RESEARCH ARTICLECRY–BARs: Versatile light-gated molecular tools for the
remodeling of membrane architectures
Received for publication, March 22, 2022, and in revised form, August 6, 2022 Published, Papers in Press, August 18, 2022,
https://doi.org/10.1016/j.jbc.2022.102388
Anna I. Wurz1, Wyatt P. Bunner2 , Erzsebet M. Szatmari2 , and Robert M. Hughes1,*
From the 1Department of Chemistry, and 2Department of Physical Therapy, East Carolina University, Greenville, North Carolina,
USA
Edited by Ursula JakobBAR (Bin, Amphiphysin, and Rvs) protein domains are
responsible for the generation of membrane curvature and
represent a critical mechanical component of cellular func-
tions. Thus, BAR domains have great potential as components
of membrane-remodeling tools for cell biologists. In this work,
we describe the design and implementation of a family of
versatile light-gated I-BAR (inverse BAR) domain containing
tools derived from the fusion of the Arabidopsis thaliana
cryptochrome 2 photoreceptor and I-BAR protein domains
(“CRY–BARs”) with applications in the remodeling of mem-
brane architectures and the control of cellular dynamics. By
taking advantage of the intrinsic membrane-binding propensity
of the I-BAR domain, CRY–BARs can be used for spatial and
temporal control of cellular processes that require induction of
membrane protrusions. Using cell lines and primary neuron
cultures, we demonstrate here that the CRY–BAR optogenetic
tool evokes membrane dynamic changes associated with
cellular activity. Moreover, we provide evidence that ezrin, an
actin and phosphatidylinositol 4,5-bisphosphate–binding
protein, acts as a relay between the plasma membrane and the
actin cytoskeleton and therefore is an important mediator of
switch function. Overall, we propose that CRY–BARs hold
promise as a useful addition to the optogenetic toolkit to study
membrane remodeling in live cells.
Membrane-bound architectures, including filopodia, lamel-
lipodia, and dendritic spines in neurons, are critical for a cell’s
ability to transmit and respond to extracellular cues. As such,
chemists and biologists have developed numerous optogenetic
and chemo-optogenetic tools to control cellular architectures
via manipulation of plasma membrane dynamics (1–3).
However, these tools have to date incorporated a relatively
small fraction of the numerous proteins involved in the
building and dismantling of these architectural features. In
particular, nonenzymatic proteins have been overlooked: while
numerous optogenetic strategies exist for enzyme-mediated
control of membrane dynamics (4–9), far fewer have incor-
porated nonenzymatic proteins as the basis of optogenetic
switch action (10). As mechanical control of membrane
architecture by nonenzymatic proteins is a critical component* For correspondence: Robert M. Hughes, hughesr16@ecu.edu.
© 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for
BY license (http://creativecommons.org/licenses/by/4.0/).of cellular signaling (11), addressing this oversight will be a
bridge to a more comprehensive understanding of cellular
dynamics and function.
In recent years, the critical role of proteins involved in
membrane curvature inducing and sensing has come to light
(12). In particular, BAR (Bin, Amphiphysin, and Rvs) domain–
containing proteins possess diverse activities at the plasma
membrane (13–19); as such, a heightened understanding of
these roles has invited recent investigations of their suitability
for control with optogenetic tools. In a recent work, Jones et al.
(20) used a two-component approach (iLID/SspB) to design a
light-activated switch enabling recruitment of the extended
Fes–CIP4 homology BAR domain from FBP17 to the plasma
membrane and a hybrid two-component approach (iLID/
SspB/pdDronpa1) enabling recruitment of the inverse BAR
(I-BAR) domain from IRSp53 to the plasma membrane. These
switches, designed to have minimal interaction with the cell
membrane in the absence of light, promoted either positive
(Fes–CIP4 homology BAR; FBP17) or negative (I-BAR;
IRSp53) membrane curvature in response to light activation.
The MTSS1 (Missing in Metastasis 1) protein is considered
the prototype of I-BAR proteins (21), but, to the best of our
knowledge, has not previously been applied in an optogenetic
context. In this work, we investigated the potential of the
MTSS1 I-BAR domain to serve as an actuator of membrane
architecture and plasma membrane dynamics. We demon-
strate that the I-BAR domain from MTSS1, in conjunction
with the Cry2 photoreceptor protein (22–25), forms the basis
of a versatile optogenetic approach (“CRY–BAR”) for con-
trolling membrane dynamics and cellular architecture. In
contrast to previous approaches, we have not sought to
minimize the interaction between our constructs and the
plasma membrane. Instead, CRY–BAR combines the intrinsic
membrane-binding affinity of the I-BAR domain (26) with the
homo-oligomerizing capability of Cry2 (22), resulting in a
membrane-localized switch that induces membrane remod-
eling in response to blue light. We also provide insight into
the mode of action of CRY–BAR by showing that ezrin, a
membrane- and cytoskeletal-relay protein, is linked to the
ability of CRY–BAR to induce light-activated membrane
remodeling and restriction of cellular dynamics by acting as a
relay between CRY–BAR activation and the actin
cytoskeleton.J. Biol. Chem. (2022) 298(10) 102388 1
Biochemistry and Molecular Biology. This is an open access article under the CC
Light-activated membrane remodelingResults and discussion
Increased MTSS1 activity has been associated with exercise-
induced enhancement of synaptic function (27). This prosy-
naptic plasticity effect of MTSS1 is attributed to the presence
of an I-BAR domain within its structure. Moreover, I-BAR
domains have also been shown to promote formation and
stabilization of dendritic spines (Fig. 1A), leading to improved
synaptic function and resilience against neurodegeneration
(16). To gain insights into the molecular mechanisms that
control membrane expansion associated with cell morphology
changes, we fused the I-BAR domain of MTSS1 with a blue
light–sensing domain (Cry2PHR; Fig. 1, B and C), creating a
light-activatable I-BAR protein (CRY–BAR). For this investi-
gation, we engineered four permutations of the CRY–BAR
switch: I-BAR–Cry2–mCh, I-BAR–Cry2–mCh–WH2, Cry2–
mCh–I-BAR, and Cry2–mCh–MTSS1 (Fig. 1C), where
I-BAR = residues 1 to 250 of MTSS1, WH2 = residues 601 to
759 of MTSS1, and MTSS1 = the intact 759 residue protein.
Each construct was confirmed by Sanger and NextGenFigure 1. I-BAR-mediated initiation of dendritic spine generation in
neurons. A, phosphoinositide (PIP2) signaling recruits I-BAR domain pro-
teins to the plasma membrane, inducing a proto-protrusion. Subsequent
recruitment of actin (brown crosshatches) and actin-binding and remodeling
proteins promotes protrusion elongation. Mature dendritic spines (purple
nodules on dendrite) are mushroom-shaped bulbous protrusions and major
sites of excitatory synaptic transmission in the mammalian brain. B, diagram
of MTSS1, an I-BAR domain–containing protein. The N-terminal I-BAR
domain (250 amino acids) is comprised of three alpha helices. MIM-S1 and
MIM-S2 represent the I-BAR domain dimerization interface as determined
from X-ray crystallography (40). C, schematic of CRY–BAR optogenetic
switches: (i) The N-terminal I-BAR domain (250 amino acids) is fused to
photoreceptor protein CRY2, which is fused to the red fluorescent mCherry
protein for visualization purposes. (ii) At the C terminus, proline-rich and
WH2 domains can be included for actin recruitment. (iii) The N-terminal I-
BAR domain is fused to the C terminus of mCherry. (iv) The intact MTSS1
protein (green, I-BAR; black, internal domains as shown in panel B; and
yellow, WH2) is fused to the C terminus of mCherry. Graphic (A) generated
with BioRender (https://biorender.com/). BAR, Bin, Amphiphysin, and Rvs; I-
BAR, inverse BAR; MTSS1, Missing in Metastasis 1; PIP2, phosphatidylinositol
4,5-bisphosphate; PRD, proline-rich domain; SRD, serine-rich domain; WH2,
Wiskott–Aldrich syndrome homology region.
2 J. Biol. Chem. (2022) 298(10) 102388sequencing and expressed predominantly at the expected
molecular weight (Fig. S1).
CRY–BAR constructs were initially probed for their
response to blue light in the presence of CIBN–CAAX, a
membrane-localized binding partner to Cry2, in human
embryonic kidney 293T (HEK293T) cells. As anticipated, light
activation of the Cry2–mCh control resulted in rapid
recruitment to the plasma membrane (Figs. 2A and 3A). Light-
activated recruitment to CIBN–CAAX was also observed for
all the I-BAR-containing constructs (Figs. 2 and 3, B and C).
Interestingly, the I-BAR domain–containing constructs
I-BAR–Cry2–mCh and Cry2–mCh–I-BAR, while exhibiting
light-activated recruitment to the plasma membrane (Fig. 2, A
and C), had significant prelocalization to the plasma mem-
brane in the dark. We attributed this to the phosphatidylino-
sitol 4,5-bisphosphate (PIP2)–binding propensity of the I-BAR
domain (readily observed in the preillumination images in
Fig. 2, A and C). Because of the significant prelocalization of
these constructs to the plasma membrane, we then investi-
gated whether they might exhibit light-activated recruitment,
via Cry2 homo-oligomerization, in the absence of CIBN–
CAAX. These experiments revealed significant light-activated
clustering/membrane recruitment (Fig. 2, B and D) in the
absence of CIBN–CAAX. By contrast, Cry2–mCh–MTSS1,
which also accumulated in nonlight responsive clusters, did
not exhibit light-activated membrane recruitment in the
absence of CIBN–CAAX (50 of 50 cells pooled from six rep-
licates; Fig. 2D). As a result, this construct was not pursued
further.
Homo-oligomerization of the CRY–BAR constructs I-BAR–
Cry2–mCh and Cry2–mCh–I-BAR results in membrane
deformation and restriction of cellular dynamics (Movie S1, A
and B). These effects are reversible in the dark. To demon-
strate this using Cry2–mCh–I-BAR, we conducted a 10 min
light activation sequence, followed by a 30 min observation
period in the absence of blue light. Rapid restriction of
membrane dynamics was observed during the 10 min blue
light activation period, with full reversibility of membrane
rounding achieved after 30 min in the absence of blue light
(Fig. 4 and Movie S2). We subsequently demonstrated that this
effect can be selectively induced using localized illumination of
the cell on a confocal microscope (Fig. 5 and Movie S3). We
also investigated whether the presence of a WH2 domain
(present in I-BAR–Cry2–mCh–WH2 but not in I-BAR–Cry2–
mCh and Cry2–mCh–I-BAR) impacted the membrane
remodeling capabilities of the optogenetic switch. Light
activation of an N-terminal I-BAR domain without a C-ter-
minal WH2 domain (I-BAR–Cry2–mCh) promoted its accu-
mulation in numerous filopodia-like protrusions that rapidly
coalesced with continued light exposure (Fig. 6A). By contrast,
a construct that paired an N-terminal I-BAR domain with a C-
terminal WH2-containing domain (I-BAR–Cry2–mCh–WH2)
did not accumulate in filopodia-like protrusions (Fig. 6). The
interaction between WH2 and monomeric actin and associ-
ated complexes (28), which would disfavor the preassociation
of I-BAR and PIP2 required for CRY–BAR function, is likely
responsible for the observed failure to accumulate at the
Light-activated membrane remodeling
Figure 2. Light-activated responses of CRY–BAR constructs. A, HEK293T cells were cotransfected with CIBN–GFP–CAAX and CRY–BAR fusions I-BAR–
Cry2–mCh or I-BAR–Cry2–mCh–WH2 or Cry2–mCh and imaged on a widefield fluorescent microscope. I-BAR labeled as IBAR in figure. B, HEK293T cells were
transfected with CRY–BAR fusions I-BAR–Cry2–mCh and I-BAR–Cry2–mCh–WH2 or Cry2–mCh and imaged on a widefield fluorescent microscope. C,
HEK293T cells were cotransfected with CIBN–GFP–CAAX and CRY–BAR fusions Cry2–mCh–I-BAR and Cry2–mCh–MTSS1 or Cry2–mCh and imaged on a
widefield fluorescent microscope. D, HEK293T cells were transfected with CRY–BAR fusions Cry2–mCh–I-BAR and Cry2–mCh–MTSS1 or Cry2–mCh and
imaged on a widefield fluorescent microscope. Images acquired every 30 s (50 ms exposure of 480 nm light and 200 ms exposure of 553 nm light every
30 s). White arrows indicate example areas of light-activated recruitment. The scale bars represent 10 microns. BAR, Bin, Amphiphysin, and Rvs; HEK293T,
human embryonic kidney 293T cell line; I-BAR, inverse BAR; MTSS1, Missing in Metastasis 1.plasma membrane. Analogously, a recent investigation of the
I-BAR protein IRSp53 revealed that the I-BAR domain of
IRSp53, expressed as a fusion to GFP, localized to filopodia,whereas the C terminus of IRSp53 was entirely cytosolic (29).
Taken together, these results illustrate the modular nature of
I-BAR proteins, where the N-terminal I-BAR-containingJ. Biol. Chem. (2022) 298(10) 102388 3
Light-activated membrane remodeling
Figure 3. Analysis of light-activated responses of CRY–BAR constructs. A, Cry2–mCh construct shows large decrease in cytosolic fluorescence intensity
in the presence of plasma membrane–anchored CIBN–GFP–CAAX (CIB) but not in its absence. B, Cry2–mCh–I-BAR construct shows a decrease in cytosolic
fluorescence intensity in both the presence and absence of plasma membrane–anchored CIB, indicative of significant homo-oligomerization at the plasma
membrane. C, I-BAR–Cry2–mCh construct shows a decrease in cytosolic fluorescence intensity in both the presence and absence of plasma membrane–
anchored CIBN, indicative of significant homo-oligomerization at the plasma membrane. Blue arrows indicate beginning of blue light illumination
(50 ms exposure of 480 nm light every 30 s); black arrows indicate end of blue light illumination. Graphs show changes in normalized cytosolic mCherry
fluorescence in HEK293T cells. Cells were initially imaged on mCherry channel (0–300 s), illuminated with 470 nm light for 10 min (330–930 s), then imaged
on mCherry channel again for 10 min (960–1560 s). D, quantitation of the fluorescence of ROIs (n = 6 cells) at the time points: immediately before light
exposure (0 min), then 1 min, 5 min, and 10 min of light exposure. Inset cell image: representative ROI inside the cytosol of HEK293T cell transfected with
I-BAR–Cry2–mCh; this image is duplicated from Figure 2A to demonstrate measurement methodology. The scale bar represents 10 microns; ROI dimensions:
4.02 × 4.02 microns. I-BAR labeled as IBAR in figure. BAR, Bin, Amphiphysin, and Rvs; HEK293T, human embryonic kidney 293T cell line; I-BAR, inverse BAR;
ROI, region of interest.domain is required for membrane binding and localization,
and the WH2 domain is required for recruitment of actin-
polymerizing components that promote protrusion forma-
tion and elongation (17). Only upon expression as isolated
entities are the competing contributions of these domains to
subcellular localization apparent. Finally, a side-by-side com-
parison of light activation of I-BAR–Cry2–mCh, I-BAR–
Cry2–mCh–WH2, and Cry2–mCh–I-BAR demonstrated that
while robust membrane recruitment is associated with light
activation of Cry2–mCh–I-BAR and I-BAR–Cry2–mCh
(Movie S4), Cry2–mCh–I-BAR induces a more robust effect
on cell membranes, as evidenced by impact on total cell area
(Fig. 4B). We attribute this to enhanced PIP2 binding and
clustering capability of the I-BAR domain when in the C-ter-
minal position of the optogenetic fusion protein.
We postulated that the dynamic membrane-remodeling
activity observed with CRY–BARs might be due to its inter-
action with proteins that link the plasma membrane with the
cytoskeleton. Ezrin is one such protein that has both lipid-
binding and cytoskeleton-binding domains (30). We hypoth-
esized that clustering of the PIP2-bound CRY–BARs might
impact dynamics by also clustering ezrin, resulting in restric-
tion of cytoskeletal-associated phenomenon such as
membrane ruffling. To investigate this, we cotransfected4 J. Biol. Chem. (2022) 298(10) 102388Cry2–mCh–I-BAR with an ezrin–GFP fusion. In the presence
of light, we observed colocalization of ezrin–GFP with Cry2–
mCh–I-BAR clusters at the plasma membrane (Fig. 7). This
effect was pronounced in the light, and absent in the dark,
indicating that CRY–BAR activation actively restricts ezrin
dynamics. CRY–BAR activation accompanied by ezrin
sequestration also resulted in increased cell thickness (Fig. 8),
with no such effect being observed in cells expressing the
Cry2–mCh control. Accompanying live-cell experiments
demonstrate that, while not exclusively so, the accumulation of
ezrin near the cell membrane largely followed that of CRY–
BAR during light activation and exhibits a close temporal
relationship with CRY–BAR activation (Fig. S2). The CRY–
BAR–ezrin link implies that CRY–BAR impacts actin dy-
namics as well. To explore this, we performed live-cell imaging
of CRY–BAR light activation in the presence of a GFP-labeled
actin (Fig. 9). Actin-rich protrusions were observed to coalesce
upon CRY–BAR activation (Fig. 9A) but were not generally
colocalized with CRY–BAR. Interestingly, in the dark, CRY–
BAR is present in filopodia tips and at lamellipodial edges
and interspersed with actin (Fig. 9A). However, upon light
activation, their spatial relationship changes, as rounded CRY–
BAR clusters and actin-rich regions at the cell membrane
become distinct (Fig. 9A, inset and Movie S5).
Light-activated membrane remodeling
Figure 4. Reversible light-activated membrane remodeling with CRY–
BAR. A, HEK293T cells transfected with Cry2–mCh–I-BAR were subjected to
blue light illumination for 10 min (activation), followed by 30 min without
blue light (dark recovery). Membrane retraction and reshaping is observed
during blue light illumination, followed by recovery in the absence of blue
light. The scale bar represents 10 microns. B, quantification of changes in
percent cell area during alternating cycles of light activation (10 min) and
dark recovery (30 min) from n = 6 cells per construct pooled from three
replicate measurements. Statistical analysis conducted with one-way
ANOVA (***p < 0.001; n.s., not significant [p > 0.05]). I-BAR labeled as
IBAR in figure. BAR, Bin, Amphiphysin, and Rvs; HEK293T, human embryonic
kidney 293T cell line; I-BAR, inverse BAR.Having investigated the CRY–BAR response in immortal-
ized cell culture, we next evaluated the capability of the sensor
to report membrane expansion dynamics in neurons. For this,
dissociated postnatal cortical neuron cultures prepared from
newborn mice were transfected with Cry2–mCh–I-BAR, or
cotransfected with CIBN–GFP–CAAX, according to protocol
we developed for expressing optogenetic sensors in primary
cultures (31). Light activation of Cry2–mCh–I-BAR, but not
Cry2–mCh, resulted in elongation of neuronal processes that
might indicate early stage spinogenesis (Fig. 10, Movies S6 and
S7). There was no apparent benefit to activation of Cry2–
mCh–I-BAR in the presence of CIBN–GFP–CAAX, providing
additional evidence that activation of Cry2–mCh–I-BAR alone
is sufficient for effecting membrane dynamics (Fig. 10C).
Accordingly, whole-cell illumination of neurons transfected
with only Cry2–mCh–I-BAR results in abundant membranous
cluster formation throughout neuronal processes (Fig. S3A).
Notably, oligomerization of I-BAR domain proteins is essential
for their membrane-remodeling activities (32). In the contextof CRY–BAR, optimal membrane remodeling is achieved via
the homo-oligomerization of Cry2. By contrast, recruitment of
CRY–BAR to CIBN–GFP–CAAX, while not completely
detrimental to CRY–BAR activity, results in less membrane
activity than activation of CRY–BAR alone (Fig. 10C). This is
presumably because of a combination of reduced CRY2 homo-
oligomerization in the presence of CIB, a phenomenon that
has been previously described (22), and possible orientation
effects of the Cry2–CIB interaction on the I-BAR domain.
CIBN–GFP–CAAX has a similar membrane distribution to
CRY–BAR and could interfere with CRY–BAR activity at the
cell membrane (Fig. S4).
Finally, using localized illumination conditions, we demon-
strated that Cry2–mCh–I-BAR can be selectively activated in
neuronal processes (Fig. 11 and Movie S8). Taken together,
this work provides evidence that the Cry2–mCh–I-BAR mo-
lecular optogenetic tool is suitable for the targeted manipula-
tion of cytoskeletal structures and dynamics at the plasma
membrane. To our knowledge, this switch represents the first
application of the I-BAR domain from MTSS1 in an
optogenetic switch. In comparison to prior work in this area,
CRY–BAR has the advantage of being a single-component
switch, which can reduce experimental complexity and facili-
tate its implementation into transgenic model organisms. We
note that the mechanisms of I-BAR domain proteins are not
completely understood, and there are structural and mecha-
nistic differences in the I-BAR domains across different pro-
teins (26). As a result, one can reasonably anticipate that the
incorporation of I-BAR domains from different I-BAR-con-
taining proteins and their orthologs could produce different
experimental phenotypes within the same optogenetic back-
ground. This intriguing possibility illustrates the value of
pursuing multiple optogenetic variations of BAR domain
proteins. Potential applications of CRY–BARs include site-
directed mutagenesis of the I-BAR domain, which could pro-
vide valuable structure–function information regarding the
mechanism of the I-BAR–plasma membrane interaction, and
investigation of membrane deformation–dependent cell
signaling pathways, such as calcium signaling (33).Conclusion
In this report, we describe the development of a family of
optogenetic switches, collectively named CRY–BAR, that
comprise a versatile platform for controlling membrane
dynamics in live cells with high spatial and temporal resolu-
tion. These switches combine homo- and hetero-
oligomerization of the Cry2–CIB photoreceptor system with
the innate PIP2-binding affinity of the I-BAR domain. As their
function varies depending on the presence of various func-
tional elements, such as WH2-binding domains, we anticipate
that a modular approach can be undertaken to adapt these
optogenetic switches for other applications, including the
recruitment of enzyme and receptor-activating and -inhibitory
domains. Finally, CRY–BARs are suitable tools to study
membrane dynamics not only in immortalized cells but also in
sensitive and difficult to transfect primary cultures, such asJ. Biol. Chem. (2022) 298(10) 102388 5
Light-activated membrane remodeling
Figure 5. Localized light activation of membrane remodeling with CRY–BAR. A, HEK293T cells transfected with Cry2–mCh–I-BAR were subjected to
restricted blue light illumination (yellow circles) using a confocal microscope. Clustering of Cry–BAR is apparent in the areas illuminated 25 frames post–blue
light stimulation. An overlay of the cell outline is shown before (green) and after (red) blue light illumination. The scale bar represents 10 microns.
B, quantification of fluorescence intensity changes in illuminated versus nonilluminated cell areas for n = 3 circular ROIs (area = 61.4 microns2) per condition.
Blue arrow indicates time of applied restricted blue light illumination (488 nm laser at 5% power). Error bars represent standard deviations. Differences are
statistically significant (one-way ANOVA, p < 0.05) beginning at the 283 s time point (*) and beyond. BAR, Bin, Amphiphysin, and Rvs; HEK293T, human
embryonic kidney 293T cell line; I-BAR, inverse BAR; ROI, region of interest.neurons. Therefore, the CRY–BAR optogenetic switches we
report here are expected to have wide applicability for inves-
tigating cellular processes associated with membrane dynamics
in a variety of experimental paradigms.Experimental procedures
Plasmids and cloning
Cloning of I-BAR–Cry2–mCh, Cry2–mCh–I-BAR, I-BAR–
Cry2–mCh–WH2, and Cry2–mCh–MTSS1 constructs was
conducted using a previously described cloning scheme (34).
Briefly, the I-BAR domain from MTSS1, the WH2 domain
from MTSS1, and MTSS1 were PCR amplified from human
MTSS1 complementary DNA obtained from the Arizona
State University DNA repository (DNASU ID:
HsCD00746054). N-terminal I-BAR genes were cloned into
Cry2PHR–mCherry (Addgene; #26866) using NheI and XhoI
restriction sites (23). C-terminal I-BAR, WH2, and MTSS1
genes were cloned into Cry2PHR–mCherry using BsrgI and
NotI restriction sites. The ezrin–GFP expression construct
was a gift from Stephen Shaw (plasmid pHJ421; Addgene;
#20680 (35)). The actin expression construct (pCAG-mGFP-
actin; Addgene; #21948) was a gift from Ryohei Yasuda.
CIBN–GFP–CAAX (pCIBN(deltaNLS)-pmGFP; Addgene;
#26867) was a gift from Chandra Tucker. Midi prep quanti-
ties of DNA of each construct were created from Escherichia
coli and collected for cell transfection.6 J. Biol. Chem. (2022) 298(10) 102388Cell lines and transfection
HEK293T cells (passage 8) used for these experiments were
purchased from American Type Culture Collection and were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal bovine serum (FBS) and 1% penicillin–
streptomycin. Cultures were transfected at 70% confluency
with the Lipofectamine 3000 reagent (Invitrogen) following the
manufacturer’s suggested protocols. Briefly, for dual trans-
fections in 35 mm glass bottom dishes for cell imaging or
6-well culture plates for lysis, plasmid DNA was combined in a
1:1 ratio (1250 ng per plasmid) in 125 ?l of Opti-MEM, fol-
lowed by the addition of 5 ?l of P3000 reagent. For single
transfections, 2500 ng of plasmid DNA was used per trans-
fection. In a separate vial, 3.75 ?l of Lipofectamine 3000 were
added to 125 ?l of Opti-MEM. The two 125 ?l solutions were
combined and allowed to incubate at room temperature for
10 min, followed by dropwise addition to cell culture. For
ezrin–GFP and actin–GFP cotransfection with CRY–BAR,
2000 ng of CRY–BAR DNA and 1000 ng of GFP DNA were
added to 100 ?l of DMEM along with subsequent addition of
3 ?l of CalFectin (SignaGen Laboratories). After 10 min of
incubation at room temperature, the solution was added
dropwise to cell cultures. Each transfection solution remained
on cells overnight. Transfected cells were maintained at 37 
C
and 5% CO2 in a humidified tissue culture incubator, in culture
medium consisting of DMEM supplemented with 10% FBS
and 1% penicillin–streptomycin.
Light-activated membrane remodeling
Figure 6. Light activation of N-terminal I-BAR–Cry2 fusions with and without a C-terminal WH2 domain. A, HEK293T cells transfectedwith I-BAR–Cry2–
mCh or I-BAR–Cry2–mCh–WH2 were subjected to blue light illumination (50 ms pulse every 30 s) using a widefield microscope. I-BAR–Cry2–mCh rapidly
coalesces into dynamic filopodial structures. The scale bars represent 10microns. B, the presence of a C-terminalWH2 domain results in a cytosolic distribution,
reducing impact on filopodial dynamics. The scale bars represent 5microns. C, average number of protrusions at time 0, 5, or 10min of light exposure from n =
10 cells pooled from three replicatemeasurements. Statistical analysis conductedwith one-wayANOVA (***p< 0.001; *p= 0.002; n.s., not significant [p> 0.05]).
I-BAR labeled as IBAR in figure. BAR, Bin, Amphiphysin, and Rvs; I-BAR, inverse BAR; HEK293T, human embryonic kidney 293T cell line.Neuron cultures and transfection
Postnatal-dissociated cortical neuron cultures were pre-
pared as previously described (31, 36), from newborn B6 mice.
Neurons were plated into 35 mm glass bottom Petri dishes at a
1 million/ml density in culture medium consisting of Basal
Medium Eagle supplemented with 10% bovine calf serum and
1% penicillin–streptomycin. On day in vitro 2 (DIV2), culture
medium was changed to Neurobasal A medium, supplemented
with B27-plus reagent (Invitrogen), Glutamax, and 1% peni-
cillin–streptomycin. Neurons were transfected with the
CryBAR optogenetic system (6 ?g plasmid/plate) on DIV5
using Lipofectamine LTX reagent. On DIV7, culture medium
was removed and neurons were placed in imaging solution
(Mg-free HEPES-buffered artificial cerebrospinal fluid (37))
Live-cell imaging was performed before and after illumination
using a Leica DMi8 Live Cell Imaging System. Animal use
protocols were approved by East Carolina University Institu-
tional Animal Care and Use Committee.Fixed and live-cell imaging preparation
Fixed cell experiments
Immediately prior to fixation, transfected HEK293T cells
were either kept in dark conditions or continually illuminatedwith LED blue light (Sunlite LED Par30 Reflector, item #80021,
4 W, 120 V, 66.21 ?mol/s/m2; placed 10 cm from cell culture
dishes) for 5 min. Cells were washed with Dulbecco’s PBS
(DPBS) (with calcium and magnesium; 1× 1 ml), then fixed for
10 min at room temperature with prewarmed 4% para-
formaldehyde solution in DPBS (37 
C; prepared from 16%
paraformaldehyde [Electron Microscopy Sciences]). Following
fixation, cells were washed with DPBS and then stored in
DPBS at 4 
C.
Live-cell experiments
Transfected HEK293T cell media were replaced with 10%
FBS in Leibovitz’s L-15 medium. Cells were allowed to equil-
ibrate in the live-cell incubation system (OKOLab) for 10 min
prior to beginning the illumination sequence.
Imaging
Confocal microscopy
Confocal images of fixed cells were collected with a
Zeiss LSM 700 laser scanning microscope using ZEN
Black 2012 software. Ezrin–GFP and actin–GFP live-cell
images were collected with a Zeiss LSM 700 laser scan-
ning microscope equipped with a stage-top incubator andJ. Biol. Chem. (2022) 298(10) 102388 7
Light-activated membrane remodeling
Figure 7. CRY–BAR activation linked to ezrin. A, HEK293T cells cotransfected with Cry2–mCh–I-BAR and ezrin–GFP were subjected to blue light illu-
mination or dark followed by fixation. Confocal microscopy reveals colocalization of CRY–BAR and ezrin–GFP as a result of CRY–BAR activation. The scale bar
represents 10 microns. B, plot of the log ratio of membrane ROI versus cytosol ROI intensity (n = 6 cells per construct), normalized intensity for each channel.
ROI dimensions: 1.4 × 1.4 microns. Statistical analysis conducted with one-way ANOVA (***p < 0.001; n.s., not significant [p > 0.05]). C, representative
images of ROIs positioned in the cytosol (cyan) and at the membrane (red). The scale bar represents 10 microns. I-BAR labeled as IBAR in figure. BAR, Bin,
Amphiphysin, and Rvs; HEK293T, human embryonic kidney 293T cell line; I-BAR, inverse BAR; ROI, region of interest.ZEN Black 2012 software. Fluorescence images were
colorized and overlaid using FIJI software (38) equipped
with Bio Formats (39).Widefield microscopy
A Leica DMi8 Live Cell Imaging System, equipped with an
OKOLab stage-top live cell incubation system, LASX software,
Leica HCX PL APO 63×/1.40 to 0.60 numerical objective oil
objective, Lumencor LED light engine, CTRadvanced+ power
supply, and a Leica DFC900 GT camera, was used to acquire
images. Exposure times were set at 200 ms (mCherry, 553 nm)
and 50 ms (GFP, 480 nm), with LED light sources at 50%
power, and images were acquired every 30 s over specified time
course.Western blotting
Transfected HEK293T cells were lysed with 200 ?l of
M-PER lysis buffer (Thermo Fisher Scientific) containing 1×
Halt protease–phosphatase inhibitor cocktail (Thermo Fisher
Scientific). After 10 min on ice, lysates were collected and
centrifuged for 15 min (94 rcf; 4 
C). The supernatants were
combined with Laemmli SDS sample buffer (Alfa Aesar) and
incubated at 65 
C for 10 min. The resulting lysates were8 J. Biol. Chem. (2022) 298(10) 102388subjected to electrophoresis on a 10% SDS-PAGE gel and
then transferred onto polyvinylidene fluoride membranes
(20 V, 800 min). Membranes were then blocked for 1 h with
5% bovine serum albumin (BSA) in 1× Tris-buffered saline
(TBS) with 1% Tween (TBST), followed by incubation with
primary antibody (anti-mCherry antibody [Cell Signaling]
1:1000 dilution in 5% BSA–TBST; anti-GAPDH antibody
[Invitrogen] 1:1000 dilution in 5% BSA–TBST) overnight at
4 
C on a platform rocker. The membranes were then
washed 3× for 5 min each with TBST and incubated with
the appropriate secondary antibody in 5% BSA–TBST (1 h;
room temperature). After washing 3× for 5 min with TBST,
the membranes were exposed to a chemiluminescent sub-
strate for 5 min and imaged with an Azure cSeries imaging
station.Statistical analyses
Statistical significance (p values; Figs. 4–8, 10, and 11) was
determined using a one-way ANOVA (Holm–Sidak method)
in SigmaPlot v. 13.0 (Systat Software, Inc). Mean ± S.D. plots
were constructed using GraphPad Prism version 8.2.1 for
Windows, GraphPad Software, San Diego, California, USA
(www.graphpad.com).
Light-activated membrane remodeling
Figure 8. CRY–BAR activation and cell thickness.HEK293T cells cotransfectedwith Cry2–mCh–I-BAR and ezrin–GFPwere subjected to blue light illumination or
dark followed by fixation. Cellular thickness was analyzed for n = 6 cells per experimental condition. A, average cellular thickness of Cry2–mCherry–I-BAR–
transfected HEK293T cells before and after illumination. B, average cellular thickness of Cry2–mCh–transfected HEK293T cells before and after illumination.
C, analysis of average cellular thickness at half height (one-way ANOVA: **p = 0.001; *p = 0.002, n.s., not significant [p > 0.05]). D, representative images of
Z-projections of ezrin–GFP distribution in Cry2–mCherry–I-BAR transfected cells before and after illumination and fixation. The scale bars represent 10microns. I-
BAR labeled as IBAR in figure. BAR, Bin, Amphiphysin, and Rvs; HEK293T, human embryonic kidney 293T cell line; I-BAR, inverse BAR.
Figure 9. Coalescence of actin-rich protrusions in response to CRY–BAR activation. A, HEK293T cells cotransfected with Cry2–mCh–I-BAR and actin–GFP
were illuminated with 488 nm light (5% power) for 10min on a confocal microscope. Individual images of channels for mCherry (top row) and GFP (second row) in
addition to an overlay (mCherry in red; GFP in green; inset inwhite box) showed a coalescence of actin-enriched protrusions in conjunction with CRY–BAR-induced
membrane rounding after 10min light exposure (arrow in left cell inset). Cry2–mCh–I-BAR is also enriched at the tips of actin-enrichedprotrusions in images before
light illumination (arrows in right cell inset). After 10min of light exposure, Cry2–mCh–I-BAR is retracted into the cell and no longer observed in protrusions (right cell
inset, fourth column). Full cell image scale bar represents 10microns. Inset image scale bar represents 5microns. B, HEK293T cells cotransfectedwith Cry2–mCh and
actin–GFPwere illuminatedwith488nmlight (5%power) for 10minonaconfocalmicroscope. Individual imagesof channels formCherry (top row) andGFP (second
row) in addition to an overlay (mCherry in red; GFP in green). Cry2–mCh and actin–GFP exhibit no change after 10min light exposure. The scale bar represents 10
microns. I-BAR labeled as IBAR in figure. BAR, Bin, Amphiphysin, and Rvs; HEK293T, human embryonic kidney 293T cell line; I-BAR, inverse BAR.
J. Biol. Chem. (2022) 298(10) 102388 9
Light-activated membrane remodeling
Figure 10. CRY–BAR plasma membrane recruitment in neurons. Pre- and 10 min-post 480 nm light illumination of processes in dissociated hippocampal
neuron cultures.A, neurons cotransfectedwithCry2–mCh–I-BARor Cry2–mChandCIBN–GFP–CAAX (GFP channel shown). B, neurons transfectedwith Cry2–mCh–
I-BARor Cry2–mCh.White arrows indicate example protrusions. The scale bars represent 10microns.C, log fold increase in lengthof protrusionsof Cry2–mCh–I-BAR
and Cry2–mCh expressing neurons with or without CIBN–GFP–CAAX (CIB) after 10 min of 480 nm light illumination (n = 10 neurons per construct; neurons from
three separate cultures; number of protrusions per neuron analyzed: Cry2–mCh–I-BAR+CIB: range 24–141, average 65; Cry2–mCh–I-BAR: range 35–75, average 50;
Cry2–mCh+CIB: range34–116, average61; Cry2–mCh: range4–17; average10. Statistical analysis conductedwithone-wayANOVA: ***p< 0.001; *p=0.04; n.s.:p>
0.05). Error bars represent standard deviations. I-BAR labeled as IBAR in figure. BAR, Bin, Amphiphysin, and Rvs; I-BAR, inverse BAR; n.s., not significant.
Figure 11. Localized light activation of CRY–BAR in neurons. A, neurons transfected with Cry2–mCh–I-BAR were subjected to restricted blue light
illumination (white circles) using a confocal microscope. Clustering of CRY–BAR is apparent in the areas illuminated 15 min post–blue light stimulation
(irradiation with 488 nm laser at 5% power at the 120 s mark). The scale bar represents 10 microns. B, a plot of fluorescence intensity from four illuminated
ROIs versus four nonilluminated regions is shown at right (error bars = standard deviation; differences are statistically significant [one-way ANOVA, p < 0.05]
beginning at the 263 s time point [*] and beyond). Blue arrow indicates time of blue light illumination. BAR, Bin, Amphiphysin, and Rvs; I-BAR, inverse BAR;
ROI, region of interest.
10 J. Biol. Chem. (2022) 298(10) 102388
Light-activated membrane remodelingData availability
Experimental data are available upon request. All CRY–BAR
plasmids will be available through Addgene, a nonprofit DNA
repository.
Supporting information—This article contains supporting
information.
Acknowledgments—We thank Mr Collin T. O’Bryant for assistance
with confocal microscopy.
Author contributions—A. I. W., W. P. B., E. M. S., and R. M. H.
methodology; A. I. W., W. P. B., E. M. S., and R. M. H. formal
analysis; A. I.W. and R.M.H. investigation; A. I.W.,W. P. B., E.M. S.,
and R. M. H. writing–original draft; A. I. W., W. P. B., E. M. S., and
R. M. H. visualization.
Funding and additional information—This work was supported by
a seed grant to R. M. H. and E. M. S. from the East Carolina Uni-
versity Biomaterials Research Cluster and by the National Institutes
of Health (grant no.: 1R15NS125564-01). The content is solely the
responsibility of the authors and does not necessarily represent the
official views of the National Institutes of Health.
Conflict of interest—The authors declare that they have no conflicts
of interest with the contents of this article.
Abbreviations—The abbreviations used are: BAR, Bin, Amphiphy-
sin, and Rvs; BSA, bovine serum albumin; DIV, day in vitro; DMEM,
Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s PBS; FBS,
fetal bovine serum; HEK293T, human embryonic kidney 293T cell
line; I-BAR, inverse BAR; MTSS1, Missing in Metastasis 1; PIP2,
phosphatidylinositol 4,5-bisphosphate; TBS, Tris-buffered saline;
TBST, TBS with Tween-20.
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