, 260 (2012);
Roger C. Hardie and Kristian Franze
Photomechanical Responses in
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grade flow of actin and myosin-2 is resisted by
friction (termed “flow-friction motor”; Fig. 3A).
In the embryo, friction will arise when the flow
velocity in the ring is different from the velocity
of the adjacent material, such as the yolk cell
plasma membrane and the yolk cytoplasm. Con-
sistent with this, we observed differential flow ve-
locities between the actomyosin in the ring and
adjacent microtubules within the YSL (fig. S6).
This flow-friction motor pulls the EVL in a di-
rection opposite to the actomyosin flow, operates
resisted flow provides additional tension in the
AV direction, consistent with the small degree of
tension anisotropy observed in the laser ablation
measured flow profiles within the EVL and the ac-
tomyosin ring, as well as the relative tensions ob-
tained from laser ablation, are accurately predicted
by our theoretical description at all stages when
(Fig.3B).Toconclude,we identifiedtwo distinct
modesofring propulsion:a cable-constrictionmo-
tor due to circumferential contraction of the YSL
to contraction along the AVaxis of the network.
We next asked if the flow-friction motor is
sufficient to drive EVL epiboly. To this end, we
took advantage of the predicted geometry depen-
dence of the cable-constriction motor. Because
the cable-constriction motor cannot exert a net
force on the EVL when positioned right at the
equator, propulsion by this motor would be hin-
dered when the yolk cell is deformed from its orig-
inal spherical geometry into a cylindrical shape.
We thus deformed the yolk cell into a cylindrical
shape by aspirating pre–gastrula-stage embryos
than that of the embryo and analyzed resulting
changes in EVL movements. To verify that the
actomyosin ring is unperturbed in cylindrical em-
bryos, we analyzed the distribution and flow of
actin and myosin-2 within the YSLof cylindrical
actin and myosin-2 in a ring-like structure adja-
cent to the EVL/YSL border and their retrograde
border were largely unaffected in cylindrical em-
bryos as compared to normal-shaped control em-
bryos (Fig. 4 and movie S10). This suggests that
the actomyosin ring remains intact in cylindrical
largely unaffected in cylindrical embryos and pro-
control embryos (2.0 T 0.2 mm/min compared to
1.9 T 0.1 mm/min at 60 to 70% epiboly; compare
Figs. 4D and 2D). This shows that the cable-
movements and indicates that the flow-friction
motor is sufficient to drive this process.
Our findings have major implications for the
function of actomyosin rings in morphogenesis.
Whereas the prevalent model of actomyosin ring
function assumes circumferential contraction as
the main force-generating process, we present
evidence that friction-resisted actomyosin flows
can represent an equally important process me-
a more general role of cortical flows in morpho-
genetic pattern formation processes (18).
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Eur. Phys. J. E 16, 5 (2005).
14. G. Salbreux, J. Prost, J. F. Joanny, Phys. Rev. Lett. 103,
15. D. Bray, J. G. White, Science 239, 883 (1988).
16. L. P. Cramer, Front. Biosci. 2, d260 (1997).
17. J. Howard, Mechanics of Motor Proteins and Cytoskeleton
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18. N. W. Goehring et al., Science 334, 1137 (2011).
Acknowledgments: We are grateful to M. Sixt, T. Bollenbach,
and E. Martin-Blanco for advice and the service facilities of
the IST Austria and MPI-CBG for continuous help. M.B., G.S.,
S.W.G., and C.-P.H. synergistically and equally developed the
presented ideas and the experimental and theoretical
approaches. M.B. and P.C. performed the experiments; G.S.
developed the theory; and R.H., F.O., and J.R. contributed to
the experimental work. This work was supported by a grant
from the Fonds zur Förderung der wissenschaftlichen
Forschung (FWF) and the Deutsche Forschungsgemeinschaft
(DFG) (I930-B20) to C.-P.H., S.W.G., and G.S.
Figs. S1 to S16
Materials and Methods
Movies S1 to S10
1 May 2012; accepted 10 September 2012
Photomechanical Responses in
Roger C. Hardie* and Kristian Franze
Phototransduction in Drosophila microvillar photoreceptor cells is mediated by a G protein–activated
phospholipase C (PLC). PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate
(PIP2), leading by an unknown mechanism to activation of the prototypical transient receptor potential
of the photoreceptor cells and modulated the activity of mechanosensitive channels introduced into
photoreceptor cells. Furthermore, photoreceptor light responses were facilitated by membrane stretch
and were inhibited by amphipaths, which alter lipid bilayer properties. These results indicate that, by
cleaving PIP2, PLC generates rapid physical changes in the lipid bilayer that lead to contractions of the
localized within tightly packed microvilli (tubu-
lar membranous protrusions), together forming a
light-guiding rod-like stack(rhabdomere; Fig.1).
After photoisomerization, rhodopsin activates a
n most invertebrate photoreceptor cells, the
visual pigment (rhodopsin) and other compo-
nents of the phototransduction cascade are
bound a subunit, which in turn activates phos-
pholipase C (PLC; Fig. 1C). How PLC activity
leads to gating of the light-sensitive transient re-
ceptor potential channels (TRP and TRPL) in the
4,5-bisphosphate (PIP2), yielding solubleinositol
which remains in the inner leaflet of the micro-
channels in Drosophila photoreceptors can be
activated by a combination of PIP2depletion and
protons (4), but it remains unclear how PIP2de-
pletion might contribute to channel gating. It has
been speculated that changes in membrane prop-
erties play a role (4, 5), and members of the TRP
controversially, implicated as mechanosensitive
channels (6, 7). This led us to ask whether cleav-
age of the bulky, charged inositol head group of
PIP2(Fig. 1C) from the inner leaflet might alter
the physical properties of the lipid bilayer in the
tribute to channel gating.
Remarkably, Drosophila photoreceptors re-
sponded to light flashes with small (<1 mm) but
rapid contractions that were directly visible in
Department of Physiology, Development and Neuroscience,
University of Cambridge, Cambridge CB2 3EG, UK.
*To whom correspondence should be addressed. E-mail:
12 OCTOBER 2012VOL 338
on October 12, 2012
Fig. 1. AFM measurements of photomechanical
responses. (A) An AFM cantilever contacts distal
tips of ommatidia in an excised Drosophila retina.
(B) An ommatidium, containing photoreceptors
(orange) and pigment cells (red). Elements of the
phototransduction cascade are contained within
microvillar rhabdomeres (two shown in longitu-
dinal section, seven in cross section), which are
rodlike stacks ~80 mm in length containing
~30,000 microvilli. Right: Electron micrograph
cross section of a rhabdomere (scale bar, 1 mm),
showing tubular microvilli, each ~50 nm in
diameter, with lumen in diffusional continuity with
the cell body. (C) Phototransduction cascade.
Rhodopsin (R) is photoisomerized to meta-
rhodopsin (M*), which catalyzes release of the Gq
protein a subunit to activate PLC. PLC hydrolyzes
PIP2(red), leaving DAG (green) in the membrane.
traces: AFM measurements of contractions (canti-
5-ms flashes, with intensity increased from ~200
to 8000 effectively absorbed photons per photo-
receptor. Blue traces: Whole-cell current-clamped
voltage responses to the same stimuli recorded
from a dissociated photoreceptor cell. (E) Contrac-
tions evoked by 5-ms flashes covering the full
intensity range (~200 to 106photons) in a wild-
type retina. (F) Same on faster time base. (G)
Response versus intensity (R/I) functions of con-
N = 13), and peak voltage (mV) recorded from
dissociated photoreceptors (blue; means T SEM,
in trpl mutant before and after (red) channel block
by 50 mM La3+and 10 mM ruthenium red (RR),
which prevents inhibition of PLC by Ca2+influx.
Blue trace: Lack of response in norpAP24(PLC
mutant) despite using flashes of higher intensity
(~2 × 105photons; N = 3). (I) R/I function of
channel block by La3+and RR (means T SEM, N = 5). For intensity calibration, see fig. S2.
Fig. 2. Light-induced modulation of gramicidin channels.
(A) Whole-cell recordings from trpl;trp mutant photo-
receptor cell lacking all native light-sensitive channels.
Perfusion with gramicidin induced a constitutive inward
current. (B) Flashes of increasing intensity (5 × 103to 4 ×
105effective photons), each 1 s (denoted by bar at upper
left), up-regulated the current. (C) Averaged responses
(TSEM) to 100-ms flashes containing 1.3 × 104(middle
trace, N = 4) and 3.7 × 104(lower trace, N = 10) effective
in the presence of La3+and RR). The same flashes delivered
before gramicidin application (controls) induced residual,
noise-free transient currents of uncertain origin. (D) R/I
function (after subtracting control responses measured
before gramicidin perfusion) expressed as a fraction of the
steady-state gramicidin current I/ISS(means T SEM, N = 4).
Dotted curve: R/I function of contractions measured by AFM
VOL 33812 OCTOBER 2012
on October 12, 2012
dissociated cells via bright-field microscopy (see
movie S1). To obtain improved temporal and
spatial resolution, we recorded these photome-
chanical responses with an atomic force micro-
on the distal tips of photoreceptors in a whole
excised retina glued to a coverslip (Fig. 1A). Con-
tact force (~100 pN) was maintained constant, so
that changes in sample height resulted in imme-
brief flashes of modest intensity, with kinetics
similar to those of electrical responses recorded
S1). The latencies of contractions induced by the
brighteststimuli(4.9T 0.9ms,meanT SEM,N=
11; Fig. 1F) were significantly shorter than the
latencies of voltage responses to the same stimuli
(6.6 T 0.6 ms, N = 6; P = 0.002, unpaired two-
tailed t test). Only a few hundred effectively ab-
sorbed photons per photoreceptor were required
to elicit detectable contractions, which saturated
overlapped with that of the electrical response
(Fig. 1G and supplementary text). Like the elec-
trical responses, the contractions were eliminated
that they too required PLC activity (Fig. 1H).
Because PLC activity is normally terminated
by Ca2+influx through the light-sensitive chan-
nels, net PIP2hydrolysis is enhanced when the
fore measured contractions in trpl mutants ex-
pressingonly TRPlight-sensitive channelsbefore
and ruthenium red (RR). Indeed, after blocking
the light-sensitive channels the contractions were
lower intensities, corresponding to only ~1 to 5
effectively absorbed photons per microvillus
(Fig. 1, H and I). In the absence of Ca2+influx,
such intensities deplete virtually all microvillar
PIP2, resulting in temporary loss of sensitivity to
light (10). After such saturating flashes, the
photomechanical response was also temporarily
(t1/2~ 40 s) similar to that of PIP2resynthesis
(10). By contrast, without channel blockers, sen-
sitivity recovered within ~10 s (fig. S3).
not result from any downstream effects of Ca2+
influx or osmotic changes caused by ion fluxes as-
sociated with the light response. Therefore, these
results indicate that the contractions result from
hydrolysis of PIP2. Although we do not exclude
other downstream effects of PLC, the speed of
anism. Cleavage of the bulky head groups from
PIP2molecules, which represent 1 to 2% of
lipids in the plasma membrane, leaves DAG in
the membrane, which occupies a substantially
brane tension, leading to shrinkage of the micro-
villar diameter, as reported for the action of PLC
on the diameter of artificial liposomes (11). In-
tegrated over the stack of ~30,000 microvilli,
such a mechanism seems capable of accounting
for the observed macroscopic contractions,which
(see supplementary text). Within each micro-
villus, we suggest that the alteration to the me-
chanical properties of the lipid bilayer may
contribute to channel gating.
To test whether phototransduction generates
sufficient mechanical forces to gate mechano-
sensitive channels (MSCs), we made whole-cell
patch-clamp recordings from dissociated photo-
receptors lacking all light-sensitive channels
(trpl;trp double mutants, or trpl mutants exposed
to La3+and RR). We then perfused the photo-
receptors with gramicidin, a monovalent (Ca2+-
impermeable) cation channel and one of the
best-characterized MSCs, which is known to be
regulated by changes in bilayer physical proper-
ties (12, 13). Incorporation of gramicidin chan-
nels into the membrane generated a constitutive
inward current that stabilized after a few minutes
(Fig. 2A). Despite having replaced the native
light-sensitive channels with MSCs, the photo-
receptors still responded to light, with a rapid
increase in the gramicidin-mediated current (Fig.
2,B and C). Like the photomechanical responses
recorded after blocking Ca2+influx through the
mediated responses inactivated slowly (Fig. 2C),
were temporarily refractory to further stimulation
To test whether the light-sensitive channels
brane tension osmotically. Channels were not
directly activated by perfusing cells with hyper-
Fig. 3. Modulation of light-sensitive channels by osmotic pressure. (A to D) Whole-cell voltage-clamped
responses to 1-ms flashes (~50 effective photons) in control bath (300 mOsm) were reversibly increased
by perfusion with 200 mOsm solution and suppressed by 400 mOsm in wild-type (A), trpl (B), and trp (C)
mutants as well as in wild-type photoreceptors recorded in Ca2+-free solutions (D). (E) Response am-
plitudes (I/I300) after hyperosmotic (400 mOsm) and hypo-osmotic (200 mOsm) challenges normalized to
controlresponses in 300 mOsm bath. Data are means T SEM; N = 4 to 8cells. All conditions plotted were
cells [P < 0.005; analysis of variance (ANOVA) followed by posttest for trend]. (F) Spontaneous TRP
channel activity (from trpl mutant) after several minutes of recording, using pipettes lacking nucleotide
additives. Perfusion with 400 or 200 mOsm (bar) reversibly suppressed and facilitated this “rundown
channel conductance (g) estimated by variance/mean ratio (means T SEM, N = 7). Although macroscopic
not significantly affected by osmotic manipulation (P > 0.2).
12 OCTOBER 2012 VOL 338
on October 12, 2012
that it would be impossible to mimic the exact
physical effects of PIP2hydrolysis, which would
include a specific combination of changes in
membrane tension, curvature, thickness, lateral
pressure profile, charge, and pH. We therefore
tested whether osmotic manipulation could en-
were rapidly and reversibly facilitated by ~50%
after perfusion with hypo-osmotic solutions (200
mOsm), which, like PIP2depletion, would be ex-
pected to alleviate crowding between phospho-
lipids, increase tension, and reduce membrane
thickness. Conversely, responses in hyperosmotic
(400 mOsm) solutions were about half those
in control solutions (Fig. 3). Analysis of single-
photon responses (quantum bumps) indicated
that modulation resulted from changes in both
quantum efficiency (fraction of rhodopsin photo-
isomerizations generating a quantum bump) and
Recordings from trp and trpl mutants showed
that both TRP and TRPL channels were modu-
lated, although facilitation of currents mediated
by TRPL channels (in trp mutants) was more
pronounced (Fig. 3). Modulation of the light re-
sponse by osmotic manipulation was at least as
pronounced in Ca2+-free bath (Fig. 3D), indicat-
ing that facilitation by membrane stretch did not
result from leakage of Ca2+into the cell from the
To test whether modulation might have been
mediated by effects on upstream components of
the cascade such as PLC (14), we measured the
activity of spontaneously active TRP channels in
recordings made with pipettes lacking adenosine
triphosphate. Under these conditions, PIP2be-
comes depleted (thereby removing PLC’s sub-
strate), sensitivity to light is lost, and the TRP
state uncoupled from the phototransduction cas-
cade (15, 16). Nonetheless, the channels were
still similarly modulated by osmotic manipula-
tion, whereas single-channel conductance, esti-
mated by noise analysis, was unaffected (Fig. 3,
F to H). These results indicate that osmotic pres-
sure directly modulated the open probability of
both TRP and TRPL channels.
MSCs such as gramicidin are sensitive to
amphiphilic compounds, which insert into the
phospholipids, cationic amphipaths insert prefer-
entially into the inner leaflet, where they increase
and decrease membrane stiffness (17, 18). We
found that four structurally unrelated cationic
of the light-induced current. Neither light-induced
PLC activity (measured using a genetically tar-
geted PIP2-sensitive biosensor to monitor PIP2
hydrolysis) nor single-channel conductance were
substantially affected (fig. S5). The 50% inhibi-
tory concentrations (IC50values) were much
higher than those of their traditional drug targets
nitude. However, after correcting for pKaand
pounds in the membrane was similar (~5 mM) in
each case (Fig. 4). Thus, their mode of action is
likely related to their physicochemical properties
rather than conventional drug-receptor interac-
tions. Because cationic amphipaths are also li-
pophilic weak bases, and because we propose
that protons are also critical for activating the
light-sensitive channels(4),an alternative butnot
mutuallyexclusive possible mechanismofaction
is as lipophilic pH buffers of the membrane
environment. We also note that polyunsaturated
fatty acids (PUFAs) such as arachidonic and lin-
TRP and TRPL (5, 19)—are not only anionic
amphipaths (predicted to have opposite effects
to cationic amphipaths) but also, as weak acids,
natural protonophores; such a dual action could
account for their agonist effect.
The mechanism of activation of the light-
sensitive channels in invertebrate microvillar
photoreceptors has long remained an enigma
(2, 3, 20). Neither InsP3nor DAG—the two ob-
vious products of PIP2hydrolysis—are reliable
from DAG, a DAG lipase with the appropriate
specificity has not been found in the photorecep-
of PLC activity—the depletion of its substrate
(PIP2) together with protons released by PIP2
the effect of PIP2depletion is mediated mechan-
ically by changes to the physical properties of the
lipid bilayer, thereby introducing the concept of
mechanical force as an intermediate or “second
messenger” in metabotropic signal transduction.
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Acknowledgments: We thank D. G. Stavenga, S. B. Laughlin,
and M. Postma for comments on the manuscript; O. Andersen
for advice on the use of gramicidin; and J. Grosche (Effigos AG)
for artwork for Fig. 1A. Supported by UK Biotechnology and
Biological Sciences Research Council grant BB/G0068651
(R.C.H.) and a UK Medical Research Council Career Development
Award (K.F.). Author contributions: project initiation, R.C.H.;
whole-cell electrophysiology experiments, R.C.H.; AFM
measurements, K.F., R.C.H.; paper written by R.C.H. with
contribution from K.F.
Materials and Methods
Figs. S1 to S5
26 March 2012; accepted 7 August 2012
inhibit the light response.
in trp mutants in the pres-
ramine (IMP), trifluoperazine
(TFP), and chlorpromazine
fore (turquoise) and after
washout (blue) are super-
imposed. (B) Dose response
functions (means T SEM:
PROC, N = 4; IMP, N = 3;
by inverse Hill curves (slope
on raw values (IC50: PROC,
3.1 mM; IMP, 27 mM; TFP,
4.9 mM; CPZ, 4.5 mM). (C)
Same as in (B) after correc-
tition coefficients, reflecting
VOL 338 12 OCTOBER 2012
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