Optical Manipulation of F-Actin with Photoswitchable Small Molecules

Article abstract of DOI:10.1021/jacs.9b12898

Cell-permeable photoswitchable small molecules, termed optojasps, are introduced to optically control the dynamics of the actin cytoskeleton and cellular functions that depend on it. These light-dependent effectors were designed from the F-actin-stabilizing marine depsipeptide jasplakinolide by functionalizing them with azobenzene photoswitches. As demonstrated, optojasps can be employed to control cell viability, cell motility, and cytoskeletal signaling with the high spatial and temporal resolution that light affords. Optojasps can be expected to find applications in diverse areas of cell biological research. They may also provide a template for photopharmacology targeting the ubiquitous actin cytoskeleton with precision control in the micrometer range.

Full text of DOI:10.1021/jacs.9b12898

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Optical Manipulation of F‑Actin with Photoswitchable Small
Molecules
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Malgorzata Borowiak, Florian Kullmer, Florian Gegenfurtner, Sebastian Peil, Veselin Nasufovic,
Stefan Zahler, Oliver Thorn-Seshold, Dirk Trauner,* and Hans-Dieter Arndt*
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ABSTRACT: Cell-permeable photoswitchable small molecules,
termed optojasps, are introduced to optically control the dynamics
of the actin cytoskeleton and cellular functions that depend on it.
These light-dependent effectors were designed from the F-actin-
stabilizing marine depsipeptide jasplakinolide by functionalizing them
with azobenzene photoswitches. As demonstrated, optojasps can be
employed to control cell viability, cell motility, and cytoskeletal
signaling with the high spatial and temporal resolution that light
affords. Optojasps can be expected to find applications in diverse
areas of cell biological research. They may also provide a template for
photopharmacology targeting the ubiquitous actin cytoskeleton with
precision control in the micrometer range.
tightly to the G-actin monomer.10 By contrast, phalloidin and
jasplakinolide are incorporated at the interface between three
actin subunits, promoting aggregation and suppressing
depolymerization.11 These small molecules and their deriva-
tives have become powerful tool compounds for research.12,13
Fluorescent derivatives of phalloidin are common reagents for
visualizing actin filaments in fixed cell microscopy.14 The
silicon-rhodamine conjugate 4, termed SiR-actin, is cell
permeable and images F-actin dynamics in living cells.15 SiR-
actin is prepared by total synthesis and features an optimized
jasplakinolide-derived structure16 that is more chemically
stable (Figure 1b).
Although actin inhibitors have potent antimigratory activity
and are toxic to cancer cells, systemic toxicity resulting from
disturbed actin dynamics in healthy tissue has prevented them
from reaching therapeutic application.17 Moreover, many
actin-dependent processes are anisotropic and time-depend-
ent,18,19 which limits the utility of globally acting inhibitors.6,12
Inhibitors that could instead be directed to modulate the actin
cytoskeleton more precisely would circumvent these drawbacks
and be extremely valuable for research and therapy.
INTRODUCTION
■
Actin is the most abundant cellular protein and an essential
component of the eukaryotic cytoskeleton.1 Its ability to
reversibly assemble into long supramolecular filaments2 drives
the definition of cell shape, migration, endocytosis, phago-
cytosis, and intracellular transport. In neurons, actin remodel-
ing configures dendritic spines, is required for synapse
function, and is a key component in axonal growth.3 In the
nucleus, nuclear actin contributes to RNA transcription and
chromatin maintenance.4 Actin also plays a critical role in cell
division as an essential component during cytokinesis.5
The actin cytoskeleton is highly dynamic and undergoes
constant remodeling in response to the physiological state and
external stimuli. From a pool of a globular monomeric form
(G-actin), a polymeric, filamentous form (F-actin) is
generated, which dynamically interconverts through assembly
and disassembly in an adenosine triphosphate (ATP)-depend-
ent and -consuming process (“treadmilling”). F-Actin consists
of two helically intertwined strands of a supramolecular protein
polymer fiber that can be hierarchically arranged by actin
binding proteins in parallel bundles, branched dendritic
networks, and stress fibers, leading to diverse microfilament
topologies of variable dynamics.1
Spatiotemporal control can be realized by incorporating
photoswitches into bioactive molecules.20,21 Molecular photo-
The key role of actin dynamics in living organisms has led to
the evolution of interfering secondary metabolites,6 including
the polyketides cytochalasin7 and latrunculin,8 as well as the
(bi)cyclic peptides phalloidin (1), chondramide (2), and
jasplakinolide (3, sometimes also referred to as “jaspamide”,
Figure 1a).9 The cytochalasins and the latrunculins retard
polymerization and enhance depolymerization by binding
Received: January 3, 2020
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Figure 1. (a) Chemical structures of F-actin stabilizing natural products. (b) Structure of SiR-actin composed of the silico-rhodamine fluorophore
(red), a linker (gray), and the optimized jasplakinolide scaffold (right). (c) Azobenzene isomerization controlled by illumination.
switches are particularly well suited to modulate dynamic,
nonlinear processes, as featured in cytoskeleton biology, in G-
protein coupled receptor pathways, and in neuronal signaling.
Indeed, photopharmacology has been successfully used in vivo
to control receptor signaling,22 tubulin dynamics,23 and to
restore visually guided behavior in blind animals.24 Azoben-
zenes that show metastable irradiation-dependent change in
their diazene configuration have been frequently applied in this
regard (Figure 1c).24,25 Here, we introduce a family of
azobenzene-based small molecules, termed optojasps, that
provide direct optical control of the actin cytoskeleton.
Design and Synthesis of the Optojasps. For designing
the optojasps, the noticeable dependence of cell permeability
and actin-staining potential on fluorophore type and linker
length of conjugates, such as 4, was key.15,16 Without any
specific mechanistic conjecture, it suggested that modulating
the activity of jasplakinolides by placing an azobenzene in the
same region as the fluorophore of 4 might be possible. Such an
“azo-extension” strategy has been successful with ion channel
blockers26 and dihydrofolate reductase inhibitors.27 For initial
screening, a panel of eight optojasps was considered, which
differed in the length of the connecting amino acid side chain
(diaminopropionate, diaminobutyrate, ornithine, lysine) and in
the geometry of the appended azobenzene photoswitch
(Figure 2a). The latter were connected in para (azo-1, Figure
2c) or ortho (azo-2) in order to probe for different interactions
exerted by the photoswitch in its respective trans or cis
isomeric state.
well as recent high-resolution X-ray crystal structures of
tubulin-bound combretastatin in cis- and trans-configuration29
suggested that any protrusion from the B-ring significantly
larger than the para methoxy group of azobenzene 18 (Figure
2c, highlighted) entirely abrogates affinity for tubulin. There-
fore, “clean” actin-selective photoswitches were expected to
result from conjugation of the extended azo-1 (16) or azo-2
(17) scaffolds.
The syntheses of optojasps then used six building blocks
each (Figure 2c).15,16,30 In brief, β-tyrosine derivative 5 was
immobilized on solid phase (6, Figure 2b) and was sequentially
extended by solid phase peptide synthesis (SPPS) with N-Me-
Trp building block 11, one of the diamino building blocks
12a−12d, and the unsaturated acid 13, to give tripeptides 7a−
7d after cleavage from the resin. Esterification with alcohol 14
provided dienes 8a−8d. Ring-closing metathesis with Grubbs’
second-generation catalyst 15 yielded the macrocyclic cyclo-
depsipeptides 9a−9d as E-configured cycloalkenes. After acid-
mediated deprotection, the ammonium salts 10a−10d were
coupled to azobenzene carboxylic acids azo-1 (16) or azo-2
(17) and deprotected to afford optojasps 1−8 (Figure 2b) in
10−20% overall yield.
Photochemical and Biological Evaluation. An initial
evaluation of the photochemical properties of optojasps 1−8
showed that all compounds were photoswitched trans→cis
with deep violet and cis→trans with green light, reaching
satisfactory photostationary ratios upon deep violet irradiation
(cis/trans typically 10:1). They were fairly stable to
spontaneous thermal cis→trans relaxation (Figure 3). The
azo-1 derived photoswitches showed very slow thermal
relaxation (t1/2 ≈ 36 h), whereas azo-2switches displayed a
relaxation half-life of ∼80 min in CH3CN at 25 °C. Not
unexpectedly, thermal relaxation was found accelerated under
physiologically relevant conditions (37 °C, determined in
aqueous buffer, see Figure 3g). Notably, photoswitching was
fully reversible, and compounds remained chemically stable
The azobenzenes finally chosen featured 4,4′-dialkoxy
substituents to ensure high bidirectional completion of
photoswitching and retarded thermal isomerization. They
were 3,4,5-trimethoxylated in the A-ring to improve solubility
by disfavoring compound aggregation (Figure 2a,c).23 Notably,
while structurally related photostatin switches (e.g., 18, Figure
2c) have been successfully used for targeting tubulin,23
extensive structure−activity relationship (SAR) studies and
successful prodrug designs of combretastatin analogues28 as
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Figure 2. (a) Structures of optojasps 1−8. (b) Chemical synthesis of optojasps. Conditions: (a) 2-Chlorotrityl chloride resin, DIPEA, CH2Cl2/
DMF; (b) 1. piperidine, DMF; 2. HATU, HOAt, 2, DIPEA, CH2Cl2/DMF; 3. piperidine, DMF; 4. HATU, HOAt, 12a, b, c, or d, DIPEA, CH2Cl2/
DMF; 5. piperidine, DMF; 6. HATU, HOAt, 4, DIPEA, CH2Cl2/DMF; 7. HFIP, CH2Cl2; (c) EDCI, DMAP, DIPEA, 5, CH2Cl2/DMF; (d)
catalyst 15, CH2Cl2, rfx.; (e) 4 M HCl in dioxane, CH2Cl2, 0 °C; (f) 1. 16 or 17, HATU, DIPEA, THF; 2. HF-pyridine, THF. (c) Building blocks
used for optojasp synthesis and structure of tubulin-selective photostatin-1 (18). See text and Supporting Information for details.
even under prolonged (>12 h) pulsed irradiations with 390
and 450 nm light (Figure 3b and Figure S3).
potency upon illumination with 390 nm (cis form predom-
inating; Figure 3g), while some stayed equipotent (optojasps 2
and 6). By contrast, the potency of optojasps 3 and 4 was
higher in the dark (trans).
To investigate the mode of action and the selectivity of
optojasps we investigated their effect on the cellular actin
cytoskeleton by fluorescence microscopy (Figure 4). We
observed that optojasps caused a light-dependent increase of
actin nucleation, resulting in formation of large aggregates, and
leading to very similar cellular phenotypes as caused by the F-
actin stabilizers 1−3. HeLa cells exposed to the cis-active
Since membrane-permeant actin inhibitors are potent
cytotoxins,12 the optojasps were evaluated in cellulo for light-
dependent cytotoxic activity in HeLa cells (Figure 3 and Figure
S1). Multi-light-emitting diode (LED) arrays of a DISCO
system23 were used to deliver pulsed illuminations at different
wavelengths. Notably, all optojasps were bioactive, indicating
that the azo extension of the parent compound does neither
compromise cell permeation nor compound binding. Com-
pared to the dark-state trans isomers, most optojasps gained
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Figure 3. Switching properties and cytotoxicity of optojasps. (a) Absorption spectra of cis and trans optojasp-1. (b) Photoswitching reversibility
monitored by absorption at 390 nm on pulsed treatment with 390 and 450 nm light. (c) Wavelength-dependent cytotoxicity of optojasp-1
determined by MTT assay. (d−f) Photoswitching-dependent cell toxicity profiles for (d) “trans-active” optojasp-3, (e) “cis-active” optojasp-5, (f)
jasplakinolide (3). (g) Photophysical and cytotoxicity parameters for optojasps 1−8, see Figure S1 for details. (h) Wavelength-dependent
cytotoxicity of cis-active optojasp-1, related to panel (a). All data were obtained with HeLa cells (45 h, pulsed illuminations of 75 ms every 15 s at
the specified wavelength or in the dark).
Figure 4. Light-dependent selective disruption of the actin cytoskeleton by optojasps. (a) HeLa cells treated with optojasps (1 μM) in the absence
or presence of 390 nm illumination, stained for F-actin (red). (b) HeLa cells untreated or treated with optojasp-1 (1 μM) in the absence or
presence of 390 nm illumination, stained for tubulin (green). (c) Cells treated with optojasp-1 (3 μM) and exposed to 390 nm light for 5 h
(“390 nm”), subsequently exposed to 475 nm light for 1 h, then kept in darkness for 12 h (“390 nm then dark”). (d) HeLa cells treated with
jasplakinolide (3, 100 nM) for 5 h in the absence or presence of 390 nm irradiation. Nuclear DNA was stained with Hoechst 33342 (blue), cells
were imaged after fixation, scale bar 20 μm.
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optojasp-1 showed induction of actin polymerization into
amorphous clumps under 390 nm illumination, while cells
maintained in the dark showed no abnormalities in the F-actin
network. In line with the photophysical data, the trans-active
optojasp-3 displayed an inverted light dependency with actin
cytoskeleton disruption occurring in the dark and with no
effect on the organization of the F-actin network under 390 nm
illumination (Figure 4a). By contrast, the microtubule network
was clearly not affected by optojasp-1 treatment (Figure 4b),
even though the trimethoxy azobenzene motif is found in
microtubule-disrupting photostatin photoswitches23 and re-
sembles a substructure motif of the tubulin-polymerization
inhibitors colchicine and combretastatin A4.28 Our data show
that, by extending the optojasp switches at the combretastatin-
like B-ring in para, affinity for tubulin is abrogated and that
actin-selective conjugates for cytoskeleton disruption are
obtained.
Incubation under actin-nucleating illumination conditions
(optojasp-1 with 390 nm light pulses over 5 h) led to a hyper-
aggregated actin cytoskeleton (Figure 4c). Subsequent
irradiation with light that promotes back-isomerization
(475 nm pulse block for 1 h) followed by incubation in the
dark for 10 h to further promote back-isomerization
phenotypically recovered the regular actin cytoskeleton
structure (Figure 4c).
To study whether the observed activities were connected to
nonspecific interactions, we studied the physicochemical
properties of the best candidate optojasp-1 as well as its
activity on target. Compound solubility remained unaffected
by irradiation, that is, both trans- and cis form of optojasp-1
were equally soluble. Furthermore, by collecting light-
scattering data at 800 nm wavelength, we could not find any
indication for compound aggregation in either cis- or trans-
configuration (see Supporting Information for details).
On-target activity was then studied by measuring the rate of
F-actin polymerization induced by optojasp-1, depending on
its configurational state, using pyrene-labeled actin (Figure
5).31 We found that both forms of optojasp-1 stimulated F-
actin polymerization, but, to a lesser extent than jasplakinolide.
Furthermore, the cis form of optojasp-1 was found significantly
more active than the trans form. Both observations are in good
agreement with the cytotoxicity data.
By contrast, the azobenzene extensions 16 or 17 (not
shown) devoid of the actin-binding ligand were incapable of
promoting F-actin polymerization and did not modify the
activity of jasplakinolide, neither in cis nor in trans form (see
Supporting Information). These data strongly suggest that it is
the direct molecular interaction of the optojasps with the F-
actin target that accounts for their properties.
Figure 5. Illumination-dependent capacity of optojasp-1 for inducing
actin-polymerization in vitro. trans-Optojasp-1, cis-optojasp-1, or
jasplakinolide (3, 20 μM each) were incubated with pyrene-labeled
actin (5 μM) under low salt, nonpolymerizing conditions.31
Polymerization was monitored by pyrene fluorescence emission
measurement at λEm = 410 nm after low-intensity excitation at λEm
=
350 nm in 60 s intervals over 4 h at 37 °C. Optojasp configuration
remained stable under these conditions (see Figure S5). Data are
shown for the initial linear phase (1 h). All measurements were
performed in duplicates in 384-well plates. RFU: relative fluorescence
units, detector response normalized to fluorescence of pyrene-labeled
G-actin in nonpolymerizing buffer. External fluorophore 16 was added
to 3 to correct for azobenzene absorbance; k: slopes of linearly fitted
curves in RFU/s; krel: comparative potential for induction of actin
polymerization, normalized to jasplakinolide (krel = 1).
cell velocity and migration distance were found significantly
reduced in a dose-dependent manner upon activation with
390 nm light. cis-Active optojasps were particularly robust in
this application, reducing motility threefold in light-targeted
cells only minutes after illumination, at doses where cells
remained unaffected in the dark (Figure 6b, Movie 3, and
Movie 4).
We also examined whether optojasps could optically control
cell division, since its successful completion relies on proper
chromosome segregation and correct functionality of the actin
contractile ring.5,19 At intermediate doses optojasp-1 was
indeed capable of controlling the completion of cell division.
While cell division proceeded with chromosome segregation to
the state of daughter cell constriction, photoswitching stalled
the separation of daughter cells. This blockade finally resulted
in their refusion into tetraploids, strongly indicative of a
dysfunctional contractile ring (Figure 6c, Movie 5, and Movie
6). These phenotypic data independently confirm that tubulin
assembly remains unaffected by optojasps, which exert
selective activity on F-actin dynamics only.
Furthermore, we investigated the ability of the optojasps to
optically control processes acutely coupled to cytoskeletal
signaling.34 The myocardin-related transcription factor A
(MRTF-A) was selected as an example, as it directly
communicates changes of cytoplasmic actin dynamics to
gene regulation.35 G-Actin-bound MRTF-A is released when
F-actin is formed. Liberated MRTF-A translocates into the
nucleus, where it activates target genes depending on serum
response factor (SRF).36 We first used a fluorescent MRTF-A
fusion protein in order to monitor its cellular localization.
Indeed, upon treatment with activated optojasps, MRTF-A
localized to the nucleus, just as observed after treatment with
With target selectivity and reversibility established, we
evaluated the potential of optojasps for photocontrol of
cellular dynamics. Actin reorganization is involved in neuronal
development and plasticity,32 cell cycle and division,19 as well
as cell motility, invasive migration,18 and endocytosis.33 When
studying cell morphology, we observed that, upon irradiation
with 390 nm, optojasp-1 caused cells to detach rapidly from
their substrate at doses that caused no morphological changes
in the dark (Figure 6a, Movie 1, and Movie 2). Motile cells
exposed to optojasp-1 immediately stopped migrating upon
illumination with 390 nm, reduced lamellipodia formation, and
finally rounded up. We then performed cell tracking experi-
ments to quantify the control of cell motility by photo-
switching optojasps. In line with the phenotypic observations,
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Figure 6. Optical control over cell shape, motility, and division. (a) Motile cells exposed to optojasp-1 (5 μM) before and after illumination with
390 nm, see Movie 1 and Movie 2. (b) Quantification of light-dependent cell motility parameters with escalating doses of optojasp-1, see Movie 3
and Movie 4. (c) Dividing cells treated with optojasp-1 (2.5 μM) in the dark or under 390 nm illumination, see Movie 5 and Movie 6). All
experiments were conducted using MDA-MB-231 cells stably expressing mCherry-Lifeact; scale bars 40 μm, time shown as hh:mm. See Supporting
Information for experimental details.
Figure 7. Optical control over MRTF-A signaling in human umbilical vein endothelial cells (HUVECs). (a) Cells expressing mCherry labeled
MRTF-A exposed to escalating doses of optojasp-5 in the dark or with 390 nm irradiation, stained for F-actin, and imaged after fixation. Scale bars
40 μm. (b) Quantification of the nuclear fraction of MRTF-A. (c) Dual luciferase reporter gene assay for SRF response elements in cells incubated
with optojasp-5 or jasplakinolide. Luciferase reporter activity is expressed in relative luminometer units (RLU) normalized to the constitutive
renilla luciferase expression level (control). Bar graph results are given as mean standard deviation (SD); statistical significance was determined
by unpaired Student t-test, p ≤ 0.05. See Supporting Information for details.
jasplakinolide. We selected optojasp-5 for quantitative
analysis, as this compound has a spontaneous cis-trans
relaxation rate in the order of the biological response
(18 min, Figure 3) and only a marginally lower efficacy
compared to the slowly relaxing optojasps-1−4. Without
illumination, labeled MRTF-A remained homogeneously
distributed in the cytoplasm (Figure 7a). When cells were
exposed to optojasp-5 and to 390 nm light, nuclear
translocation of MRTF was significantly enhanced when
compared to nonilluminated cells (Figure 7a). The effect was
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most prominent at intermediate concentrations (250−
750 nM).
Quantification of the nuclear fraction of MRTF-A confirmed
the significance of these data (Figure 7b). These results
indicate that MRTF-A can be specifically activated by optojasp
photocontrol, further validating F-actin assembly as the direct
optojasp target.
In addition, we studied the function of MRTF-A induction
by using a luciferase reporter gene regulated by SRF. Cells
treated with optojasp-5 fully induced reporter gene expression,
with strong optical control in the medium concentration range
(Figure 7c). Jasplakinolide-treated control cells carrying the
same reporter displayed strong, but illumination-independent,
induction of luciferase activity upon compound treatment.
Finally, we investigated optojasps’ ability to exert both time-
and cell-specific control over actin-dependent processes in
cellulo. After illumination bursts were delivered to regions of
interest (ROIs) comprising single selected cells within a cell
population, cellular response was individually monitored. For
this experiment the slowly relaxing optojasp-1 worked again
well (Figure 8, Movie 7, and Movie 8). We reproducibly
optojasps directly work on F-actin as target and are capable of
acutely blocking actin dynamics on the minute time scale with
high spatial precision.
Taken together, our data show that optojasps are a robust
and powerful tool to optically control the actin cytoskeleton
and processes that depend on it. They can be used
noninvasively in live cells, with exquisite spatial and temporal
precision at the cellular level, and enable optical control over
actin nucleation and cytotoxicity in cells, as well as over cell
motility and cytoskeletal signaling.
DISCUSSION
■
Optical control of biological activity by using caged
compounds, optogenetics, and more recently photopharmacol-
ogy has advanced significantly in recent years. Photouncaging
is by now routinely applied for spatiotemporal control in
biology, particularly in neuroscience, where caged neuro-
transmitters are standard tools.37 However, to our knowledge,
reversibly controllable actin modulators have not yet been
introduced for biological studies.
Precision control of cytoskeleton-associated proteins with
light has been recently demonstrated by using optogenetics
involving proteins that indirectly act on the actin cyto-
skeleton.38−40 On the one hand, engineering actin itself has
been rather difficult due to the strong influence even small
modifications have on its proper functioning. On the other
hand, small-molecule photoswitches for the direct optical
control of the microtubule cytoskeleton by tubulin polymer-
ization inhibition have been developed23,41 and have already
led to applications in neuroscience, morphogenesis, and in vivo
applications in embryology.42−44 Photoswitchable tubulin
stabilizers were recently reported as well.45 By analogy, we
expect that the small-molecule optojasps introduced here as
photoswitchable F-actin modulators will provide a powerful
tool for studying actin biology in a spatiotemporally controlled
manner. In particular, we surmise that the ability to easily
phototune the inhibitory activity of the optojasps by varying
the wavelength of illumination (“photodosing”, Figure 3) to
sensitively set the isomer ratio above or below its activity
threshold offers a robust and adaptable method for complex
biological experiments, which is particularly advantageous
when compared to irreversible photouncaging approaches.
Optojasps retain most of the potency of the parent
compound jasplakinolide and can be expected to bind to the
same binding site on F-actin, considering all functional and
phenotypic data acquired. However, the discovery of both cis-
active and trans-active compounds alongside with compounds
with barely photoswitchable bioactivity is intriguing consider-
ing their rather similar chemical structures. It suggests a
specific molecular interaction of the optojasps with their target
that directly involves the configurationally bistable azobenzene.
The optojasp design is based on an azo-extension of the
jasplakinolide structural scaffold. Recent structure elucidations
of F-actin bound with jasplakinolide and its Lys analogue by
using cryo-electron microscopy showed that both bind at
regular intervals inside the F-actin filament contacting three
adjacent actin subunits.46,47 They reinforce the fiber by
stabilizing a state of partial ATP hydrolysis that is only
transient during regular F-actin turnover.47 Interestingly, the
lysine side chain of the bound analogue projects toward the
interior of F-actin and extends into an inner cavity of moderate
volume that is open to the outside (see Figure S2).
Figure 8. Time-lapse imaging series of MRTF-A:mCherry-expressing
HUVEC cells following treatment with 750 nM optojasp-1 and cell-
specific, localized illumination (+UV, white rectangle ROI, 405 nm
irradiation). Scale bars 20 μm.
observed that light activation triggered actin-dependent
MRTF-A translocation to the nucleus in exactly the defined
ROI, with cell-by-cell spatial control applied side-by-side. The
response that was both stable and rapid, and cross-talk to
neighboring cells, was not observed. In nonilluminated cells,
MRTF-A remained in the cytosol. Recovery was not evident
on the time scale of the experiment, probably because
stimulated F-actin aggregation needs longer time to recover
(cf. Figure 4c). Nevertheless, these data strongly suggest that
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The macrocycle’s binding pose can be expected to be
retained for jasplakinolide conjugates. The azo-extension will
then interact with the interior of the cavity and affect the
affinity of the optojasps to F-actin. As the space in this cavity is
confined, the ability to stabilize the F-actin fiber would become
dependent on switch topology and its configuration. Such a
model could tentatively explain the “switchability” and the
ability to generate “cis-active” (optojasp-1) and “trans-active”
modulators (optojasp-3), which would result from the
individually different optojasp topologies and conformational
preferences. Furthermore, altered conformational preferences
of the optojasp macrocycle could contribute to switchability as
well.
ity monitoring, materials and methods including
solubility and aggregation data, chemical synthesis and
characterization, optojasp HPLC traces, 1H NMR
spectra, UV−vis spectra (PDF)
Movie 1. Cell morphology, optojasp-1 in the dark (AVI)
Movie 2. Cell morphology, optojasp-1 upon UV
exposure (AVI)
Movie 3. Cell migration, optojasp-1 in the dark (AVI)
Movie 4. Cell migration, optojasp-1 upon UV exposure
(AVI)
Movie 5. Cell division, optojasp-1 in the dark (AVI)
Movie 6. Cell division, optojasp-1 upon UV illumination
(AVI)
By contrast, changes in general compound properties are
likely not strongly contributing to compound activity. Our
experiments showed that solubility does not depend on switch
configuration; neither was aggregation observed. While not
investigated for all optojasps in detail, the single cell
irradiation experiments conducted for studying MRTF-A
signaling (Figure 8) as well as experiments with pre-irradiated
compounds (data not shown) indicated that both the active
and the inactive state should have comparable cell perme-
ability. If this was not the case, local concentration reached by
irradiation would probably be insufficient for observing any
compound activity localized in the extremely small volume of a
single cell (∼10−14 m3). Furthermore, “spilling” of activity to
neighboring cells, indicative of a highly diffusible active state
populated by irradiation, was also not observed during these
experiments. Finally, if the azobenzene configuration (cis or
trans) would generally affect cell permeability, a concordant
activity profile for all optojasps should emerge in cellulo
which is not the case. Together with the F-actin polymerization
experiment, which supported direct on-target activity, these
data strongly suggest that a direct structural interaction of the
switch with the binding cavity is likely eliciting the observed
properties.
Movie 7. MRTF nuclear translocation, UV illumination
control (AVI)
Movie 8. MRTF nuclear translocation, optojasp-5 with
local UV exposure (AVI)
AUTHOR INFORMATION
Corresponding Authors
■
Dirk Trauner − Department of Chemistry, New York University,
New York 10003 New York, United States; orcid.org/0000-
0002-6782-6056; Email: dirktrauner@nyu.edu
Hans-Dieter Arndt − Institute for Organic Chemistry and
Macromolecular Chemistry, Friedrich-Schiller-University, Jena
D-07743, Germany; orcid.org/0000-0002-0792-1422;
Email: hd.arndt@uni-jena.de
Authors
Malgorzata Borowiak − Department of Pharmacy, Ludwig-
Maximilians University, Munchen D-81377, Germany
̈
Florian Kullmer − Institute for Organic Chemistry and
̈
Macromolecular Chemistry, Friedrich-Schiller-University, Jena
D-07743, Germany
Florian Gegenfurtner − Department of Pharmacy, Ludwig-
Maximilians University, Munchen D-81377, Germany
̈
Sebastian Peil − Institute for Organic Chemistry and
Macromolecular Chemistry, Friedrich-Schiller-University, Jena
D-07743, Germany
CONCLUSION
■
Further mechanistic characterization and functional optimiza-
tion notwithstanding, we expect that optojasps will become
highly useful research tools for deciphering the complex role of
actin in cell biology with the high temporal and spatial
resolution that only light can provide. Importantly, our
experimentation showed that switching the activity of the
optojasps results in highly localized, acute activity on F-actin.
This activity can be confined to a single cell. Since actin is
highly conserved among all eukaryotes, the optojasps should
be applicable in a variety of cell types originating from different
organisms, provided they can be illuminated appropriately.
Lastly, the optojasps’ ability to inhibit actin dynamics only in
selected cells could open new vistas for functional research in
cancer biology and neuronal regeneration. Optojasps should
hence prove a versatile and powerful tool for enabling high-
precision cytoskeletal control inaccessible to other methods.
Veselin Nasufovic − Institute for Organic Chemistry and
Macromolecular Chemistry, Friedrich-Schiller-University, Jena
D-07743, Germany
Stefan Zahler − Department of Pharmacy, Ludwig-Maximilians
University, Munchen D-81377, Germany; orcid.org/0000-
̈
0002-5140-7287
Oliver Thorn-Seshold − Department of Pharmacy, Ludwig-
Maximilians University, Munchen D-81377, Germany;
̈
orcid.org/0000-0003-3981-651X
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.9b12898
Author Contributions
#M.B. and F.K. contributed equally.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b12898.
■
sı
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health (R01 GM126228 to D.T.), the Deutsche Forschungs-
gemeinschaft (SFB1032 to D.T., O.T.-S., and S.Z.; SFB152
and Emmy Noether grant to O.T.-S.), and the TMWWDG
(Grant No. 43-5572-321-12040-12 to H.-D.A.). H.-D.A. is a PI
MTT assays, binding pocket region in F-actin, extended
photoswitching stability trace of optojasp1, raw data of
F-actin polymerization experiments, isomerization stabil-
H
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