor (BDNF) coated on a soft substrate. Structurally, axonal growth cones of
rodent hippocampal neurons grown on 0.1 kPa hydrogels possess an expanded T zone in which drebrin is
highly integrated with both F-actin and microtubules. Biochemically, we identify paxillin as interacting with
drebrin in cells grown on 0.1 kPa hydrogels but not on glass coverslips. When grown on 0.1 kPa substrates,
growth cones asymmetrically exposed to BDNF-bound stripes exhibit enhanced paxillin-drebrin interaction
on the side facing the stripes, an activity that is PKA and AAK1 dependent but independent of Src kinase.
Functionally, we show that BDNF-induced growth cone turning and force generation on soft substrates
require drebrin phosphorylation and paxillin-drebrin association.

INTRODUCTION
Axon pathfinding activity requires orientation of growth cones in
brain tissue, where the elastic modulus can be as low as 0.1–
1 kPa (Balgude et al., 2001; Koser et al., 2016; Tyler, 2012).
Growth cone attraction or repulsion on a soft substrate may
not necessarily employ the same set of force transduction ma-
chineries as those functioning in motility of non-neuronal cells
on more rigid substrates (Koch et al., 2012; Koser et al., 2016).
However, in vitro mechanistic studies of growth cone dynamics
have mostly been performed on tissue culture plastic or on glass
coverslips whose mechanical properties do not resemble those
of embryonic brain tissues. Hence, mechanisms that drive force
generation and directed movements of highly mobile growth
cones in response to chemical factors bound to soft-tissue-like
environment remain to be addressed.
Mechanistically, growth cone movement behaviors evoked by
chemical and physical instructive cues require actin filopodia
stability and microtubule capture at the leading edges (Dent
and Gertler, 2003; Geraldo and Gordon-Weeks, 2009; Kolodkin
and Tessier-Lavigne, 2011; Lowery and Van Vactor, 2009; Ros-
ner et al., 2007). Structurally, growth cones are divided into three
subregions: (1) the central domain (C domain), located at or near
the distal end of the axonal shaft, which is composed of microtu-
bule bundles emanating from the axonal shaft; (2) the peripheral
domain (P domain), distributed around the leading edge and
mainly composed of lamellipodia and filopodia; and (3) the tran-
sition zone (T zone), representing the interface between C and P
domains (Lowery and Van Vactor, 2009) and containing microtu-
bule-actin crosslinking molecules such as non-muscle myosin II
and developmentally regulated brain protein, or drebrin (Bur-
nette et al., 2008; Dent and Gertler, 2003; Geraldo and Gor-
don-Weeks, 2009; Shin et al., 2014; Shirao et al., 2017; Shirao
and Obata, 1985). Drebrin belongs to a class of neuron-specific
F-actin side binding/bundling proteins and has two major iso-
forms, namely, embryonic-type drebrin E and adult-type drebrin
A (Geraldo et al., 2008; Ishikawa et al., 1994; Shirao, 1995; Shirao
et al., 2017). Drebrin A accumulates within dendritic spines to
enable postsynaptic protein assembly (Hayashi et al., 1996;
Mizui et al., 2005; Takahashi et al., 2003), while drebrin E is a
typical T-zone-residing molecule expressed during neurogene-
sis and facilitating neuronal motility (Dun et al., 2012; Geraldo
et al., 2008; Mizui et al., 2009; Trivedi et al., 2017). Actin-stabiliz-
ing drebrin binds to the microtubule plus-end tracking protein
EB3 (Geraldo et al., 2008; Merriam et al., 2013), which may
modulate consumption of chemical energy by microtubule
extension and/or non-muscle myosin II-mediated contraction
(Ketschek et al., 2007; Mizui et al., 2009; Rosner et al., 2007).

Cell Reports 40, 111188, August 16, 2022 ª 2022 The Author(s). 1
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These actions represent the molecular basis for growth cone
dynamics.
To address whether and how the common force transmission
machinery of growth cones functions in a soft-tissue-like hydro-
gel, we examined differential cytoskeletal integration of growth
cone molecules on 0.1 kPa polyacrylamide (PA) hydrogels
compared with glass coverslips. Morphologically, we observed
a compact fist-like actin-based structure for growth cones of
hippocampal neurons grown on 0.1 kPa hydrogels, unlike open
palm-like structures exhibited by growth cones grown on cover-
slips. Growth cones on 0.1 kPa hydrogels exhibited significantly
greater T-zone integration with the cytoskeletal P domain than
conventional cultures on coverslips. Drebrin knockdown (KD)
in newly differentiated rat layer 2/3 cortical neurons promoted
relative shortening of commissural axons. Mechanistically, out
of eight factors associated with cytoskeletal dynamics exam-
ined, paxillin and the brain-derived neurotrophic factor (BDNF)
receptor TrkB displayed substrate rigidity-specific enrichment
at the growth cone T zone. Paxillin directly associated with dre-
brin, and their binding affinity increased when neurons were
grown on 0.1 kPa hydrogels. KD of either paxillin or drebrin in
hippocampal neurons lowered the stress exerted on 0.1 kPa hy-
drogels, and corresponding growth cones failed to respond to a
steep BDNF gradient coated on the 0.1 kPa surface. Importantly,
BDNF-elicited force generation and growth cone turning, which
requires expression, phosphorylation, and interaction of paxillin
and drebrin, was not dependent on Src kinase activity, which
is required for motility of growth cones on glass coverslips.
Together, our findings suggest that an extremely soft surface
transforms growth cone structures in a manner that favors
drebrin-paxillin interactions, which then enable generation of in-
ternal mechanical force required for efficient growth cone
orientation.
RESULTS
The growth cone T zone on brain-tissue-mimicking
hydrogels exhibits alternative molecular architecture
In conventional Banker’s neuronal culture on coverslips (Dotti
et al., 1988), the classical F-actin retrograde flow model de-
scribes two types of mechanical force transmission linking sepa-
rate cytoskeletal systems of growth cones: (1) a contractile force
driven by the motor protein myosin II in the T zone and (2) a thrust
force driven by F-actin polymerization in the P domain (Betz
et al., 2011; Forscher and Smith, 1988; Katoh et al., 1999; Le
Clainche and Carlier, 2008; Medeiros et al., 2006; Pollard and
Borisy, 2003; Woo and Gomez, 2006). However, it had been un-
clear whether this model applied to rodent hippocampal or
cortical neurons grown in a compliant native tissue. To answer
this question, we performed super-resolution structured illumi-
nation microscopy (3D SIM; Figures 1A and 1B) to analyze
morphology of growth cones of hippocampal neurons cultured
2–3 days in vitro (DIV). Polyacrylamide (PA) hydrogel (0.1 kPa)
was designed to mimic mechanical properties of embryonic
brain (E = 0.1–1 kPa). At 2 DIV, most neurons grown on
0.1 kPa exhibited a polarized morphology, with one minor neurite
longer than the others. By contrast, neurons grown on coverslips
showed an approximately 1-day delay in reaching the same
extent of polarization relative to neurons grown on 0.1 kPa gels
(Figure S1). Thus, we acquired images from DIV 1–2 cultures
on 0.1 kPa gels and from DIV 2–3 cultures on coverslips and
focused on comparing growth cones of either the nascent
axon or the most extended or enlarged minor neurites. Analysis
of cytoskeletal organization of growth cones on 0.1 kPa hydro-
gels revealed a fist-like, compact, F-actin-rich P domain and
an enlarged T zone (defined by drebrin staining) extensively over-
lapping the C domain (Figures 1B and 1C). By contrast, growth
cones of neurons grown on glass coverslips exhibited a wide P
domain with significantly reduced spatial overlap among cyto-
skeletal components (Figures 1B and 1C). Furthermore, when
we examined the arrangement of microtubules in 16-h neuronal
cultures using expansion microscopy (Figure S1), the direction of
microtubule bending near the cell periphery was outward on
0.1 kPa and inward in coverslip cultures. These observations
support the idea that native brain tissue or 0.1 kPa substrate,
which resembles the stiffness of brain tissue, is a more permis-
sive environment for microtubule protrusions from the cell
body than is a glass coverslip.
To assess the function of growth cone T-zone molecules in
terms of axonal outgrowth of newborn neurons in neonatal

Figure 1. Axonal growth cones of hippocampal neurons grown on 0.1 kPa hydrogels exhibit a distinct T zone with a high degree of paxillin-
drebrin co-localization
(A) Top and side views of 3D SIM images of hippocampal neurons grown on 0.1 kPa PA hydrogels or glass coverslips for 2 days in vitro (DIV), immunostained for
the T-zone factor drebrin (green in merge panels), microtubules (blue), and phalloidin for F-actin (red). Scale bars, 5 mm.
(B) Traces of staining intensity reveal a larger drebrin-positive growth cone T zone (gray rectangles) in 0.1 kPa hydrogels than on glass coverslips.
(C) Pie charts show percentage of drebrin, phalloidin, and microtubule co-localization based on signal intensity at the growth cone C domain or T zone of neurons
grown on substrates of varying stiffness.
(D) Fluorescence images of P3 rat cortices transfected in utero at E17 with IRES constructs harboring mScarlet and scrambled shRNA (SC shRNA) or drebrin
shRNA (DBN shRNA). Scale bars, 200 mm. The bottom plot shows relative mScarlet level of transfected cortical axons between the initial position (-2) and indi-
cated positions (dashed boxes) of the corpus callosum (±SEM, n = 3 independent experiments, >2 cortices for each drebrin shRNA and littermate control groups;
***p < 0.001, multiple t test).
(E) Images showing hippocampal neurons grown on 0.1 kPa hydrogels and coverslips, immunostained by antibodies against drebrin (top panel), paxillin (top
panel), TrkB (bottom panel), non-muscle myosin II (NMII; bottom panel), and phalloidin at 2 DIV. Scale bars, 5 mm.
(F) Summary scatterplot showing Pearson’s coefficient for protein co-localization with drebrin on 0.1 kPa culture and coverslips, as indicated (±SEM, n > 7 growth
cones each groups; ****p < 0.0001, t test).
(G) Images of hippocampal neurons grown on 0.1 kPa hydrogels and coverslips, immunostained with drebrin and paxillin (PXN) antibodies at 2 DIV. Repre-
sentative surface rendering 3D images sampled from the growth cone showing the co-localization surface (yellow). Volume thickness is 3 mm. Scale bars, 5 mm.
(H) Histograms showing the percentage of drebrin co-localizing with cytoskeletal proteins (left panel) and paxillin (right panel) in the growth cone of neurons grown
on 0.1 kPa hydrogels than for those grown on glass coverslips (±SEM, n = 7–13 cells for each set of experiments; **p < 0.01; ****p < 0.0001 by unpaired t test).

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cortex in vivo, we performed in utero electroporation to express
constructs encoding short hairpin RNA (shRNA) against drebrin
or scrambled control in a subpopulation of neural progenitor
cells of 17-day rat embryos (E17; Figure 1D). To extend analysis
of growth cone phenotypes, we obtained brain slices from
newborn (postnatal day 3 [P3]) rat cortex when most commis-
sural axons have crossed the midline. Nearly all (
100%)
newborn cortical neurons at P3 arriving at cortical layer 2/3 ex-
hibited a neurite-bearing, polarized morphology (Figure 1D),
with dendritic arbors oriented toward the pial surface and an
axon oriented radially in the cortical plate. At the same time,
most (
76%) control commissural axon tips (namely, those ex-
pressing mScarlet tracer) had reached the contralateral side of
the corpus callosum (Figure 1D). By contrast, drebrin KD cortical
neurons exhibited retardation of axon growth after crossing
the midline. Quantitative analysis indicated that drebrin KD
decreased axon length by
20% on the contralateral side of
corpus callosum at P3 (Figure 1D). The shortened axon pheno-
type was also observed in drebrin KD cortical neurons at P6 (Fig-
ure S2). Consequently, the extent of branching and innervation of
drebrin KD callosal axons to contralateral cortical layer 2/3 at
P16 was significantly less than that seen in control axons (Fig-
ure S2). These results suggest that T-zone factors are important
for proper growth and targeting of axonal projections of newly
generated cortical neurons in vivo.
To identify compliant substrate-dependent T-zone accessory
factors, we assessed differential expression in growth cones on
substrates with varying rigidity of eight factors linked to growth
cone movement or environmental sensing (Figure S3). Among
them we found that paxillin modulates actin dynamics, and the
BDNF receptor TrkB preferentially accumulated at the T zone
of neurons grown on 0.1 kPa hydrogels, a pattern not seen
when cells were grown on coverslips (Figures 1E–1H). Despite
the fact that T-zone formation is not dependent on substrate
elasticity (Figure 1E), levels of drebrin co-localizing with
the F-actin-stained P domain, the microtubule end-stained C
domain, and paxillin were significantly higher (
3-fold) on
0.1 kPa hydrogels than on coverslips (Figures 1G and 1H). These
enhanced molecular associations may be due in part to the
compact architecture seen at the tip of 0.1 kPa growth cones
(compare paxillin staining in Figures 1E and 1G). These findings
suggest that a compliant environment ensures proper engage-
ment and coordination of T-zone factors (such as myosin II, dre-
brin, and paxillin) with the growth cone cytoskeleton.
T-zone protein drebrin associates with paxillin in a
substrate elasticity-dependent manner
Given that paxillin accumulates at the T zone of growth cones on
0.1 kPa hydrogels (Figures 1E and 1G), we postulated that pax-
illin physically and functionally interacts with factors modulating
growth cone dynamics in such environments. Others had previ-
ously reported that embryonic drebrin is neuron specific (Shirao
and Obata, 1986), and we had previously identified drebrin as a
paxillin-associating factor via liquid chromatography-tandem
mass spectrometry analyses (Chang et al., 2017). Thus we
examined physical association of paxillin with drebrin in rat em-
bryonic brain lysates and in vivo co-immunoprecipitation (co-IP)
of cortical tissue lysates or lysates obtained from neurons
4 Cell Reports 40, 111188, August 16, 2022
Article
cultured on substrates of varying stiffness. Our in vivo co-IP anal-
ysis detected paxillin associating with drebrin in immunoprecip-
itates from brain lysates, but not from hepatic or cardiac tissues
(Figure 2A). Interestingly, paxillin and drebrin exhibit a similar
expression time course in the developing brain cortex, both
peaking at P0 and then progressively declining (Figure 2B), sug-
gesting they are temporally and spatially associated with each
other at early stages of neuronal development.
Next, we asked whether the degree of paxillin-drebrin associ-
ation reflects mechanical stiffness of substrates. Levels of dre-
brin immunoprecipitated by an anti-paxillin antibody from neu-
rons grown on 0.1 kPa hydrogels were significantly higher
than that seen in comparable analysis of neurons grown on cov-
erslips (Figure 2C), suggesting higher binding affinity in a soft
environment. Moreover, histidine-tagged drebrin co-purified
with GST-tagged paxillin in an in vitro binding assay, suggesting
direct interaction (Figure 2D). In vitro domain-mapping analysis
indicated that the paxillin C terminus, which harbors the LIM
domains, is required and sufficient for drebrin interaction
(Figures S4A and S4B). Notably, the paxillin N-terminal LD motif
alone is insufficient for drebrin interaction but had a modulatory
effect on binding affinity of the paxillin LIM domain for drebrin
(Figure S4). Domain-mapping experiments showed that the dre-
brin N-terminal actin-depolymerizing factor homology (ADFH)
domain is required for paxillin association (Figures S4C and
S4D). Importantly, confocal imaging of drebrin KD or control
neurons grown on 0.1 kPa hydrogels indicated a relative loss
of immunostaining for paxillin at T zone in drebrin KD samples
(Figure 2E), suggesting that drebrin is required for paxillin accu-
mulation at the growth cone T zone on these substrates.
Together, these findings indicate that substrate softness enables
drebrin to recruit neuronal paxillin to the growth cone T zone.
Expression of paxillin and drebrin is necessary for
traction force generated by hippocampal neurons grown
on 0.1 kPa hydrogels
To confirm functional association between paxillin and drebrin,
we assessed growth cone force generation and motion in soft
environments. We took advantage of the fact that 0.1 kPa hydro-
gels preserve structural features of advancing growth cones to
analyze force transduction upon cell-substrate interactions.
First, we compared the degree of integration of the core compo-
nent of the force generation machinery, namely, non-muscle
myosin II (NMII), at the growth cone T zone (Shin et al., 2014)
with P- and C-cytoskeletal domains in neurons grown either on
0.1 kPa hydrogels or coverslips (Figure 3A). We observed signif-
icantly higher (
3-fold) levels of NMII co-localizing with F-actin in
growth cones in neurons grown on 0.1 kPa hydrogels relative to
coverslips (Figure 3A3), a trend comparable with that seen with
drebrin (Figures 1A–1C). These observations suggest more effi-
cient growth cone motility in soft environments.
To investigate that possibility, we used traction force micro-
scopy (TFM) (0.5 frames per second over 10 min), which quan-
tifies particle displacements due to stress generated by growth
cones pressing against a 0.1 kPa hydrogel surface, to evaluate
changes in force transduction (Figure 3B). The average applied
force measured from control hippocampal neurons (at 2 DIV)
was 0.4 pN/m2 for the area of the cell body and growth cone of
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Figure 2. Paxillin directly binds drebrin in neurons grown on soft substrates

(A) Paxillin-associated complexes were immunoprecipitated (IP) from lysates of indicated E17.5 rat tissues using paxillin antibody (a-PXN) and detected by
western blot analysis. Normal rabbit immunoglobulin G (IgG) served as a negative control. Histograms show an increasing binding preference of paxillin toward
drebrin on soft (0.1 kPa) versus rigid (glass) substrates. Data represent mean intensity ± SEM (n > 3 independent experiments; ***p < 0.001; ****p < 0.0001 by
t test).
(B) Western blots (top panel) of lysates showing drebrin and paxillin protein expression at indicated developmental stages of cerebral cortex. Quantification
(bottom panel) of data shown in blots above. Data represent mean ± SEM from more than three independent experiments.
(C) Similar to (A), except cell lysate was obtained from cortical neuronal cultures grown on indicated substrates.
(D) Western blot showing direct paxillin-drebrin interaction. Bacterially expressed drebrin-His was purified by fast protein liquid chromatography and subjected to
a GST pull-down assay using GST-fused paxillin (GST-PXN) or GST alone. Precipitates were analyzed by western blotting with drebrin antibodies. Histogram
quantifies pull-down data (±SEM, n = 3; normalized to corresponding GST-PXN or GST inputs; ***p < 0.001 by t test).
(E) Representative confocal images of 2-DIV neurons on 0.1 kPa hydrogels incubated with scrambled control (SC) or drebrin siRNAs 30 min after cell plating,
followed by co-immunostaining for paxillin (PXN) and drebrin, and phalloidin staining for F-actin. Scale bars, 5 mm. Traces (bottom left) and histograms (bottom
right) showing the paxillin staining intensity in various cell compartments and the relative paxillin level between growth cone subdomains grown on 0.1 kPa hydro-
gels (±SEM; n = 20–25 cells for each group from three independent experiments; *p < 0.05; ns, not significant by ANOVA with multiple comparison test).

the longest neurite (Figure 3B2). After pretreating cells with
the NMII inhibitor blebbistatin (50 mM), those stress values
slightly but significantly decreased over the entire cell area
(Figures 3B2 and 3B3). However, pharmacological block of in-
tegrin-regulated FAK-Src signaling by pretreatment with the
Src kinase inhibitor PP2 (50 nM) did not alter the average force
generated by neuronal cell bodies or growth cones for the
10-min interval (Figures 3B2 and 3B3). These findings indicate
that cells on 0.1 kPa hydrogels exert forces in an NMII activity-
dependent but FAK-Src activity-independent manner. More-
over, drebrin or paxillin KD by respective small interfering
RNAs (siRNAs) significantly reduced stress (applied force) gen-
eration on 0.1 kPa hydrogels (Figures 3B4 and 3B5). Ectopic
expression of a drebrin construct harboring a phospho-
mimicking mutation (YFP-DBNS142D) restored force generation
in drebrin KD growth cones (Figures 3B4 and 3B5), supporting
the idea that FAK-Src signaling-independent force generation
requires paxillin and drebrin expression.
We propose that neurons grown on stiff versus soft substrates
differ in force generation and neurite extension modes. To
examine this possibility, we assessed neurite growth of neurons
grown on substrates of varying rigidity in the presence and
absence of NMII inhibitor blebbistatin (Figure S5). Blebbistatin-
treated hippocampal neurons grown on coverslips exhibited a

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Figure 3. Myosin II-mediated force generation of growth cones grown on 0.1 kPa hydrogels requires paxillin and drebrin expression, but not
Src kinase activity
(A) (A1) Myosin II distribution at the growth cone of 2-DIV hippocampal neurons grown on 0.1 kPa PA hydrogels or glass coverslips. Scale bars, 5 mm. Traces of
staining intensity reveal a compact growth cone with greater overlap of T-zone myosin II (NMII; marked by gray rectangles) with cytoskeletal proteins in neurons
grown on 0.1 kPa hydrogels compared with when grown on glass coverslips. (A2 and A3) Traces showing the staining intensity of a single cell (A2) and the aver-
aged intensity (n > 25 cells for each group; A3) of the indicated molecules in various cell compartments of neurons grown on 0.1 kPa hydrogels and coverslips.
Data represent mean ± SEM (*p < 0.05; ns, not significant by ANOVA with multiple comparison test).
(B) Myosin II-mediated contractile force measured by traction force microscopy. (B1) Schematic showing traction force microscopy and force-field visualization.
PIV, particle image velocimetry. FTTC, Fourier transform traction cytometry. (B2 and B4) Representative TFM images of vector fields plotted over magnitudes of
displacement (presented as pseudocolor maps in a linear scale) of 2-DIV hippocampal neurons grown on 0.1 kPa hydrogels in the presence or absence of phar-
macological inhibitors (B2; 50 mM myosin II inhibitor (±)-blebbistatin [(±)-Bleb] or 50 nM Src kinase inhibitor PP2) or siRNA-mediated knockdown of paxillin (PXN)
or drebrin (DBN) expression, and/or ectopic expression of phosphorylation mimicking YFP-DBNS142D (B4), as indicated. Scale bars, 20 mm. (B3 and B5) Histo-
grams summarizing averaged stress exerted by the entire image field (Whole Field), growth cone (Growth Cone), and cell body (Soma). Data represent mean ±
SEM (n > 10 cells for each set of experiments; *p < 0.05; **p < 0.01; ns, not significant by t test).

collapse of filopodia on growth cones as well as more curved and
elongated processes (both dendrites and axons) relative to un-
treated control neurites on coverslips (Figure S5), suggesting a
relaxation of the NMII-generated actin arc barrier that restricts
microtubule protrusion. In contrast, growth cone morphology
in cells grown on 0.1 kPa gels was little changed by blebbistatin
treatment, and neurite length of those neurons was significantly
shortened (Figure S5), implying impaired regulation of microtu-
bule dynamics. These findings also support the notion that those
mechanisms controlling neurite extension are different in neu-
rons grown on 0.1 kPa gel from those grown on coverslips.
Concomitantly, drebrin and/or paxillin KD in neurons grown on
0.1 kPa hydrogels resulted in shortening of axons relative to con-
trol (scrambled siRNA) neurons (Figure S6). Overall, these find-
ings indicate that proper neurite extension and mechanical activ-
ity of growth cones on a soft substrate requires T-zone factors
and paxillin.
Asymmetric exposure to BDNF biases paxillin
distribution and growth cone turning on soft substrates
independent of Src
Next, we asked whether the turning direction of growth cones
grown on soft substrates is dictated by paxillin and drebrin. To
do so, we established a turning assay for growth cones grown
on substrates of varying rigidity (Figure 4). We counter-printed
the chemotropic factor BDNF (Gallo et al., 1997; Oberstar
et al., 1997) and/or fluorescent tag-conjugated BSA stripes
(50 mm wide, spaced 100 mm apart) onto surfaces of 0.1 kPa hy-
drogels or glass coverslips and then analyzed chemotropism of
growth cones approaching the stripes as well as their molecular

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Figure 4. Different signaling pathways mediate BDNF-induced growth cone attraction for neurons grown on 0.1 kPa hydrogels or glass
coverslips
(A) (A1) Schematic showing procedure used for BDNF/BSA-stripe printing onto surface of 0.1 kPa hydrogels. (A2) Fluorescent image of a stripe coated with
BDNF-AlexaFluor647. Right panels show fluorescence intensity across the stripe boundary. Scale bar, 3 mm.
(B) Images of growth cones growing near a stripe coated with fluorescently conjugated BDNF or control BSA on substrates of various elastic moduli, immu-
nostained with drebrin and paxillin antibodies (presented as pseudocolor maps in a linear scale) and phalloidin for F-actin at 2 DIV. Dashed line marks the central
axis of growth cones. Scale bars, 5 mm.
(C) Schematic diagrams (top panel) depict turning angles of the growth cone near BDNF or control BSA-coated stripes (blue rectangle). Plots show similar
percentages of growth cones turning toward (positive angles) BDNF stripes for neurons grown on substrates of varying stiffness. Only neurons with growth cones
located near the stripe boundary were counted.
(D) Quantitative measurement showing the percentage of drebrin co-localizing with paxillin on the side of growth cones facing toward or away from the BDNF or
BSA-coated stripes on substrates on varying stiffness. Data were calculated by dividing the co-localized volume on the indicated side by the total drebrin stained
volume, and presented as mean ± SEM (>18 cells for each group from three independent experiments; **p < 0.01; ns, not significant by t test).
(E) Similar to (D), except that preferential pattern of drebrin-paxillin co-localization (denoted as ‘‘PXNXdrebrin’’) is assessed based on a co-localization index (CI)
formula, as indicated. Data represent mean ± SEM (>18 cells for each group from three independent experiments; *p < 0.05; ns, not significant by t test).
(F) Similar to (C) and (E), except cells were subjected to bath application of pharmaceutical inhibitors of AAk1, PKA (KT 5720), or Src tyrosine kinase (PP2), as
indicated. Data represent mean ± SEM (>10 cells for each group from three independent experiments; **p < 0.01; ***p < 0.001; ns, not significant by t test).
(G) Similar to (F), except cells were cultured on BDNF-stripe-coated glass coverslips. Only neurons with growth cones located near the stripe boundary were
counted.

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repositioning toward BDNF-coated stripes (Figures 4A–4E). As
shown in Figures 4B and 4C, BDNF-coated stripes significantly
attracted growth cones grown on both 0.1 kPa hydrogels and
glass coverslips, with most (>70%) turning toward BDNF stripes.
In contrast, turning behavior of growth cones on 0.1 kPa gels or
coverslips relative to control BSA-coated stripes was more
random (Figures 4B and 4C). When we compared protein distri-
butions on sides of the growth cone (i.e., facing toward stripes
versus away from them), we observed preferential paxillin and
drebrin co-localization on the side facing BDNF stripes on
0.1 kPa hydrogels (Figures 4D and 4E; co-localization index > 1),
suggesting that asymmetric exposure to BDNF on soft surface
promotes paxillin-drebrin association. We did not, however,
observe the same effects for growth cones grown on coverslips
(Figures 4D and 4E), implying that the paxillin-drebrin association
is weak in neurons grown on glass.
Given that newborn neurons grown on compliant substrates
(E < 1 kPa) switch the relative dominance of endocytosis over
adhesion signaling for neurite initiation (Chang et al., 2017), we
reasoned that molecular mediators and signaling pathways
used for BDNF-induced chemotaxis differ among neurons grown
on microenvironments of varying elasticity. To test this hypothe-
sis, we pretreated neuronal cultures grown on 0.1 kPa hydrogels
or coverslips with inhibitors of the focal adhesion kinase Src
(PP2), the endocytosis kinase AAK1, or the growth cone turning
signaling kinase (Song et al., 1997) PKA (KT5720) (Figures 4F and
4G), and measured the BDNF-directed growth cone turning.
Indeed, although Src kinase activity was required for growth
cone turning on coverslips (Figure 4G), attraction of a growth
cone to BDNF on 0.1 kPa hydrogels was abolished by blocking
either AAK1 or PKA activities but not Src kinase activity (Fig-
ure 4F). We also evaluated preferential paxillin/drebrin co-local-
ization on the side of growth cone facing BDNF stripes, and its
dependence on endocytic activity. Consistently, BDNF was
insufficient to promote paxillin/drebrin co-localization in growth
cones moving on hydrogels in the presence of an AAK1 inhibitor
or of KT5720, resulting in uniform paxillin distribution across the
growth cone (Figure 4F, co-localization index = 1). In contrast,
neither directed growth cone turning nor paxillin/drebrin co-
localization (Figure 4F) in hydrogel-grown growth cones was
altered by Src kinase inhibition. These findings indicate that pax-
illin/drebrin distribution in neurons responds to BDNF stimuli in a
matrix compliance-specific manner, independent of activity of
Src kinase.
Paxillin-drebrin interaction and drebrinS142
phosphorylation coordinate BDNF-induced growth cone
turning behavior on 0.1 kPa hydrogels
To quantify in situ direct paxillin-drebrin interaction in the pres-
ence of BDNF, we used a proximal ligation assay (PLA) to assess
proximity (<40 nm apart) of paxillin and drebrin proteins at the
growth cone T zone. In this assay, a change in the number of
PLA puncta observed reflects paxillin-drebrin interaction after
bath application of BDNF. As shown in Figure 5A, PLA signal
density in growth cones of neurons grown on 0.1 kPa hydrogels
was significantly greater following BDNF treatment relative to un-
treated samples, an effect eliminated by pretreatment with
K252a, which inhibits the BDNF receptor TrkB.
8 Cell Reports 40, 111188, August 16, 2022
Article
Using PLA analysis, we also detected direct interaction be-
tween TrkB and paxillin in the cell body of neurons at 2 DIV grown
on 0.1 kPa gels in the absence of BDNF treatment (Figure S6).
Interestingly, treatment of 0.1 kPa cultures with BDNF increased
paxillin association (based on increases in PLA puncta density)
with TrkB in both cell bodies and growth cones relative to con-
trols (Figure S7). To determine whether this activity resulted in
phosphorylation of paxillin-drebrin complexes, we treated
cortical neurons cultured on 0.1 kPa gels with and without
BDNF and then subjected cell lysates to immunoprecipitation
with an anti-paxillin antibody, followed by immunoblotting with
antibodies against potential paxillin-associated factors,
including CIP4, vinculin, and drebrin, as well as S142 phosphor-
ylated drebrin (p-drebrinS142). Indeed, levels of drebrin and
p-drebrinS142 co-immunoprecipitated with paxillin increased in
BDNF-treated cells (Figures S8A and S8B). While we also
observed greater association of paxillin with CIP4 in BDNF-
treated cells, we did not detect vinculin/paxillin association in
0.1 kPa cultures before or after BDNF treatment (Figures S8A
and S8B). These findings agree with our previous report that pax-
illin expressed in neurons grown on 0.1 kPa substrates does not
preferentially bind focal adhesion molecules (Chang et al., 2017).
In contrast, pretreating 0.1 kPa cultures with the TrkB inhibitor
K252a abolished BDNF-induced drebrin phosphorylation
(Figures S8A and S8B), confirming that BDNF signaling induces
phosphorylation of paxillin-drebrin complexes.
To determine whether BDNF-induced paxillin association re-
quires drebrin phosphorylation at serine 142, we transfected
N2a cells with YFP-tagged wild-type (WT) drebrin or a tagged
phosphorylation-deficient drebrinS142A construct (DBNS142A)
and treated both groups with and without BDNF. Co-IP of cell ly-
sates with an anti-YFP antibody revealed increased levels of
paxillin and S142-phosphorylated WT drebrin in BDNF-treated
cells (Figures S8C and S8D), while control and BDNF-treated
cells transduced with the DBNS142A mutant showed comparable
paxillin association (Figures S8C and S8D). Because this residue
enables microtubule coupling to F-actin in growth cone filopodia
(Worth et al., 2013), p-drebrinS142 likely contributes to attraction
to BDNF stripes by growth cones on 0.1 kPa hydrogels. To
assess this possibility, we calculated the percentage of axons
that moved forward on or off BDNF stripes coated on 0.1 kPa hy-
drogels, assessing only neurons with somata located off the
stripes (Figures S8E and S8F). At 3 DIV,
70% of neurons ex-
pressing WT drebrin exhibited axon tips moving along the
BDNF-coated surface, while neurons expressing DBNS142A ex-
hibited randomly distributed axon terminals (50% on and

50% off BDNF-coated stripes) (Figures S8E and S8F), suggest-
ing reduced attraction to BDNF stripes.
Finally, we asked whether BDNF-induced growth cone turning
required drebrin-paxillin interaction. We found that preferential
turning of 0.1 kPa hydrogel-grown growth cones toward BDNF
stripes (76% toward/on versus 24% off the stripes; Figures 5B
and 5C), as well as preferential accumulation of paxillin on the
sides of growth cones facing BDNF stripes, was absent in dre-
brin KD neurons (53% toward/on versus 47% off the stripes;
Figures 5B and 5C). Importantly, attraction of growth cones
turning on 0.1 kPa hydrogels to BDNF stripes was abrogated
in neurons expressing a paxillin LIM3-4 deletion variant, which
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Figure 5. BDNF-induced growth cone turning requires association of paxillin with drebrin
(A) BDNF promotes paxillin-drebrin interaction at the growth cone on 0.1 kPa gels. Left panel: schematic describing PLA analysis used to assess paxillin-drebrin
interaction. Middle panels: representative images of 2-DIV hippocampal neurons stained with phalloidin for actin (red in merge) plus PLA detection of paxillin and
drebrin antibody signals (green in merge) after 30 min of BDNF (20 nM) treatment, with or without 30 min of K252a (200 nM) pretreatment. Scale bars, 5 mm. Right
panel: histogram shows number of PLA puncta per 50 mm2 of growth cone area from all experiments (±SEM, n > 25 cells for each group from three independent
experiments; ****p < 0.0001 by Student’s unpaired t test). Growth cone area was defined by thresholding phalloidin-stained images and tracing growth cone out-
lines (dashed lines in top middle panels). Only puncta located inside or on the phalloidin-stained growth cone area were counted.
(B and C) BDNF-induced chemoattraction on 0.1 kPa hydrogels requires drebrin expression. Similar to Figure 4B, except that cells grown on BDNF-stripe-coated
0.1 kPa hydrogels were treated with scrambled siRNA (SC-siRNA) or drebrin siRNA (DBN-siRNA), and immunostained with drebrin and paxillin antibodies and
phalloidin for F-actin at 3 DIV, as indicated. Scale bars, 5 mm. Only neurons located off the stripe with neurite initiation off the stripe were counted.
(D) Quantitative assessment of paxillin asymmetry (as calculated by the formula shown at the top) showing that drebrin KD eliminates asymmetric paxillin
accumulation on the side of growth cones facing BDNF-coated stripes on 0.1 kPa hydrogels. Data represent mean ± SEM (>14 cells for each group from three
independent experiments; **p < 0.01 by t test).
(E–G) Similar to (B–D), except cells were transfected with plasmid encoding full-length (PXN_FL) or the LIM 3–4 domain deletion variant (PXN_DLIM3-4) of paxillin.
Data represent mean ± SEM (>10 cells for each group from three independent experiments; *p < 0.05 by t test). Scale bars, 5 mm.

Cell Reports 40, 111188, August 16, 2022 9

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10 Cell Reports 40, 111188, August 16, 2022

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cannot interact with drebrin (Figures 5E and 5F). Similarly, pref-
erential paxillin/drebrin accumulation on the growth cone side
facing BDNF stripes was suppressed in neurons following
ectopic expression of the paxillin LIM3-4 deletion variant (Fig-
ure 5G). Consistently, axons of neurons misexpressing a drebrin
deletion mutant lacking the ADFH domain, a construct that
cannot bind paxillin (Figures S4C and S4D), exhibited no prefer-
ence for growth on BDNF-coated stripes in 0.1 kPa cultures
(45% on versus 55% off the stripes; Figures S4E and S4F). These
findings support the idea that paxillin-drebrin interaction medi-
ates the effect of BDNF on growth cone motion.
BDNF-elicited traction force requires paxillin and
drebrin expression
Our observations indicate that paxillin-drebrin interaction at the
growth cone T zone is facilitated by BDNF (Figure 5), which in
turn elicits a pushing force in the growth cone against the
0.1 kPa substrate. To investigate this possibility, we used TFM
to examine changes in stress induced by growth cones on
0.1 kPa hydrogels in response to BDNF stimuli. Bath application
of 20 nM BDNF significantly enhanced stress generated by the
cell body and growth cone in neurons grown on 0.1 kPa hydro-
gels (Figure 6A), an effect significantly blocked by K252a pre-
treatment (Figure 6A), supporting the idea that BDNF signaling
is sufficient to generate neuronal mechanical force on a soft sub-
strate. Finally, we asked whether BDNF-elicited neuronal force
contributes to growth cone motion on 0.1 kPa hydrogels, where
substrate adhesion is minimized. Since paxillin is critical for
growth cone force generation (Figures 3B4 and 3B5) and turning
behavior (Figure 5E) on 0.1 kPa hydrogels, we postulated that
paxillin functions in the force generation response to BDNF. To
confirm this possibility, we performed paxillin KD in neurons
and assessed growth cone force generation capacity. Growth
cones of control neurons generated a differential force, with
stronger stress emanating from the side facing toward BDNF
stripes, on 0.1 kPa hydrogels (Figure 6B). In contrast, paxillin
or drebrin KD neurons exerted much less force on 0.1 kPa hydro-
gels and their growth cones facing toward BDNF stripes ex-
hibited no preferential activity (Figure 6B), suggesting that paxil-
lin expression is required for BDNF-induced force generation by
neurons on 0.1 kPa hydrogels. This activity likely requires paxil-
lin-drebrin interaction, as neurons expressing paxillin DLIM3-4
did not exhibit BDNF-induced force generation (Figures 6A3
and 6B3). Together, these findings indicate that paxillin in the
growth cone links chemical guidance signals to generation of
physical force, which then drives movement of axonal tips on
soft environments.
DISCUSSION
Axonal pathfinding in its most energy-efficient form is instructed
by the chemical and physical context of tissue environments
conducive to long-range neuronal targeting to designated brain
areas. Here, we assessed major force-generating components
and functional structures of axonal growth cones of hippocam-
pal neurons cultured on 0.1 kPa hydrogels. We observed fist-
like architectures of growth cones on 0.1 kPa hydrogels, strongly
resembling the typical morphology of rapidly extending axons
in vivo or reported in three-dimensional (3D) cultures (Balgude
et al., 2001). This compact structure enhances the degree of
actin and microtubule engagement, as well as physical interac-
tions between the T-zone factor drebrin and the accessory pro-
tein paxillin. We found that direct drebrin-paxillin interaction is
dependent on the physical environment, since binding affinity
of paxillin for drebrin is significantly greater in brain tissue and
on 0.1 kPa hydrogels than on stiffer 20 kPa hydrogels. Moreover,
we found that BDNF stripes bound to 0.1 kPa hydrogel surfaces
exert a chemoattractive turning and force generation effect on
growth cones, actions that required paxillin and drebrin expres-
sion and interaction but not the activity of Src kinase. These find-
ings demonstrate that the force generation mechanism of neu-
rons growing in soft substrates differs from that of neurons
grown on coverslips and is mediated by paxillin-drebrin interac-
tion at the T zone of the axonal growth cone (Figure 6C).
Several in vitro culture systems—such as synthetic hydrogel
platforms, microfluidic systems, paper-based culture platforms
(Ng et al., 2017; Pelham and Wang, 1997; Santos et al., 2020;
Taylor et al., 2005), or stem cell-based brain organoids (Gopalak-
rishnan, 2019)—have been engineered to recapitulate various
stages of neuronal/brain development. By increasing the elastic-
ity of PA hydrogels to a range resembling the stiffness of
brain tissue (0.1–1 kPa), and with proper surface coating with

Figure 6. BDNF is sufficient to induce asymmetric force generation by growth cones of neurons growing on 0.1 kPa hydrogels via a
mechanism mediated by paxillin-drebrin interaction
(A) BDNF signaling increases force exerted by growth cones of neurons grown on 0.1 kPa hydrogels. (A1) Representative TFM images of vector force fields
plotted over magnitudes of the displacement (presented as pseudocolor maps in a linear scale) for 2-DIV hippocampal neurons growing on 0.1 kPa hydrogels and
subjected (or not) to bath application of 20 ng/mL BDNF and/or pretreatment of the TrkB inhibitor K252a (200 nM). (A2) Histograms summarizing averaged stress
exerted (±SEM, n = 7–15 cells for each set of experiments; *p < 0.05; **p < 0.01; ns, not significant by t test) by the entire image field (Whole Field), growth cone
(Growth Cone), and cell body (Soma). (A3) Similar to (A2), except cells were transfected with plasmid encoding full-length (PXN_FL) or the LIM 3–4 domain deletion
variant (PXN_DLIM3-4) of paxillin. Histograms summarizing averaged stress exerted (±SEM, n = 21 cells; ns, not significant by t test) by the entire image field
(Whole Field), growth cone (Growth Cone), and cell body (Soma).
(B) Asymmetric exposure to BDNF biases the force distribution exerted by growth cones on 0.1 kPa hydrogels. (B1) Representative TFM images of growth cones
approaching BDNF- or control BSA-coated stripes (blue rectangle) on 0.1 kPa hydrogels. Cells were treated with scrambled siRNA (SC-siRNA), paxillin siRNA
(PXN-siRNA), or drebrin siRNA (DBN-siRNA). Scale bars, 5 mm. (B2 and B3) Quantitative measurement of force-field asymmetry in a 2.5 mm 3 2.5 mm area (dashed
boxes in B2) covering half of a growth cone facing toward versus away (stoward/saway) from BSA- or BDNF-coated stripes and pretreated with or without 200 nM
K252a, as indicated. Data represent mean ± SEM (n = 7–20 cells for each group from three independent experiments; *p < 0.05; **p < 0.01 by t test). Only neurons
with growth cones located near the stripe boundary were counted.
(C) Schematic illustration showing organization of growth cones on soft substrates and asymmetric distribution of T-zone factors associated with paxillin that
enable growth cone force generation toward a BDNF gradient, in a Src kinase-independent manner.

Cell Reports 40, 111188, August 16, 2022 11

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OPEN ACCESS
positively charged macromolecules, hippocampal neurons
grown on hydrogels can attain structural properties of growth
cones undergoing active extension, as seen in classic ‘‘open-
book’’ preparations or by DiI tracking of spinal commissural
axons ex vivo (Bovolenta and Dodd, 1990; Stoeckli and Land-
messer, 1995). Inverse durotaxis has been shown for growth
cones on PA hydrogels displaying a gradient of rigidity, with
axon bundles of retinal ganglion cells (RGCs) preferentially ex-
tending toward the softer side of the gel (Koser et al., 2016).
This feature has been demonstrated in vivo for zebrafish RGC
axons extending along the optic tract (Koser et al., 2016).
Here, we replicated the stiffness of brain environment using
0.1 kPa PA hydrogels with minimal surface coating of laminin
and showed that the intracellular T-zone components drebrin
and paxillin, in the context of a particular cytoskeletal architec-
ture, function like a counter-shaft to transmit force for bound
chemical guidance cues.
Despite the fact that most cell types grown on stiff (>10 kPa)
substrates use integrin-dependent pathways for directed cell
migration (Kerstein et al., 2017; Lo et al., 2000; Robles and Go-
mez, 2006), other cell types such as neurons and tumor cells
adapt to low-rigidity environments and do not rely on mature
focal adhesion for migration in response to gradients of bound
chemical cues (Tyler, 2012). Our findings reveal an alternative
type of cell mechanics specifically evoked on compliant environ-
ments, one that exhibits three features enabling growth cone
advancement and navigation. First, the compact architecture
of the growth cone in soft substrates means greater mechanical
proximity of the T zone and C and P domains. Second, the
T-zone molecule drebrin selectively associates with paxillin on
soft surfaces to generate stress or pressing force on the sub-
strate. Third, chemotaxis induced by bound neurotrophic factors
on soft surfaces is independent of that exerted by the integrin-
FAK-Src signaling. Conversely, the primary model of growth
cone mechanics in soft substrates does not follow the actin-
based treadmill model for the leading edge of migrating cells
on rigid substrates (Forscher and Smith, 1988; Lowery and Van
Vactor, 2009; Mitchison and Kirschner, 1988). Instead, neurons
grown on a soft substrate express extremely low levels, if any,
of the focal adhesion protein vinculin relative to neurons grown
on stiffer substrates (Chang et al., 2017), implying that the integ-
rin-dependent pathway is dispensable in compliant physical
contexts in terms of directing growth cone turning or force
generation.
The force exerted by rat cortical neurons (in the range of 0.5–1
pN/mm2) is exceedingly small compared with that exerted by
many other cell types, such as fibroblasts, which exert force in
the range of 10 nN/mm2 (Dembo and Wang, 1999). Our findings
indicate that growth cone attraction on soft surfaces requires
drebrin and paxillin, an association that leverages the actin-
microtubule coordination/interface with NMII to induce force
generation in the T zone. Accordingly, we postulate that upon
contact with soft surfaces, neurons undergo a structural and mo-
lecular transformation to maximize efficiency and growth cone
sensitivity and movement. Neurons grown on stiff substrates in-
crease levels of RhoA/Rock and form point contacts at filopodia
tips (Chang et al., 2017; Woo and Gomez, 2006). In that case,
NMII located at the T-zone actin arc mediates actin contraction,
12 Cell Reports 40, 111188, August 16, 2022
Article
flattens lamella, and obstructs microtubules protruding into the P
domain (Luo, 2000; Santos et al., 2020). Thus, neurite overexten-
sion observed in coverslip cultures after NMII inhibition can be
attributed to: (1) reduction in RhoA/Rock-activated, NMII-gener-
ated contractile forces acting on cortical actin along the axonal
shaft; and (2) relaxing the NMII-generated actin arc barrier that
restricts microtubule protrusion (Bradke and Dotti, 1999; Dupraz
et al., 2019; Santos et al., 2020). The same NMII inhibition ac-
counts for lack of neurite overextension on 0.1 kPa PA-gel cul-
tures, in agreement with previously reported data in 0.2 kPa
collagen 3D cultures (Bradke and Dotti, 1999). We argue that
growth cone mechanics of neurons grown on 2D 0.1 kPa sub-
strates resemble those seen in 3D soft matrices, in which there
is a relatively low level of integrin-actin linkage (adhesion clutch
engagement), endowing growth cones with an amoeba-like
movement independent of FAK-Src activity. Accordingly, NMII-
mediated contractility may ensure that growing axons of
0.1 kPa neurons form a compact actomyosin arc to gather the
ends of microtubule bundles and allow growing axons to
press/push aside obstacles in their path. Although we applied
the same coating solution (a mixture of poly-L-lysine [PLL] and
laminin) to coverslips and 0.1 kPa PA gels, resulting surface
PLL and laminin levels may differ. Thus, different adhesion sub-
strates and elasticity could have altered the NMII distribution
and/or function (Ketschek et al., 2007; Turney et al., 2016). We
also noted that a snapshot of growth cone zip-zap behavior
does not accurately indicate the direction in which the growth
cone is going on a uniform substrate. However, while the growth
cone is exposed to a gradient of attractants or moves up a
gradient, it does exhibit a longer continuous movement and a
more significant turning angle toward attractants (Mai et al.,
2009; Ming et al., 1997). Since the attractant gradient (such as
BDNF stripes) biases growth cone behavior, comparison of the
sides of the growth cone facing both toward and away from
the BDNF stripe allows us to analyze potential asymmetric distri-
bution of T-zone molecules and force generation underlying
growth cone steering.
It is intriguing that compliant environments evoke systematic
changes in axonal growth cones, not only in terms of structure
and protein localization but in signaling required for force exer-
tion, which are all distinct from those observed in neurons grown
on rigid substrates. These differences reflect the low-adhesive,
high-surface-tension characteristics of neuronal mechanics in
brain. Here, we present a potential molecular mechanism that
underlies how axonal tips in the brain orient their movement
and force toward trace amounts of a chemical guidance cue,
namely through low-rigidity and specific interaction between
neuronal paxillin and drebrin. Looking forward, our proposed
model, which integrates structural changes in axonal tips with
molecular interaction of T-zone factors, describes a synergistic
mechanism that drives microtubule-to-actin engagement and
force generation enabling growth cone movement in the context
of an extremely soft environment.
Limitations of the study
Acquiring high-resolution F-actin images of growth cones on
our 0.1 kPa PA hydrogels (
200 mm thick and watery) using
stimulated emission depletion microscopy remains technically

Article
challenging, possibly due to the inhomogeneous refractive index
between the cell and scaffold.
Although our findings focus on paxillin/drebrin and their integ-
rin-independent regulation of neurons grown on soft substrates,
it is important to recognize that post-translational modification
(either phosphorylation or acetylation) of paxillin can be induced
by FAK or Src activity and is known to be essential for movement
of cells/growth cones on rigid surfaces coated with high concen-
trations of positively charged PLL (Koch et al., 2012; Myers and
Gomez, 2011). Also, apart from paxillin’s association with T-zone
factors, other paxillin functions mediated by various interacting
partners—such as receptor-mediated endocytosis, trafficking,
or nuclear translocation—could play important roles in neuronal
differentiation in a compliant microenvironment (Chang et al.,
2017; Dong et al., 2009; Dubois et al., 2017; Sathe et al., 2016;
Xu et al., 2019). In addition to growth cone dynamics, physical
environmental factors may also modulate strength and efficacy
of cell-cell contacts along axons and dendrites, providing
another mechanism by which neuronal maturation can be sys-
tematically controlled.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Plasmids, antibodies, and inhibitors
B Cell culture, protein lysate preparation, and immuno-
staining
B Expression of GST- and His-tagged proteins, and
in vitro and in situ binding assays
B Polyacrylamide gel preparation
B In utero electroporation
B Microfabrication and substrate patterning
B Expansion microscopy
B Traction force microscopy, image acquisition, and im-
age processing
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2022.111188.
ACKNOWLEDGMENTS
We thank Mu-Ming Poo and Hwai-Jong Cheng for discussions and critical
reading of the manuscript. We thank the IMB Imaging Core Facilities (Institute
of Molecular Biology, Academia Sinica) for SIM imaging and TFM analysis.
This work was supported in part by a grant (MOST 110-2628-B-001-028-)
from the Ministry of Science and Technology of Taiwan, and Career Develop-
ment Award (AS-CDA-107-L04) from Academia Sinica, Taiwan.
ll
OPEN ACCESS
AUTHOR CONTRIBUTIONS
C.C., C.-H.C., Y.C., and T.-Y.C. performed, analyzed, and validated biochem-
istry experiments, immunofluorescence staining, plasmid construction, and
hydrogel preparation, and co-wrote the manuscript. S.-W.C. and P.-L.C. per-
formed the IUE experiment. S.-Y.L. performed primary neuronal cultures.
Y.-C.T. and B.-C.C. supervised, performed, analyzed, and validated expan-
sion microscopy. H.-L.T. supervised TFM analysis. P.-L.C. supervised,
conceptualized, and co-wrote the manuscript. All authors read and contrib-
uted to the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
We worked to ensure sex balance in the selection of non-human subjects. One
or more of the authors of this paper self-identifies as a member of the LGBTQ+
community. One or more of the authors of this paper received support from a
program designed to increase minority representation in science. While citing
references scientifically relevant for this work, we also actively worked to pro-
mote gender balance in our reference list.
Received: September 12, 2021
Revised: March 23, 2022
Accepted: July 20, 2022
Published: August 16, 2022
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
anti-FLAG M2 Sigma-Aldrich Cat#F1804; RRID:AB_262044
chicken polyclonal bIII tubulin (AB9354) Merck Millipore Cat# AB9354; RRID:AB_570918
anti-Actin (MAB1501) Merck Millipore Cat# MAB1501; RRID:AB_2223041
anti-alpha Tubulin (ab89984) Abcam Cat# ab89984; RRID:AB_10672056
anti-non-muscle Myosin IIB [3H2] (ab684) Abcam Cat#ab684
anti-drebrin (ab11068) Abcam Cat# ab11068; RRID:AB_2230303
anti-paxillin clone 349 (612405) BD Biosciences Cat# 612405; RRID:AB_647289
Paxillin Polyclonal Antibodies( ThermoFisher Cat# PA5-34910;
Scientific/ Invitrogen RRID: AB_2552260
c-Myc Monoclonal Antibody (9E10) Invitrogen Cat#MA1-980; RRID: AB_558470
Piezo1 Antibody Novus Biologicals Cat#NBP2-10504
Piezo2 Antibody Novus Biologicals Cat#NBP1-79321; RRID:AB_11030107
TrkB Polyclonal Antibody ThermoFisher Scientific Cat# PA5-78405; RRID:AB_2736725
CD171 (L1CAM) Polyclonal Antibody ThermoFisher Scientific Cat# PA5-85876; RRID:AB_2802677
YAP Antibody (63.7) Santa Cruz Biotechnology Cat# sc-101199; RRID:AB_1131430
Anti-Integrin b1 Antibody, activated, clone HUTS-4 Millipore Cat# MAB2079Z; RRID:AB_2233964
Talin (C-20) Santa Cruz Biotechnology Cat# sc-7534; RRID:AB_661610
Anti-GFP/YFP antibody (ab6556) Abcam Cat# ab6556;
RRID: AB_305564
Anti-phospho Drebrin (Ser142), clone 3C14 Antibody Millipore Cat#MABN833
Bacterial and virus strains
pLenti-C-mGFP-PXN OriGeneTechnologies Cat#PS100071
pGFP-C-shLenti shRNA Vector OriGeneTechnologies Cat#TR30023
ECOS101 DH5a competent cell Yeastern Biotech Co. Cat#FYE678
Biological samples
Sprague Dawley timed-pregnant rats BioLASCO N/A
Chemicals, peptides, and recombinant proteins
Recombinant Human/Murine/Rat BDNF PeproTech Cat#450-02
BSA Alexa Fluor 647 conjugate Invitrogen Cat#A34785
Forskolin, 7-Deacetyl-7-[O-(N-methylpiperazino)- Calbiochem Cat#344273
g-butyryl]-, Dihydrochloride
KT5720 Calbiochem Cat#420320
Pim1/AKK1-IN-1, also named LKB1/AAK1 dual inhibitor MedChemExpress Cat#HY-10371
PP2 Abcam Cat#ab120308
PP3 Abcam Cat#ab120617
K252a Abcam Cat#ab120419
Gem21 NeuroPlexTM GEMINI Bio-products Cat#400-160
poly-L-Lysine Sigma Aldrich Cat#P2636-1G
laminin Corning Cat#354232
SYLGARD 184 SILICONE ELASTOMER KIT Dow Corning Material#1673921
N0 -N0 -methylenebisacrylamide solution Sigma Aldrich Cat#M1533
Acrylamide solution Sigma Aldrich Cat#A4058
(3-aminopropyl)triethoxysilan Sigma Aldrich Cat#A3638
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Critical commercial assays
DuolinkR In Situ Detection Reagents Red, Anti-Rabbit MINUS, Sigma Aldrich Cat#DUO92008, DUO92005, DUO92001
Anti-Mouse PLUS
Deposited data
Mendeley DATA https://data.mendeley.com https://doi.org/10.17632/cpb3skbrhn.4
Experimental models: Cell lines
Human Embryonic Kidney 293T cells ATCC Cat# CRL-3216; RRID:CVCL_0063
Oligonucleotides
paxillin siRNA C: 50 -GAUGAGCAGUCCACAGCGA -30 This paper N/A
paxillin siRNA D: 50 -CAGCCUAGUGGUUCCAGAU -30 This paper N/A
drebrin siRNA #1: 50 -GCACGAAAUUUAAAACAUG-30 This paper N/A
drebrin siRNA #2: 50 -CCAACCUUCUUAAUUUCGA-30 This paper N/A
drebrin siRNA #3: 50 -CUUUCAGGCCACUUCGAGA-30 This paper N/A
drebrin siRNA #4: 50 -GCAGUGUGGAGGAUAUCGA-30 This paper N/A
Acell non-targeting Pool Dharmacon DAMD-001910-10-20
Recombinant DNA
Paxillin-pEGFP Laukaitis et al. (2001) RRID:Addgene_15233
drebrin-YFP Geraldo et al. (2008) RRID:Addgene_40359
S142A drebrin-YFP Geraldo et al. (2008) RRID:Addgene_58335
S142D drebrin-YFP Geraldo et al. (2008) RRID:Addgene_58336
drebrin-his Geraldo et al. (2008) RRID:Addgene_40363
GatewayTM pDONR/Zeo Vector and GatewayTM pDESTTM15 ThermoFisher Scientific Cat#11803-012, Cat#K37120, Cat# K37020
Vector, GatewayTM Cloning System
Software and algorithms
ImageJ Schneider et al. (2012) https://ImageJ.nih.gov/ij/
Imaris 9.3.1 (Bitplane) Oxford Instruments https://imaris.oxinst.com
Other
HisTrap column GE Healthcare Cat#17-5247-01
Glutathione Sepharose 4B GE Healthcare Cat#17-0756-01
square-wave electroporation generator BTX Inc. model ECM 830
carboxylate-modified FluoSpheresTM microspheres (0.2 mm, ThermoFisher Scientific Cat#F8805
blue fluorescing (365/415)

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Pei-Lin
Cheng (plcheng@imb.sinica.edu.tw).

Materials availability
All unique/stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

d Original western blot images have been deposited at Mendeley and are publicly available as of the date of publication. The DOI
is listed in the key resources table. Microscopy data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Cell Reports 40, 111188, August 16, 2022 e2

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EXPERIMENTAL MODEL AND SUBJECT DETAILS

Hippocampal and cortical neurons were prepared from embryonic day 17.5 (E17.5) Sprague–Dawley (SD) rat embryos (Dotti et al.,
1988) and cultured in neurobasal medium supplemented with Gem21 NeuroPlexTM (GEMINI Bio-products, West Sacramento, CA)
on 0.1 kPa PA hydrogel substrate or glass coverslips coated with poly-L-lysine (Sigma Aldrich, St. Louis, MO) and laminin (Corning
Incorporated, Corning, NY). Timed (14 days) pregnant 6-month-old SD rats were housed in IMB animal facility under standardized
conditions (20–24 C room temperature, 55% humidity and a day/night cycle of 12:12 h, light turned on at 7 am every morning,
receiving pelleted standard diet and drinking water) util the day of experiment. Animal protocols were approved by the Academia
Sinica Institutional Animal Care and Utilization Committee. Human Embryonic Kidney 293T cells (HEK293T; ATCC Cat# CRL-
3216) for biochemical analysis were cultured in Dulbecco’s Modification of Eagle’s Medium (ThermoFisher Scientific, Waltham,
MA) supplemented with 10% fetal bovine serum (Biological Industries, Beit Haemek, Israel). Hippocampal and cortical neurons
were maintained in an incubator with 5% CO2 at 37 C for 5–72 h. Transfection was performed using Lipofectamine 2000
(ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Cortical cultures at high density (>1 x 106
per 42mm gel/coverslips) were used to obtain a sufficient number of cells for biochemical assays.

METHOD DETAILS

Plasmids, antibodies, and inhibitors

Paxillin full-length and truncated variants were subcloned from pLenti-C-mGFP-PXN (OriGene Technologies, Rockville, MD) into
plasmids pCAGGS-GFP by PCR-based methods. Paxillin-pEGFP plasmid (Plasmid # 15,233; (Laukaitis et al., 2001), drebrin-YFP
(Plasmid # 40,359; Geraldo et al., 2008), and drebrin-his (Plasmid # 40,363; Geraldo et al., 2008) were obtained from Addgene (Water-
town, MA). FLAG-tagged drebrin was generated by cloning a PCR product containing the coding sequence of drebrin-YFP into
pLenti-C-Myc-DDK-CIP4 (Chang et al., 2017). Paxillin siRNA pool, a mixture of two siRNAs (paxillin C: 50 -GAUGAGCAGUCCA
CAGCGA -30 , paxillin D: 50 -CAGCCUAGUGGUUCCAGAU -30 ), drebrin siRNA pool (drebrin siRNA #1: 50 -GCACGAAAUUUAAAA
CAUG-30 , drebrin siRNA #2: 50 -CCAACCUUCUUAAUUUCGA-30 , drebrin siRNA #3: 50 -CUUUCAGGCCACUUCGAGA-30 , and drebrin
siRNA #4: 50 -GCAGUGUGGAGGAUAUCGA-30 ), and a scramble siRNA (Acell non-targeting siRNA #1: 50 -UGGUUUACAUGUCGA
CUAA-30 ) were purchased from Dharmacon (Lafayette, CO). GST-fusion protein expression plasmid encoding full-length or trun-
cated versions of paxillin were cloned into GatewayTM pDONR/Zeo Vector and GatewayTM pDESTTM15 Vector (ThermoFisher
Scientific, Waltham, MA) using a GatewayTM Cloning System. All constructs were sequenced to verify sequence integrity. Sources
of antibodies and chemicals are as follows: mouse monoclonal FLAG M2 (F1804) was purchased from Sigma-Aldrich (St. Louis, MO);
chicken polyclonal bIII tubulin (AB9354) and anti-Actin (MAB1501) antibodies were from Merck Millipore (Burlington, MA); anti-drebrin
(ab11068), anti-alpha Tubulin (ab89984), and anti-non-muscle Myosin IIB (ab684) were from Abcam (Cambridge, MA); anti-paxillin
clone 349 (612,405) was from BD Biosciences (Franklin Lakes, NJ); mouse c-Myc tag monoclonal antibody, Alexa Fluor 647 phalloi-
din, and Alexa Fluor 647 phalloidin were from ThermoFisher Scientific (Waltham, MA); secondary antibodies conjugated with Alexa
Fluor 488, Alexa Fluor 546, or Alexa Fluor 633 were purchased from ThermoFisher Scientific; Forskolin and KT5720 were purchased
from Calbiochem (San Diego, CA); Pim1/AKK1-IN-1, also named LKB1/AAK1 dual inhibitor, was purchased from MedChemExpress
(Monmouth Junction, NJ); and PP2 and K252a were from Abcam (Cambridge, MA).

Cell culture, protein lysate preparation, and immunostaining

To prepare cell lysates, protein was extracted with M-PERTM (Mammalian Protein Extraction Reagent, ThermoFisher Scientific,
Waltham, MA) containing protease inhibitor cocktail (Roche, Basel, Switzerland) and the phosphate inhibitor PhosSTOP (Roche,
Basel, Switzerland). To prepare tissue lysates, we briefly rinsed the brain, heart, and liver of rat E17 embryos with HBSS
(ThermoFisher Scientific, Waltham, MA) and then lysed tissues in RIPA buffer (Sigma Aldrich, St. Louis, MO) with a protease inhibitor
cocktail and PhosSTOP.
For immunostaining, cultured hippocampal or cortical neurons were fixed for 12 min in 4% paraformaldehyde, permeabilized for
12 min in 0.3% Triton X-100, and blocked with 3% BSA for 1–2 h. Fixed cells were processed further for immunostaining according to
standard procedures and imaged with a confocal microscope (Carl Zeiss LSM700) equipped with a 403 water-immersion objective
(NA1.1; Carl Zeiss). Brightness and contrast adjustments of confocal images in figures were performed using ImageJ software (NIH;
(Schneider et al., 2012). For quantitative measurement of co-localization, we acquired images using a super-resolution structured
illumination microscopy (SR-SIM) system (Zeiss ELYRA PS.1) equipped with a Plan Apo 633 oil-immersion objective (NA 1.4,
Carl Zeiss). The axial step size was set to 125 nm. Surface rendering of three-dimensional images was performed using ZEN software
(Zeiss) and Imaris software (Bitplane) without Z-correction. Images were analyzed and processed for presentation in figures using
brightness and contrast adjustments in ImageJ software (NIH) and following the guidelines of (Rossner and Yamada, 2004).

Expression of GST- and His-tagged proteins, and in vitro and in situ binding assays
For protein expression and purification, GST- or His-tagged proteins were expressed in E. coli BL21(DE3) induced by 0.4 mM IPTG
overnight at 30 C in LB medium. Bacteria were lysed by sonication in short pulses of 15 s in lysis buffer [(50 mM Tris-HCl pH = 7.4,
50 mM NaCl, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100 with 1 mM EDTA, 1 mM dithiothreitol and protease

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inhibitors (Complete EDTA-free; Roche)]. Cell debris was removed by centrifugation at 9000 g for 10 min at 4 C. The resulting super-
natant was applied onto a Glutathione Sepharose 4B or HisTrap column (GE Healthcare). After washing, GST- and His-tagged pro-
teins were eluted in buffer containing 10 mM reduced glutathione and 300 mM imidazole, respectively. We added 1 M imidazole to
samples shortly after elution to prevent precipitation of His-tagged protein. Protein concentrations were measured using the Bio-Rad
DC Protein Assay. Protein purity was further assessed by fast protein liquid chromatography, followed by SDS-PAGE and Coomassie
blue staining.
For GST pull-down assays, cell lysate/His-tagged protein and GST fusion proteins were incubated with glutathione-agarose
beads. Complexes recovered from beads were resolved by SDS-PAGE and analyzed by Western blotting. In situ detection of protein
interaction at the growth cone was performed using Duolink PLA technology (Sigma Aldrich, St. Louis, MO), following the manufac-
turer’s instruction manual. PLA signals were visualized and quantified from confocal images.

Polyacrylamide gel preparation

Polyacrylamide gels with tunable mechanical stiffness were generated using a protocol slightly modified from that previously pub-
lished (Chang et al., 2017). Briefly, uniform polyacrylamide gels were fabricated in a three-layer assembly of 200 mm thickness. The
bottom layer was a hydrophilic amino-silanized coverslip prepared according to the following protocol. A thin film of sodium hydrox-
ide was allowed to form on the coverslips at approximately 90 C. The entire surface of each coverslip was then immersed in a suf-
ficient volume of (3-aminopropyl) triethoxysilane (Sigma-Aldrich, Saint Louis, MO) for 5 min. The (3-aminopropyl) triethoxysilane was
then completely rinsed off to prevent precipitation before adding 0.5% (v/v) glutaraldehyde (Sigma-Aldrich, Saint Louis, MO) in PBS
onto the silanized coverslips for 30 min. Fluids were removed by suction before the amino-silanized coverslips were air-dried and
sterilized with 70% ethanol for 16 h prior to gel preparation. The top layer was a laminin-coated coverslip prepared by sterilizing
an acid-washed coverslip with 70% ethanol and then coating it with 5 mg/mL of poly-L-Lysine (Sigma Aldrich, Saint Louis, MO)
and 0.08 mg/mL of laminin (Corning, NY) by absorption at 37 C for 1 h. For the middle layer of 0.1 kPa hydrogels, a prepolymer mixture
(75 mL 4% acrylamide, 25 mL 2%N0 -N0 -methylenebisacrylamide, and 900 mL deionized water) was prepared. Polymerization of the
mixture was carried out by adding 10 mL of 10% ammonium persulfate, 2 mL of TEMED (Bio-Rad Laboratories, Hercules, CA),
and sufficient deionized water to yield a final volume of 1000 mL. The resulting prepolymer-catalyst mixture was dropped onto hydro-
phobic amino-silanized coverslips. The three-layer assembly was formed by transferring pre-coated poly-L-Lysine and laminin cov-
erslips to the surfaces of the polyacrylamide gels during polymerization. After 30 min, the top layer was gently peeled off and washed
three times with HEPES solution to remove unreacted monomers and excess coating.

In utero electroporation
In utero electroporation followed previously described procedures (Saito and Nakatsuji, 2001), with minor modifications. Timed-
pregnant Sprague-Dawley rats were anesthetized at E17.5 with isoflurane, and the uterine horns were exposed by way of a laparot-
omy. Saline solution containing the expression plasmid of interest (2 mg/mL) together with the dye Fast Green (0.3 mg/mL; Sigma-
Aldrich, Saint Louis, MO) was injected (1–2 mL) through the uterine wall into one of the lateral ventricles of the embryos. The embryo’s
head was electroporated by tweezer-type circular electrodes across the uterus wall, and five electrical pulses (50 V, 50 ms duration at
100 ms intervals) were delivered with a square-wave electroporation generator (model ECM 830, BTX Inc.). Uterine horns were then
returned to the abdominal cavity, the wall and skin were sutured, and embryos continued normal development. Control embryos
were electroporated with the mScarlet construct together with the GFP construct (1:2 ratio), and experimental embryos were electro-
porated with the mScarlet construct (Kindly provided by Dr. Shu-Ling Chu, ICOB, Academia Sinica) together with drebrin shRNA
(#1- #4), or control scramble-shRNA construct. Control and experimental cortices (at P3 or P16) were obtained from the same litter
and the injections were always made into the left and right ventricles, respectively, for later identification. Maximum intensities of the
z-projection of tile images (20 mm thickness) were attained from spinning-disk confocal microscopy (Zeiss). Animal protocols were
approved by the Animal Care and Use Committee of Academia Sinica.

Microfabrication and substrate patterning

Microfabrication and substrate coating methods followed those previously described (Chang et al., 2017; Hsu et al., 2005). Briefly,
the poly(dimethylsiloxane) (PDMS) cuboids used to generate microchannels were prepared from Sylgard 184 base and curing agent
(Dow Corning, Midland, MI). These were polymerized on a silicon wafer etched with parallel stripes (50 mm width each) spaced
100 mm apart. Solution containing substrate factors was filled into the microchannels formed by placing the PDMS cuboids over
the poly-L-lysine-coated glass coverslip, and overnight incubation allowed the substrate factor to be coated onto the coverslip. Sub-
strate solutions were prepared with 0.5 ng/mL fluorescently-conjugated BDNF. In all coating solutions, 5 mg/mL of fluorescently-con-
jugated BSA was added as a marker of stripes.

Expansion microscopy
Lattice light sheet microscopy and sample preparation for expanded neuronal cultures were slightly modified from a previously re-
ported protocol (Tsai et al., 2020). Briefly, 0.1 kPa polyacrylamide gels were soaked in ExM monomer solution [1 3 PBS, 2 M NaCl,
8.625% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.15% (w/w) N,N0 -methylenebisacrylamide) at 4 C overnight. Full gelation
was achieved further by adding freshly prepared ExM monomer solution plus ammonium persulfate and tetramethyl ethylenediamine

Cell Reports 40, 111188, August 16, 2022 e4

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[up to 0.2% (w/w) each] onto the sample, incubated at room temperature for 20 min followed by 1 h of incubation at 37 C. The LLS
microscopy configuration and image processing were performed as described in (Tsai et al., 2020), with optical resolution in x, y, z =
1024 3 1024 3 201, voxel size in x, y, z = 0.103 mm 3 0.103 mm x 3 mm, field of view = 1024 3 0.103 mm = 105.472 mm.

Traction force microscopy, image acquisition, and image processing

Hippocampal neurons were grown on 0.1 kPa PA hydrogels embedded with carboxylate-modified FluoSpheres microspheres
(0.2 mm, blue fluorescing (365/415), ThermoFisher Scientific, Waltham, MA) for traction force microscopy (TFM) experiments. The
concentration of fluorescent beads is 4% of the hydrogel precursor mixture. A time series of z stack images [300 two-plane (2 mm
axial step) stacks over 10 min] was acquired at 0.5 Hz by DeltaVision Core Deconvolution Microscopy (General Electric, Boston,
MA) using an Olympus IX71 microscope and a 60x oil-immersion objective (NA 1.42, Olympus). Analysis of TFM images was con-
ducted as previously described (Tseng et al., 2012). The bead displacement field was calculated and visualized using the ImageJ
plugin for iterative particle image velocimetry (PIV), followed by the Fourier transform traction cytometry (FTTC) plugin to reconstruct
the force vector field.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed by one-way ANOVA or unpaired two-tailed Student’s t-test. Statistical significance was deter-
mined as p < 0.05. Statistical analysis was conducted using GraphPad Prism 9 (GraphPad, San Diego, CA).

e5 Cell Reports 40, 111188, August 16, 2022