Membrane Tension Maintains Cell
Polarity by Confining Signals to the
Leading Edge during Neutrophil Migration
Andrew R. Houk,1Alexandra Jilkine,2,6Cecile O. Mejean,3Rostislav Boltyanskiy,4Eric R. Dufresne,3Sigurd B. Angenent,5
Steven J. Altschuler,2Lani F. Wu,2and Orion D. Weiner1,*
1Cardiovascular Research Institute and Department of Biochemistry, University of California San Francisco, San Francisco, CA 94143, USA
2Green Center for Systems Biology and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas,
TX 75390, USA
3Department of Mechanical Engineering and Material Science
4Department of Physics
Yale University, New Haven, CT 06520, USA
5Department of Mathematics, University of Wisconsin, Madison, WI 53706, USA
6Present address: Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA
Little is known about how neutrophils and other
cells establish a single zone of actin assembly during
migration. A widespread assumption is that the
leading edge prevents formation of additional fronts
by generating long-range diffusible inhibitors or by
sequestering essential polarity components. We
use morphological perturbations, cell-severing ex-
periments, and computational simulations to show
that diffusion-based mechanisms are not sufficient
for long-range inhibition by the pseudopod. Instead,
plasma membrane tension could serve as a long-
tension doubles during leading-edge protrusion, and
increasing tension is sufficient for long-range inhibi-
tion of actin assembly and Rac activation. Further-
more, reducing membrane tension causes uniform
actin assembly. We suggest that tension, rather than
diffusible molecules generated or sequestered at the
leading edge, is the dominant source of long-range
inhibition that constrains the spread of the existing
front and prevents the formation of secondary fronts.
The ability of cells to generate polarized distributions of signaling
molecules enables numerous biological processes including
asymmetric cell division, neurite specification, tissue formation,
and cell motility. The Rac GTPase drives actin polymerization
and protrusion at the leading edge in a wide range of migrating
cells (Ridley et al., 1992; Sun et al., 2004). Efficient migration
requires confining Rac activity to the leading edge: spatially
uniform Rac activation abolishes movement (Allen et al., 1998;
Inoue and Meyer, 2008; Srinivasan et al., 2003).
In neutrophils, Rac activity is highly polarized both in response
to external gradients and in the presence of uniform chemoat-
tractant (Gardiner et al., 2002; Weiner et al., 2007). Linked posi-
types to polarize during chemotaxis or random migration (Jilkine
and Edelstein-Keshet, 2011; Meinhardt, 1999; Neilson et al.,
2011; Turing, 1952; Xiong et al., 2010). Positive feedback
amplifies small, transient fluctuations into large, temporally per-
sistent asymmetries. GTPase and/or phosphoinositide-based
positive feedback loops have been implicated in the polarization
neurons (Fivaz et al., 2008), Dictyostelium (Sasaki et al., 2004),
and budding yeast (Butty et al., 2002; Wedlich-Soldner et al.,
2003). Positive feedback loops require a balancing mechanism
of inhibition to prevent them from overtaking the entire cell.
The positive feedback reaction can limit itself by generating
long-range inhibition, which constrains the spread of the existing
front and prevents the formation of secondary fronts. The inhibi-
tion is thought to arise either from the production of rapidly
diffusing inhibitory molecules by the front (Figure 1A) or from
the sequestration of limiting polarity components by the front
(Figure 1B). These mechanisms of long-range inhibition depend
on rapid diffusion of signaling components through the cytosol.
In contrast to the components that participate in the positive
feedback loops at the leading edge, the molecules responsible
for long-range inhibition are unknown. It has not even been
experimentally determined whether this inhibition is a diffusion-
Signaling-centered positive and negative feedback loops
are not the only potential mechanisms of polarization. A model
consisting entirely of mechanical interactions between the actin
cytoskeleton, myosin, and plasma membrane accurately pre-
dictsthe polarized morphologies ofkeratocytes(Kozlov and Mo-
gilner, 2007) as well as the relation between cell shape and
speed (Keren et al., 2008) without considering upstream signals.
This model is insufficient to explain neutrophil polarity because
cytoskeletal polarization and migration require Rac to be
Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc. 175
polarized (Inoue and Meyer, 2008; Srinivasan et al., 2003). Thus,
for force to play a dominant role in neutrophil polarity, it must
participate in the spatial patterning of signaling cascades, for
example by acting as a long-range inhibitor of Rac activation
A significant challenge in discriminating between the many
classes of models for cell polarity is that many of the underlying
tified, and even for the known components, the key biophysical
parameters are unknown. We performed experiments that
discriminate between models of long-range inhibition without
requiring detailed knowledge of the underlying molecular com-
ponents. Weusedmorphological perturbations and cellsevering
to push neutrophils into situations where existing diffusion-
based long-range inhibition models break down, as verified by
computational simulations. Mechanical tension is one mode of
(A) Diffusible inhibitor. An autocatalytic activator (A, green)
produces an inhibitory molecule (I, red) that diffuses
throughout the cytoplasm to act as a long-range inhibitor
of leading-edge formation.
(B) Limiting component. An autocatalytic activator in the
front inhibits activation elsewhere by consuming essential
substrates (S, gold) of the positive feedback loop, rather
than generating a diffusible inhibitor (as in A).
(C) Mechanical tension. Protrusion at the leading edge
generates mechanical tension (T, depicted as red springs)
in either the plasma membrane or the underlying cyto-
skeleton. This tension acts as a long-range inhibitor of
our observations on stretched and severed
cells. Consistent with this hypothesis, we find
that membrane tension nearly doubles during
leading-edge protrusion, tension increases suf-
fice for long-range inhibition of Rac activation,
and reducing membrane tension activates actin
assembly throughout the cell. Our data suggest
that long-range inhibition is not solely based on
diffusible molecules generated or sequestered
at the leading edge (as has been widely
assumed) but rather requires protrusion-based
increases in plasma membrane tension to con-
strain the spread of the existing front and
prevent the formation of secondary fronts.
Distinguishing between Neutrophil
Polarity Mechanisms with Cell Stretching
We sought to distinguish between long-range
detailed knowledge of the molecular players
involved in the process. Due to their widely
proposed roles in developmental patterning
(Gierer and Meinhardt, 1972; Nakamura et al., 2006; Sick et al.,
of the numerous hypothetical inhibition mechanisms in polarizing
cells (Jilkine and Edelstein-Keshet, 2011). These usually involve
a positive feedback reaction that either produces inhibitory mole-
cules (Figure 1A) or depletes essential polarity components (Fig-
ure 1B). Importantly, to generate cell polarity, the inhibitory mole-
cules or limiting components must diffuse more rapidly than the
products of the positive feedback reactions. These models were
typically developed for cells with spherical morphologies or
assumed a one-dimensional (1D) spatial geometry to denote the
‘‘front-back’’ axis. To test whether existing mathematical models
are consistent with cell behavior, we devised novel experimental
settings in which cellular morphologies attenuate diffusion.
If long-range inhibition depends on diffusion, then we can
interfere with it by creating a thin neck between the pseudopod
176 Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc.
and the rest of the cell. In contrast, tension-based inhibition
should still function in this context. Following brief heat shock
in the presence of uniform chemoattractant, neutrophils adopt
a stretched, growth-cone-like morphology (Malawista and De
Boisfleury Chevance, 1982) (Figure 2A). Despite this altered
morphology, a single zone of actin assembly (tethered pseu-
dopod) is observed with the cell body remaining inactive in
91% of these cells during the 250 s period of observation (n =
31; Figure 2B; Movie S1 available online). The inactivity of the
cell body is remarkable given that its communication with the
pseudopod is restricted by a tether that is typically 1 micron in
diameter and 25 microns long.
We simulated how three classes of diffusion-based polarity
models responded in a tethered morphology: Turing (Otsuji
et al., 2007), wave-pinning (WP) (Mori et al., 2008), and neutral
drift (Altschuler et al., 2008; Jilkine et al., 2011). These models
are conceptual, rolling many as-yet poorly characterized details
of the polarity network into generic mechanisms of interaction
and feedback. They share several common properties, namely
the following: the total amount of molecules is assumed to be
constant during the observed duration of polarization; molecules
transition between two states, either at the membrane (active) or
at the cytosol (inactive); and diffusion on the membrane is much
slower than in the cytosol. In our simulations, recruitment of
molecules to the membrane is autocatalytic and ultimately con-
strained by depletion of the cytoplasmic species. All three
models predict that the cell body will remain inactive despite
the tethered morphology (Figure 2C, left panels; Figure S1).
This either could reflect ongoing long-range inhibition of the
tion that occurred prior to tether formation and persisted indefi-
nitely due to the lack of resynthesis of limiting components. To
distinguish between these possibilities, we analyzed the
behavior of the models when the cell body is severed from the
pseudopod (Figure 2C, right panels; Figure S1). All three models
predict that the cell body remains inactive despite being discon-
not due to ongoing inhibition from the pseudopod. If it were, the
cell body would have reanimated upon pseudopod removal.
All of the long-range inhibition mechanisms under consider-
ation make strong predictions about the behavior of the cell
body after it is severed from the pseudopod (Figure 3A). Diffu-
sion-based competition for a fixed pool of molecules (limiting
component) predicts that the cell body will remain inactive after
pseudopod removal unless the limiting component is rapidly
resynthesized. Similarly, if diffusible inhibitory molecules are
responsible for long-range inhibition, then the cell body will
also remain inactive unless the inhibitory molecules have a short
the cell body will remain inactive after severing unless the resyn-
thesis/turnover rate is high. In contrast, a tension-based inhibi-
tion mechanism would allow the cell body to reanimate because
the cell could relax into a reduced-tension morphology after
severing. Thus, severing experiments discriminate between
current long-range inhibition mechanisms.
The tethered morphology enabled us to cut the tether with
a focused laser beam without destroying either the cell body or
the pseudopod. We find that the cell body becomes highly
protrusive within 70 s of pseudopod removal in 47% of the cells
(n = 36; Figure 3B; Movie S2). Two sets of controls demonstrate
that the reanimation of the cell body is not due to laser-induced
generation ofchemoattractant. Usingcellscontainingasensitive
readout of actin assembly (SCAR/WAVE complex recruitment;
Weiner et al., 2007), we observed activation of adjacent cells
upon cell destruction with lasers but no detectable response
following tether severing (Figure S2). We also studied sponta-
neous cleavage of tethers, which occurs at a low frequency in
the absence of laser cutting. Similar to laser-based severing,
spontaneous cleavage of the tether also resulted in reanimation
of the cell body within 1 min of severing in 26% of the cells
(n = 64; Figure 3C; Movie S2). Rapid reanimation of the cell
body after severing is inconsistent with an inhibition mechanism
involving slowly resynthesized limiting components. Our experi-
ments suggest that the pseudopod continuously inhibits the cell
body by either sequestering rapidly synthesized limiting compo-
nents, producing short-lived diffusible inhibitory molecules, or
generating mechanical tension.
Tethered Morphologies Dramatically Attenuate
Diffusion-Based Exchange between Pseudopod
and Cell Body
We reasoned that the tether should severely attenuate diffu-
sion-based communication between the pseudopod and cell
body. We used fluorescence recovery after photobleaching
(FRAP) to experimentally determine the mixing rate between
the pseudopod and the cell body (Figure 4). We first measured
the rate of recovery following GFP photobleaching in the cell
bodies of tethered cells (Figure 4B; Movie S3). To control for
reversible bleaching and new GFP synthesis, we also per-
formed photobleaching experiments in cells that lacked a teth-
ered pseudopod (Figure S3). We subtracted the recovery rate
due to reversible bleaching from the overall recovery rate for
tethered cells to determine the amount of recovery that was
due to diffusion of GFP from the pseudopod through the tether
(Figures 4B and S3). Our data demonstrate that the tethered
morphology slows down the exchange of GFP between pseu-
dopod and cell body by approximately 840-fold (n = 24, Fig-
ure 4C) compared to untethered cells. The experimentally
determined mixing rates correlate very well with our computa-
tionally predicted mixing rates for measured cell tether lengths
and diameters (R2= 0.8, Figure 4D; Extended Experimental
We investigated whether inclusion of synthesis and degrada-
tion of the limiting component could enable this mechanism to
replicate our experimental observations in tethered and severed
cells. We chose to analyze the limiting component in the context
of the neutral drift model because it is the most analytically trac-
table. For the cell body to reanimate within 35 s of severing, the
limiting component must be resynthesized at a rate of at least 6
particles/s (Extended Experimental Procedures). For the cell
body to remain quiescent in the tethered state in the absence
of severing, resynthesis must be balanced by loss through the
tether. Because the tethered morphology dramatically reduces
the rate at which molecules diffuse from the cell body to the
pseudopod, the limiting component would require a diffusion
coefficient of 330 mm2/s to maintain quiescence of the cell
Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc. 177
0 s 60 s 120 s 180 s
Tether formation in heat-treated HL-60 cells
Maintenance of polarity in tethered HL-60 cells
Simulation of published diffusion-based inhibition models following cell stretching and
Wave Pinning Model
0 s20 s 40 s 60 s 80 s100 s120 s
of active species
Neutral Drift Model
Figure 2. Maintenance of Polarity in Tethered Cells
(A) Tether formation in heat-treated HL-60 cells. The cell initially forms a pseudopod (black arrowhead). The pseudopod crawls away from the fixed cell body,
causing a tether (white arrowhead) to form between them. The scale bar is 5 microns.
(B) Maintenance of polarity in tethered HL-60 cells. Left: The cell body (black arrow) remains completely fixed as the pseudopod (black arrowhead) migrates
a significant distance. The asterisk in the first frame denotes a neighboring cell that lacks a tether. Right: Cell outlines from successive time points are depicted in
blue, green, orange, and red, respectively. The morphology of the cell body stays constant over the 250 s observation time in 91% of the uncut cells (n = 27). The
scale bar is 5 microns.
(C) Simulation of published diffusion-based inhibition models following cell stretching and severing perturbations. The top, middle, and bottom panels depict
simulation results of a WP model (top), Turing model (middle), and neutral drift model (bottom) following cell stretching or severing. The concentration of the
activator species (u) is represented as grayscale with black being the highest concentration. In the left panels, spherical or cylindrical cells were allowed to
develop polarized signals. We simulated the subsequent time evolution of this polarized signaling distribution in cells that were stretched into dumb-bell
geometries, similar to our experimental tethers. In the right panels, the signals in spherical or cylindrical cells were polarized as before. We simulated the time
evolution of the signals in cells that were severed into two equal halves. Steady-state distributions of membrane-bound activators for all three models are shown.
See also Figure S1 and Movie S1.
178 Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc.
body (Extended Experimental Procedures). This diffusion rate is
an order of magnitude larger than those reported for cytosolic
proteins (Swaminathan et al., 1997) and approximately 2-fold
greater than that for metabolites in the cytoplasm (Garcı ´a-Pe ´rez
et al., 1999). Similar constraints make a diffusible inhibitor
emanating from the pseudopod unlikely because the inhibitor
requires a short lifetime to enable rapid reanimation from the
cell body following severing, but such a short-lived inhibitor
cannot survive the slow journey through the tether to prevent
cell body activity in the tethered morphology.
C Pseudopod production after spontaneous tether cleavage
B Pseudopod production after laser severing
-20s0s 20s40s 60s 80s100s120s 140s-40s
A Outline of Severing Experiments
Diffusion-based inhibition without resynthesis
Diffusion-based inhibition with resynthesis
Figure 3. Cells Generate a New Pseudopod after Severing
(A) Outline of severing experiments. Following cell polarization, the pseudopod is removed, and the behavior of the cell body is observed. If the pseudopod had
sequesteredanonregenerating limitingcomponentrequiredforpolarization,thecellbodyshould nothavethematerial toreanimate.Reanimationofthecellbody
following severing of the pseudopod would be consistent with a short-lived inhibitor generated at the leading edge. This short-lived inhibitor could be due to
mechanical tension, a rapidly synthesized limiting component, or a diffusible inhibitor with a short half-life.
(B)Pseudopodproductionafterlaser severing. DIC images showing atethered HL-60 cell thatis severedwith alaser beam justbefore 0s. Following severing, the
previouslyquiescentcellbodygenerates anewpseudopod (whitearrowhead).Thecellbodymakesapseudopodaftersevering in47%ofcells (n=36).Thescale
bar is 2.5 microns.
(C) Pseudopod production after spontaneous tether cleavage. Phase images of a cell whose pseudopod (black arrowhead) spontaneously breaks free from the
cell body (black arrow) at 0 s. The cell body makes a new pseudopod within 50 s of severing (white arrowhead) and begins to migrate. The asterisks denote
neighboring cells. There is significant reanimation of the cell body following spontaneous tether cleavage in 26% of cells (n = 62). The scale bar is 10 microns.
See also Figure S2 and Movie S2.
Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc. 179
In summary, the reanimation of the cell body after severing
demonstrates that the pseudopod actively inhibits protrusions
elsewhere. However, our FRAP experiments on tethered cells
indicate that diffusion is too highly attenuated for efficient diffu-
sion-based long-range inhibition. Even with resynthesis, the
neutral drift model requires the limiting component to diffuse at
Outline of FRAP experiment Typical FRAP profile for a tethered cell
0 50 100 150200250
01 0.25 0.75 0.5
Overlaid FRAP profiles for tethered cells
Predicted vs. measured mixing rates between
pseudopod and cell body for tethered cells
Predicted Dmix (sec-1)
Observed Dmix (sec-1)
Figure 4. The Tethered Morphology Dramatically Attenuates Diffusion
(A) Outline of FRAP experiment. A GFP-expressing HL-60 cell is heated to generate a tethered pseudopod. We bleached the GFP in the cell body, and the
recovery in the cell body was measured to monitor diffusion-based mixing through the tether. Retraction of the pseudopod causes the contents of the cell body
and the pseudopod to mix completely.
(B) Typical FRAP profile for a tethered cell. The graph shows the normalized fluorescence recovery due to diffusion for the cell whose GFP fluorescence is shown
in the inset images (with cell outlines in yellow). There is slow linear recovery until 160 s, when the tethered pseudopod retracts, and the GFP from the pseudopod
rapidly mixes with the cell body.
(C) Overlaid FRAP profiles for tethered cells. The measured fluorescence recoveries for all of the tethered cells during the first second after bleaching are overlaid
in red. The expected initial fluorescence recovery for a nontethered spherical cell (mixing rate constant = 1.2/s) is shown in blue.
(D) Predicted versus measured mixing rates between pseudopod and cell body for tethered cells. Each black dot represents the diffusion-based mixing rate
constant for an individual photobleached cell. The y coordinate for each cell is the experimentally measured mixing rate constant (Dmix, obs). The x coordinate for
each cell is the predicted mixing rate constant using the formula:
Dmix;pred=DGFP=Vtether=L2Vcell, where Dmix,predis the predicted mixing rate constant; DGFPis the known diffusion coefficient of GFP in cytoplasm (27 mm2/s;
Swaminathan et al., 1997); L is the tether length; and Vcelland Vtetherare the volumes of the cell and the tether, respectively. The values L, Vcell, and Vtetherwere
measured for each cell from bright-field images. The predicted mixing rates correlate with the measured values (R2= 0.8, n = 24, red line is y = x). The tethered
geometry reduces mixing rate by 134- to 4472-fold for all of the cells in the experiment.
See also Figure S3 and Movie S3.
180 Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc.
a rate that is over an order of magnitude larger than with cyto-
plasmic proteins. In fact, the necessary diffusion coefficient is
comparable to that of water itself. Our data are inconsistent
withlong-range inhibitionmechanismsbasedsolely ondiffusion.
The leading edge must inhibit activation of the cell body through
more rapid modes of communication such as active transport,
propagating waves, or mechanical forces.
Because mechanical propagation of information (such as
tension) and diffusion-based communication do not share the
same geometrical limitations, we hypothesized that pseudopod
protrusion could generate tension in the plasma membrane that
rapidly propagates to inhibit protrusive activity in the rest of the
tion. Consistent with this hypothesis, aspiration experiments on
suspended neutrophils have shown that the cellular cortical
tension increases during polarization (Zhelev et al., 1996).
However, it is unknown whether the cortical tension represents
tension in the cytoskeleton, plasma membrane, or both (Hoch-
muth, 2000). We used optical traps (Dai and Sheetz, 1999) to
measure plasma membrane tension during chemoattractant-
stimulated polarization (Figure 5; Movie S4).
Addition of chemoattractant caused a significant increase in
cell protrusion accompanied by a significant increase in mem-
brane tension (n = 8, Figures 5B and 5C, p = 0.0006; Movie S4).
On average, the pulling force nearly doubled (from 8.5 to
16.6 pN) during protrusion (Figure 5D) in a manner that closely
correlated with cell spreading (Figure 5B, inset). We suggest
that cellular protrusion is responsible for the significant (roughly
4-fold) increase in plasma membrane tension. The pulling force
was not a response to the mechanical forces associated with
fluid exchange (n = 8; Figure 5C). Pretreatment with blebbistatin,
which causes a highly elongated morphology, also causes basal
membrane tension to increase (Figure S4).
Cellular Deformation Induces Long-Range Inhibition
of Protrusion Signals
Iftension isalong-range inhibitor of protrusion and leading-edge
signaling, then increases in cell tension (even when they are not
a result of pseudopod generation) should inhibit protrusion and
leading-edge signals throughout the cell. To test this hypothesis,
we used micropipette aspiration to mimic the mechanical in-
creases in tension that accompany protrusion (Herant et al.,
2005). This perturbation likely increases tension in both the cyto-
skeleton and plasma membrane when performed on migrating
neutrophils. We used total internal reflection fluorescence
(TIRF)-based imaging readouts of Rac activity and SCAR/WAVE
complex recruitment to assay the effects of tension increases
on cell morphology as well as leading-edge signals. Rac and
the SCAR/WAVE complex localize to the leading edge of neutro-
2004; Weiner et al., 2006).
We brought a micropipette into contact with the cell surface
and applied suction to cause the cell to bulge into the micro-
pipette, thereby increasing cell tension (Figure 6A). Within
seconds, this mechanical deformation inhibited leading-edge
protrusion. Following aspiration, the pseudopod retracts into
the cell body, leading to a significant reduction in spread area
(Figure 6B; Movie S5). Aspiration also inhibited SCAR/WAVE
complex recruitment (Figure 6C; Movie S5) and Rac activation
(Figure 6D; Movie S5).
Aspiration-induced inhibition of protrusion and leading-edge
signals was reversible (Figures 6B and 6D, late time points;
Movie S5). Thus, inhibition was not due to trivial reasons such
as irreversible cell damage following aspiration. We also moni-
tored plasma membrane integrity during aspiration with cyto-
plasmic dyes. We did not detect leakage of cytosolic GFP or
the entrance of extracellular rhodamine phalloidin, indicating
that our aspirations left the plasma membrane intact (Fig-
ure S5). Thus, mechanical tension suffices to act as a long-range
inhibitor of protrusion and leading-edge signals in migrating
Membrane Tension Restricts Signals to the Front
of Migrating Neutrophils
Our aspiration data show that tension increases are sufficient for
long-range inhibition of protrusion, but two important questions
remain. First, does the cell require tension to spatially confine
signals to the front? Second, which structures transmit the inhib-
itory tension: cytoskeleton, plasma membrane, or both? To
answer these questions we monitored leading-edge signaling
while reducing membrane tension with hypertonic buffers
(Keren, 2011) or cytoskeletal tension with myosin inhibition
(Lee et al., 2011; Pasternak et al., 1989). Treating neutrophils
to decrease (Lee et al., 2011) but plasma membrane tension to
increase (Figure S4). Blebbistatin treatment caused cell elonga-
tion due to a defect in tail retraction but did not cause an expan-
sion of signals beyond the leading edge (Figures 7A and 7C).
Elongation often led to reduced SCAR/WAVE complex recruit-
ment at later time points (Figures 7A and 7C), possibly because
of increased membrane tension. After several minutes of bleb-
bistatin treatment, the cells often developed a stellate mor-
phology with multiple arm-like projections that appeared to be
leading edges. However, close inspection revealed that only
one of these projections actively protrudes at a time (Figure S6);
the others are inanimate husks of previous leading edges that
were not retracted after they died. The addition of hypertonic
buffer (150 mM sucrose) to blebbistatin-treated cells caused
an immediate and spatially uniform accumulation of SCAR/
WAVE complex at the plasma membrane and resulted in a loss
of cell polarity (Figures 7B and 7C; Movie S6), whereas the
addition of hypotonic buffer resulted in cell rounding and a
disappearance of SCAR/WAVEcomplex recruitment (FigureS6).
Hypertonic buffer in the absence of blebbistatin also caused
SCAR/WAVE complex accumulation, although the effect was
smaller (Figures 7C and S6). At later time points, neutrophils in
hypertonic buffer often spread uniformly with wider leading
edges and exhibited multiple pseudopodia for long periods of
time (Figure S6). These behaviors are consistent with ectopic
leading-edge signaling throughout the cell. Based on the signif-
icant expansion of SCAR/WAVE complex recruitment in cells
treated with hypertonic buffer but minimal effect of blebbistatin
alone, we conclude that membrane tension plays the dominant
role in restricting signals to the leading edge.
Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc. 181
Schematic outline of membrane tension
Low pulling force on bead
High pulling force on bead
Pulling force over time for individual cells following
buffer addition or fMLP stimulation
Pulling force at different stages of the experiment
p = 0.0001
p = 0.0006
Pulling force over time for a representative cell
0 1020 3040 5060 708090
Pull membrane tube
Extend membrane tube
Pull 1Pull 2 Hold 1Hold 2 Pre-spread Post-spread
182 Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc.
Weusedmicrodissectionand perturbations ofcell morphologyto
push neutrophils into regimes where existing diffusion-based
polarity models break down. Our results support a polarity mech-
sion-based inhibition, tension can effectively propagate through
the cell even when the cellular cross-section is small. This ability
could be physiologically important as leukocytes frequently have
small cross-sections as they crawl through tight spaces in vivo,
We suggest that tension acts as a long-range inhibitor in the
following manner. First, pseudopod protrusion increases tension
in the plasma membrane (as we observe with our optical trap
measurements in Figure 5). This tension rapidly propagates
throughout the cell to act as a long-range inhibitor of leading-
edge formation. In support of this hypothesis, increases in
tension suffice for long-range inhibition of Rac activation and
protrusion (Figure 6), and decreases in tension expand leading-
edge activities (Figure 7). During cell polarization, tension only
becomes significant after the front has formed, by which point
positive feedback enables the existing front to maintain itself.
Furthermore, because the front is the source of tension, any fluc-
tuations in front size are immediately balanced by compensatory
changes in tension levels. The observation that tension spatially
restricts signals such as Rac to the leading edge differentiates
our model from purely mechanical (signaling-independent)
models of polarity.
the membrane (due to exocytosis) dissipates tension over time,
fail. Importantly, others have shown that neutrophil membrane
tension remains high long after stimulation (Shao and Xu, 2002)
and that persistent deformations cause persistent increases
in membrane tension (Herant et al., 2005). Resting neutrophils,
like many immune cells, have numerous small wrinkles in their
plasma membrane that act as a reservoir that doles out plasma
membrane as membrane tension increases during cellular defor-
unfolding of wrinkles is energetically unfavorable yet reversible,
membrane tension remains high as the cell tries to restore its
wrinkles (Herant et al., 2005). Thus, the membrane can transmit
long-range inhibitory tension in the neutrophil as in other highly
motile cells like keratocytes (Keren et al., 2008).
Tension Could Collaborate with Diffusion-Based
Systems to Guide Migration
We have shown that membrane tension restricts protrusion to
the leading edge, thereby allowing the neutrophil to polarize
and migrate. However, it is likely that other signaling systems
align migration with external cues. Latrunculin-treated cells,
which cannot polymerize actin and protrude, can still align
internal signals such as PIP3 with external chemoattractant
gradients (Janetopoulos et al., 2004; Servant et al., 2000).
Thus, cells can interpret chemical gradients without protrusion-
based increases in tension. However, neutrophils require F-actin
to polarize leading-edge signals such as PIP3 production in
response to uniform chemoattractant (Wang et al., 2002). Fur-
thermore, multiple patches of front signals coexist in latruncu-
lin-treated Dictyostelium cells exposed to multiple sources of
chemoattractant, whereas a single patch of activity dominates
in untreated cells (Devreotes and Janetopoulos, 2003; Janeto-
poulos etal.,2004).Thus,althoughgradient alignmentcan occur
without actin, important parts of the polarity program (polarity in
response to uniform chemoattractant, single site of polarity in
reflecting the role of tension. Although diffusion-based inhibitors
may collaborate with tension for polarity in response to uniform
chemoattractant, we suggest that tension is the dominant inhib-
anisms fail (tethered cells), and decreasing tension interferes
with the restriction of protrusive signals to the leading edge.
Tension Antagonizes Protrusion in Many Cell Types
Both cytoskeletal and membrane tension are capable of trans-
mitting forces over long range (Dai and Sheetz, 1999; Keren
Figure 5. Membrane Tension Increases during Protrusion
(A) Schematic outline of membrane tension measurement experiment. The tension in the plasma membrane can be measured by pulling a thin tube of membrane
from the cell surface with an adhesive polystyrene bead in an optical trap. Increases in membrane tension result in higher pulling forces on the bead. We
hypothesized that cell spreading, induced by uniform fMLP addition, should cause the membrane tension to increase. As a control, we flow in buffer, which does
not induce spreading and should not increase membrane tension.
(B)Pulling force over time for arepresentative cell.For primary human neutrophils, thetube wasfirst pulled to alength of ?2microns (pull1,arrow, light green bar)
andheldtherebriefly(hold1,light bluebar).Thetubewasthenextended toalengthof?10microns(pull2,arrow,darkgreenbar) andheldthere(hold2,dark blue
bar) before fMLP (arrow) was flowed in. The colored bars denote the time period over which the forces were averaged for the graph in (D); these regions were
selected to avoid sudden force jumps. Addition of fMLP caused the cell to spread and the pulling force to increase dramatically (red bar). The inset graph shows
response. Bright-field images of the cell, with the outline superimposedin yellow,are shown below. The tether position, determined with afluorescent membrane
dye (DiI), is also superimposed in yellow. The scale bar is 5 microns.
(C) Pulling force over timefor individual cellsfollowing buffer addition or fMLP stimulation.The leftpanel shows theforce traces of tubes held atconstantlengthas
buffer is flowed through thechamberto control for theeffects of flow on theforce measurements. The right panel shows the force traces of tubes held at constant
length as the cells were stimulated by flowing fMLP through the chamber. In both panels, flow begins at the beginning of each trace.
force values and standard errors, respectively. After fMLP addition, the cell spreads and the force increases dramatically (p = 0.0006) and briefly plateaus (post-
spread, red bar in B) before the tube detaches from the bead.
See also Figure S4 and Movie S4.
Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc. 183
Figure 6. Increasing Tension with Aspiration Reversibly Inhibits Leading-Edge Protrusion and Signaling
(A) Outline of experiment. Schematic showing the predicted results of aspiration experiments for a long-range inhibitor based on cell tension. The deformation of
the cell due to aspiration increases tension, which would be predicted to inhibit protrusion and reduce SCAR/WAVE complex recruitment.
184 Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc.
et al., 2008; Mayer et al., 2010) to inhibit cell protrusion.
Membrane tension is the loading force that growing actin fila-
ments fight in order to protrude the membrane (Keren et al.,
2008). Tension in the cytoskeleton (arising from myosin contrac-
tion) can also antagonize actin-based protrusion by pulling
actin filaments away from the membrane, thereby reducing the
amount of protrusion generated by a given amount of actin
assembly (Cai et al., 2010).
In fibroblasts, both cytoskeletal and membrane tension limit
cell protrusion. Increasing membrane tension halts spreading,
whereas decreasing membrane tension enhances the rate of la-
mellipodial protrusion and transiently causes uniform spreading
(Raucher and Sheetz, 2000). Decreasing cytoskeletal tension
(through myosin inhibition) causes faster spreading and a
larger finalspreadarea (Cai etal.,2010).Furthermore, increasing
tension with biaxial cellular stretching downregulates Rac
activity (Katsumi et al., 2002).
For fish keratocytes, membrane tension acts as a long-range
coupling mechanism for cell polarization (Keren et al., 2008;
Kozlov and Mogilner, 2007). Protrusion in one location pro-
motes retraction in other locations and vice versa due to
changes in membrane tension. Actin polymerization in a mem-
brane bag reproduces the wide range of morphologies ob-
served by keratocytes and accurately predicts quantitative
relationships between migration speed and morphology without
requiring free parameter fitting (Keren et al., 2008). Decreases in
cytoskeletal tension (through the myosin inhibitor blebbistatin)
do not destroy keratocyte polarity and only slightly reduce their
speed, suggesting that the plasma membrane (not the
cytoskeleton) carries the important tension in this system.
Although keratocyte motility has been primarily considered
as a pure mechanical system (with no need for signaling inputs),
a graded distribution of actin assembly is necessary for the ex-
isting models of keratocyte motility (Barnhart et al., 2011; Keren
et al., 2008). In light of our findings, it will be interesting to
examine leading-edge signals in keratocytes and deter-
mine whether membrane tension restricts them to the front
In Dictyostelium, cytoskeletal tension plays an important role
in restricting signals to the leading edge. Traction-force micros-
copy experiments have identified large myosin-based cytoskel-
etal tension increases during Dictyostelium migration (Meili
et al., 2010). Genetic deletion of Myosin II reduces cytoskeletal
tension dramatically (Pasternak et al., 1989) and increases
lateral pseudopod production (Wessels et al., 1988) and Ras
activation (Lee et al., 2010). These data strongly support a role
for cytoskeletal tension in Dictyostelium polarity. Whether
plasma membrane tension also plays a role in Dictyostelium
polarity is unknown.
In neutrophils, membrane tension appears to be the dominant
inhibitory mechanism for cell polarization. We find that mem-
brane tension increases during neutrophil protrusion and that
decreasing membrane tension results in the expansion of
leading-edge signals and a loss of polarity. In contrast, we find
that decreasing cytoskeletal tension with myosin inhibition has
no effect on leading-edge signals, though myosin inhibition
potentiates leading-edge signaling increases observed in hyper-
redundant with membrane tension in restricting leading-edge
Possible Mechanism of Tension Sensing
Our experiments suggest that membrane tension acts as a long-
range inhibitor of protrusion in migrating neutrophils. How do
cells sense and respond to tension? Tension-gated ion channels
in the plasma membrane could transduce membrane tension
into an inhibitory signal. Another potential mode of tension sen-
sation relies on the properties of the actin nucleation machinery
in neutrophils. The SCAR/WAVE complex forms multiple propa-
gating waves that organize the neutrophil leading edge (Weiner
et al., 2007) and extinguish if mechanical barriers prevent them
from protruding. Increases in membrane tension could similarly
extinguish waves by antagonizing protrusion. As the SCAR/
WAVE complex is required for Rac activation in neutrophils
(Weiner et al., 2006), the destruction of waves by membrane
tension also inhibits Rac activation. Because cytoskeletal ten-
sion also antagonizes protrusion, it might inhibit signaling via
SCAR/WAVE complex dynamics as well. Why don’t protrusions
at the leading edge extinguish themselves through increases in
tension? Perhaps waves at the leading edge preferentially
survive because of their high density, which could enhance
survival by activating Rac or by creating a strong actin network
that can protrude against the load provided by membrane
tension, similar to how membrane tension limits protrusion to
areas of high actin density in keratocytes (Keren et al., 2008).
Tension-based polarization mechanisms appear to operate in
a wide range of migratory cells, although though the sources
of the tension (protrusion versus contraction) and structures
bearing the tension (membrane versus cytoskeleton) can vary
from one cell type to the next.
(B) Aspiration induces pseudopod retraction. Aspiration of the trailing edge acts as a long-range inhibitor of protrusion. Left: a graph of the spread area over time
during aspiration. The spread area decreases dramatically upon aspiration and then eventually rebounds after release. Tick marks indicate bright-field frames
shown at right. Right: bright-field images of the same cell. The tip of the aspirated cytoplasm is shown with ablack arrowhead.The pseudopod (white arrowhead)
dies and retracts shortly after aspiration. When the aspirated cytoplasm is released, a new pseudopod forms with a delay of about 100 s.
(C) Aspiration inhibits SCAR/WAVE complex recruitment. Top: A crawling neutrophil expressing the SCAR/WAVE complex reporter Hem-1-YFP is shown before
(?15 s and 0 s) and during micropipette aspiration. The black arrowhead in the bright-field image denotes the portion of the cell aspirated into the pipette.
experiments (n = 10); aspiration begins at frame seven (arrow). Error bars depict the standard errors of the mean (SEM).
(D) Aspiration inhibits Rac activity. A crawling neutrophil expressing the Rac activation reporter PAK-PBD-YFP is shown. The fluorescence channel shows PAK-
PBD-YFP visualized in TIRF mode. Each bright-field frame shows the portion of the cell aspirated into the pipette for the current (black arrowhead) and previous
(white arrowhead) frames. Aspiration-mediated increases in cell tension result in a dramatic decrease in Rac activation in 85% of cells (n = 27). Rac activation
returns upon the release of aspiration pressure 65% of the time. The micropipette diameter is approximately 3 microns.
See also Figure S5 and Movie S5.
Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc. 185
C Quantification of tension reduction effects on signaling
0 1020 3040 5060
Fold increase in wave area
Blebbistatin + Hypertonic
B Combination of hypertonic buffer and blebbistatin causes uniform SCAR/WAVE complex recruitment
A Blebbistatin treatment causes cellular elongation but no enhancement of leading edge signaling
Figure 7. Membrane Tension Reduction Causes Expansion of Leading-Edge Signaling
(A) Blebbistatin treatment causes cellular elongation but no enhancement of leading-edge signaling. Top: A crawling neutrophil expressing the SCAR/WAVE
reduces cytoskeletal tension. The cells become elongated, but SCAR/WAVE complex recruitment does not expand beyond the leading edge. SCAR/WAVE
complexrecruitment decreases atlatertime points,likelyduetoelongation-induced increases inmembranetension. Bottom:Bright-fieldimages ofthesamecell
to visualize morphology. The scale bar is 10 microns.
(B) Combination of hypertonic buffer and blebbistatin causes uniform SCAR/WAVE complex recruitment. Top: A blebbistatin-treated (100 mM) neutrophil ex-
pressing the SCAR/WAVE complex reporter Hem-1-YFP (visualized in TIRF mode, shown as a heat map) is shown before (?20 s and ?10 s) and during (5 s, 15 s
and 475 s) application of hypertonic buffer (150 mM sucrose + 100 mM blebbistatin), which reduces membrane tension. Reduction in membrane tension causes
SCAR/WAVE complex recruitment throughout thecell. Bottom:Bright-field imagesofthesamecelltovisualizemorphology. Notetheuniformspreadingbetween
the 15 s and 475 s time points. The scale bar is 10 microns.
(C) Quantification of tension reduction effects on signaling. Quantitation of Hem-1-YFP recruitment during treatment with either hypertonic buffer + blebbistatin
(red, n=24),hypertonicbufferalone(blue,n=28),orblebbistatinalone(green,n=12).ThenumberofpixelscontainingHem-1signal werequantifiedateachtime
point (see Experimental Procedures) and normalized to the pretreatment signal. Error bars depict SEM.
See also Figure S6 and Movie S6.
186 Cell 148, 175–188, January 20, 2012 ª2012 Elsevier Inc.
HL-60 cells were generated, cultured, and differentiated as described
in Weiner et al. (2007). Primary neutrophils were obtained by pinprick as
described in Weiner et al. (2006).
Cell Severing and FRAP
Tethered cells were generated by brief heat shock as in Malawista and De
Boisfleury Chevance (1982). Cell severing and FRAP were performed with
a 435 nm dye cell laser (Manually Controlled Micropoint System, Photonic
Cell aspiration was performed with a heat-polished microneedle (3 mm diam-
eter) positioned with a Narishige MM-89 micromanipulator with fine hydraulic
control. Suction pressure was controlled with a Narishige IM-300 microinjec-
Bright-field, epifluorescence, and TIRF experiments were performed on Nikon
TE-2000 and Ti microscopes.
Membrane Tension Measurements
Membrane tubes were pulled with 2 mm ConA-coated beads positioned by
the distance between the bead and the center of the trap (see Extended
Cytoskeletal and Membrane Tension Perturbations
For hypertonic treatment experiments, we added an equal volume of buffer +
300 mM sucrose. To inhibit myosin, we added blebbistatin to 66 mM
final concentration. For combined treatment, we pretreated with 100 mM bleb-
bistatin for 10 min prior to adding hypertonic buffer. For hypotonic treatment
experiments, we added an equal volume of hypotonic buffer (H2O + 1 mM
MgCl2+ 1.2 mM CaCl2).
Described in the Extended Experimental Procedures.
Data are presented as mean ± standard error of the mean (SEM), and
a Student’s t test (two-tailed distribution, two-sample unequal variance) was
used to calculate p values.
Supplemental Information includes Extended Experimental Procedures, six
figures, and six movies and can be found with this article online at doi:10.
We thank our labs, Henry Bourne, John Clements, Robert Hochmuth, Dyche
Mullins, and Michael Springer for helpful discussion and a critical reading of
the manuscript. Support: AHA Predoctoral Fellowship (A.R.H.), NSERC
PostdoctoralFellowship (A.J.), DBI-0619674(E.R.D.), U54 RR022232 (O.D.W.,
E.R.D.), NSF-DMS0705431 (S.B.A.), GM081549 and Welch Foundation I-1644
(L.F.W.), R01 GM071794 and Welch Foundation I-1619 (S.J.A.), and Searle
Scholars Award and GM084040 (O.D.W.).
Received: June 17, 2011
Revised: September 15, 2011
Accepted: October 24, 2011
Published: January 19, 2012
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