Fisher JL, Levitan I, and Margulies SS. Plasma membrane surface increases with tonic stretch of alveolar epithelial cells. Am. J. Resp. Cell Mol Biol 2004:31:200-208.

Fisher JL, Levitan I, and Margulies SS. Plasma membrane surface increases with tonic stretch of alveolar epithelial cells. Am. J. Resp. Cell Mol Biol 2004:31:200-208.
Plasma Membrane Surface Increases with Tonic Stretch of
Alveolar Epithelial Cells
Jacob L. Fisher, Irena Levitan, and Susan S. Margulies
Department of Bioengineering, Department of Pathology, and Institute for Medicine and Engineering, University of Pennsylvania,
Philadelphia, Pennsylvania
Cyclic stretch stimulates numerous responses in alveolar epithelial
cells—some beneficial, some injurious—often through mechanosensitive membrane-associated proteins such as stretch-activated
ion channels. Tonic stretch, in contrast, stimulates only some of
these responses. In this study, we hypothesized that the plasma
membranes of alveolar epithelial cells expand during tonic stretch,
not only through cell surface unfolding, but also through recruitment of additional phospholipids. Such plasma membrane expansion would reduce membrane tension and decrease stimulation of
mechanosensitive membrane proteins. Primary rat alveolar epithelial cells were isolated, cultured for 48 h, and stretched between 3
and 40% change in basal membrane surface area. Gross changes in
total cell surface area were obtained from stacks of thin fluorescent
confocal micrographs; fine changes in plasma membrane area were
measured via whole cell capacitance. A 1:1 correspondence linked
changes in basal and total cell surface area, implying that cell surface
area change is dominated by stretch of the attached basal surface.
We also found that plasma membrane increased proportionally
with surface area within 5 min of tonic stretch, showing that, given
time to occur, plasma membrane expansion via lipid recruitment
preponderates the changes in cell surface shape and size demanded
by stretching the cell. Similarly, in cells tonically stretched 10 min
to allow lipid insertion and then returned to an unstretched state,
reabsorption of excess lipid occurred within 5 min. Finally, we found
that lipid insertion induced by tonic stretch was unaffected by
F-actin disassembly, ATP depletion, and calcium deprivation.
Stimulated by cyclic stretch, alveolar epithelial cells remodel
their actin cytoskeletons (1), release cytokines (2), traffick ionpumping proteins to the cell surface (3), secrete surfactant (4),
accelerate type 2 to type 1 differentiation (5), modulate their
gene expression, induce PKC activation and DNA synthesis (6),
and decrease occludin and tight junction protein (7). Although
the intracellular signaling pathways associated with these responses have been explored at length, they are diverse and complex, and generally remain to be fully elucidated (8). Equally
unclear is how cells initially perceive the mechanical strain that
triggers these signaling cascades. Nevertheless, two obvious
hypotheses are widely considered: (i ) mechanical stimuli open
stretch-activated ion channels (SACs) in the plasma membrane,
triggering intracellular signal cascades; and/or (ii) mechanical
signals are transduced directly through focal adhesions and the
cytoskeleton, stimulating intracellular processes.
(Received in original form June 16, 2003 and in revised form March 10, 2004)
Address correspondence to: Susan S. Margulies, Ph.D., Department of Bioengineering, University of Pennsylvania, 3320 Smith Walk, Philadelphia, PA 19104-6392.
E-mail: margulie@seas.upenn.edu
Abbreviations: change in basement membrane surface area, ⌬BSA; change in gross
cell surface area, not including membrane folds, ⌬CSA; cell surface area, coarsely
measured, not including membrane folds, CSA; mechanosensitive membraneassociated protein, MMAP; stretch-activated channel, SAC; ventilator-induced
lung injury, VILI.
Am. J. Respir. Cell Mol. Biol. Vol. 31, pp. 200–208, 2004
Originally Published in Press as DOI: 10.1165/rcmb.2003-0224OC on March 11, 2004
Internet address: www.atsjournals.org
Given currently available tools and technology for probing
mechanically stimulated cells, SAC-associated signaling has been
studied more often in alveolar epithelial cells, a trend continued
in this communication. By blocking SACs with toxins such as
gadolinium, investigators have inhibited calcium and sodium
influxes known to trigger numerous stretch-associated signaling
pathways in alveolar epithelial cells (8, 9). Studies have also
shown that whereas some stretch-induced functions such as surfactant release respond to either cyclic (4) or static stretch (10),
other effects, such as stretch-induced cell death (11) or stretchinduced Na⫹/K⫹-ATPase trafficking (3), are much less responsive to tonic or do not respond to tonic stretch at all. These
findings indicate that some stretch-induced responses are not
transduced as potently during tonic stretch as in cyclic stretch.
Perhaps membrane tension arising from cyclic deformations rises
and falls too quickly for the plasma membrane to remodel. Although we do not test this possibility directly in this study, we
test an alternative hypothesis that, with tonic stretch, the plasma
membrane gradually expands by insertion of additional phospholipid membrane from an intracellular reservoir, reducing membrane tension below the threshold for stimulating particular
membrane-dependent stretch-induced responses.
Plasma membrane vesiculation (to restore tension) and vesicle recruitment (to relieve tension) are already known to occur
in a variety of cell types and artificial bilayer vesicles. In artificial
liposomes, a flaccid bilayer will bud and vesiculate until it becomes a sphere, minimizing surface area, the liquid/liquid interface, and hence free energy (12). Red blood cells also become
flaccid, vesiculate, and fragment after cytoskeletal disruption
(13). On the other hand, high membrane tension favors fusion
of shed vesicles with the parent membrane to lower the total
energy of the system (13). Confirming this theory, Raucher and
Sheetz identified a plasma membrane reservoir in chick embryo
fibroblasts (14). When membrane tethers were pulled, plots of
tension versus tether elongation reveal what the investigators
labeled an initial phase, an elongation phase, and an exponential
phase. In another study, Dai and coworkers found that membrane tension and cell surface area interact in a tightly regulated
feedback loop (15). In some cell types membrane remodeling
is rapid, and one can see vesicles merging with or being absorbed
from the plasma membrane with simple light microscopy (15).
In other studies lipid trafficking is slower and subtler, and has
been tracked by measuring total cell capacitance or visualized
with lipid dyes, such as FM 1–43 (16–20) or various sphingolipids
(21–23), and captured using confocal fluorescent microscopy.
In many of the studies cited above, total cell capacitance was
the preferred method of tracking changes in plasma membrane
area. In essence the lipid bilayer of the cell membrane is an
insulator that can bear a capacitive charge on its surface directly
proportional to its surface area. Thus, by piercing a cell with a
micropipette and measuring capacitance between the cytosol
and the extracellular milieu, one can assess the surface area of
the plasma membrane and changes in it with great sensitivity
(24, 25). Frequently this technique has been used to observe
endo- and exocytosis rates in secretory cells, as vesicles merge
Fisher, Levitan, and Margulies: Tonic Stretch Expands Plasma Membrane
with and retract from the plasma membrane (26–29), because
it is sensitive to small changes in cell surface area. Further,
because electrical charge distributes throughout even the smallest spaces, capacitance is an excellent means of measuring surface area regardless of the shape of the cell and cell membrane
ruffling. This independence of whole cell capacitance from the
ruffling or smoothness of the cell membrane is especially important in determining the membrane size of animal cells, which
generally possess a stable excess of membrane area used precisely for buffering rapid deformation and tension changes (13).
Other cells respond to deformation in two phases with initial
plasma membrane unfolding, followed by vesicular recruitment
(13, 30). Studies of bladder epithelial cells, for example, found
that the apical cell surface unfolds during initial bladder filling,
but accommodates further volume changes during the latter
phase of filling by insertion of cytoplasmic vesicles (30). In each
of these studies, measurement of total cell capacitance served
as an invaluable means of distinguishing between cell surface
expansion via unfolding and expansion via lipid insertion. With
this technique, a cell that swells or changes shape by membrane
unfolding will not change its capacitance, but its apparent area
will increase in light micrographs. On the other hand, if lipid
bilayer is added to the cell membrane during events like cell
stretch or exocytosis, capacitance, as well as apparent surface
area, will increase.
Alveolar epithelial cells can potentially expand via either
mechanism. On one hand, electron photomicrographs show that
alveolar epithelial cells possess a ruffled membrane (20). Furthermore, under normal physiologic function these cells are repeatedly stretched and released at a rate greater than known
lipid recruitment could take place, yet they do not rupture,
indicating that the deformation has likely been accommodated
by the more rapid mechanism of membrane unfolding. On the
other hand, Vlahakis and colleagues, using lipid staining methods, found that lipid trafficking occurred in statically deformed
alveolar type 2 cells (20). However, using these methods, increases in plasma membrane surface area cannot be assessed,
for the measured increase in lipid insertion may be accompanied
by increased lipid recycling. Indeed, tonically stretched alveolar
type 2 cells have been reported to increase rates of both exocytosis (20) and endocytosis (31). But to remodel the cell surface
in a way that relieves membrane tension, exocytosis must outpace endocytosis and the plasma membrane must undergo a net
expansion.
In this study we have hypothesized that the increase in alveolar epithelial cell lipid insertion observed during static stretch
results in net plasma membrane expansion, and we set out to
quantify this expansion. We rationalized that given the broad,
flat shape of alveolar epithelial cells, the cell surface area must
increase during imposed alveolar epithelial stretch to accommodate a 25% increase in basal surface of area. We assume that
this increase in cell surface area likely occurs through a combination of fast membrane unfolding and slower lipid insertion. The
studies presented here quantify changes in cell size, cell surface
area, and plasma membrane area to distinguish between plasma
membrane growth and unfolding during tonic stretch. This is
done by imaging stretched and unstretched cells using fluorescent laser confocal microscopy, calculating changes in cell volume and surface area, and measuring change in capacitance over
the plasma membranes of stretched and unstretched alveolar
epithelial cells. Additionally, we explore potential pathways and
dependencies of stretch-induced plasma membrane expansion
by depleting cellular ATP, disrupting the actin cytoskeleton with
latrunculin, eliminating extracellular calcium and sequestering
intracellular calcium. These broad spectrum disruptions were
chosen because ATP reserves, a dynamic, functional cytoskele-
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ton, and calcium transients have been shown to play critical roles
in other cellular insertion pathways.
Some of the results of these studies have been previously
reported in the form of an abstract (32).
Materials and Methods
Cell Isolation
Alveolar type 2 cells were isolated from male, Sprague-Dawley rats
(Charles River, Wilmington, MA) according to a previously described
protocol (11) approved by the University of Pennsylvania IACUC.
Briefly, animals were anesthetized with sodium pentobarbital (50 mg/kg,
injected intraperitoneally). The trachea was cannulated, the lungs mechanically ventilated, and the animal exsanguinated per abdominal aortotomy. The heart was pierced and the lungs were perfused via the
pulmonary artery. The lungs were subsequently excised and type 2 cells
were isolated using an elastase digestion adapted from Dobbs and
coworkers (33), in which the lungs are instilled and incubated with an
elastase solution (3 U/ml; Worthington Biochemical, Lakewood, NJ)
and then minced with a tissue sectioner (Sorvall, Newtown, CT). Cells
were filtered through a series of sterile progressively finer Nitex mesh
(Crosswire Cloth, Bellmawr, NJ), and plated on a suspension culture
dish coated with rat IgG (3 mg IgG per 5 ml Tris-HCl incubated
overnight and rinsed; Sigma, St. Louis, MO). After a 1-h incubation at
37⬚C, gentle panning lifted type 2 cells from macrophages and other
contaminating cells preferentially adhered to the culture dish. Finally,
cells were spun down and resuspended in MEM supplemented with
Earle’s salts, 10% fetal calf serum, and 25 ␮g/ml gentamicin (Life
Technologies, Rockville, MD).
Cell Culture
Alveolar type 2 cells were seeded at a density of 1 ⫻ 105 cells/cm2
on fibronectin-coated (42 ␮g/ml; Boehringer Mannheim Biochemicals,
Indianapolis, IN), flexible Silastic membranes (Specialty Manufacturing, Saginaw, MI) mounted in custom-made wells, which could be used
for cell stretching on a microscope stage. This created a nonconfluent
culture so that whole cell capacitance could be measured in individual
cells without the concern of conductive cell-cell connections artificially
inflating capacitance values. Cultures were maintained for 48 h at 37⬚C
under 5% CO2 in MEM supplemented with Earle’s salts, 10% fetal
bovine serum, and 25 ␮g/ml gentamicin (Life Technologies).
Laser Confocal Microscopy and Image Analysis
Before imaging, cells were loaded with calcein AM, a fluorescent cytosolic dye (30 min, 37⬚C, 5% CO2) in Dulbecco’s modified Eagle’s medium without NaHCO3, supplemented with 1% penicillin (1,000 U/ml)/
streptomycin (10 mg/ml) (Life Technologies) and 20 mM HEPES
(Sigma). Wells (n ⫽ 3) were then mounted in a custom-made stretching
device, and image slices were captured through the depth of 3–5 randomly selected unstretched cells in the field of view (per well) using a
z-step of 0.25–0.3 ␮m on a Nikon TE300/Bio-Rad Radiance 2,000 laser
confocal fluorescent microscope, a Nikon 40⫻/0.75 Plan-Fluor objective
and Bio-Rad LaserSharp software (Nikon Corporation, Melville, NY;
Bio-Rad Laboratories, Hercules, CA). Wells were then stretched to
25%⌬SA by means of a computer-controlled stepper motor pushing
an annular indenter against the Silastic membrane, on the side opposite
the attached cells. The same cells previously imaged in the unstretched
state were relocated and imaged again in the stretched position. Using
Scion Image (Scion Corporation, Frederick, MD) slice images were
median filtered to reduce background noise and to smooth noisy cell
boundaries. Cells were then traced and their cross-sectional area and
perimeter were measured using the Scion Image Particle Analysis function. From each image stack, cell volume was calculated by numerical
integration (Simpson’s Rule [34]) of cell cross-sectional areas over cell
height. Total cell surface area (CSA) was calculated by numerically
integrating conic section surface areas from each image slice over cell
height. From paired image stacks of the same cell stretched and unstretched, a relationship was determined between the change in basal
surface area (⌬BSA), determined from the area change of the largest,
most basal image slice, and the change in total cell surface area (⌬CSA)
calculated here.
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Capacitance Measurements and Comparisons
Previous studies have demonstrated that cell capacitance scales linearly
with plasma membrane area (25, 35). Using this relationship, investigators have tracked mechanically induced lipid insertion via whole-cell
capacitance measures in a variety of cell types (15, 25, 36–38). Some
investigators have used high resolution patch-clamp capacitance recording in the femto- and attofarad range to detect individual exocytotic
events (39, 40) and even to discriminate among different sized exocytotic vesicles according to capacitance step magnitude (41). Together
with confocal microscopy studies of cells before and after stretch, cell
capacitance data let us distinguish between cell surface expansion via
plasma membrane unfolding or net plasma membrane accumulation
via lipid insertion.
In preparation for experiments in room air, MEM was aspirated
from cells and replaced with Dulbecco’s modified Eagle’s medium without NaHCO3, supplemented with 1% penicillin (1,000 U/ml)/streptomycin (10 mg/ml) (Life Technologies) and 20 mM HEPES (Sigma). All
cells were kept at 37⬚C until the moment wells were mounted on the
microscope stage for measuring capacitance. Unstretched control wells
were mounted in the stretching device but not stretched. Wells in the
experimental stretch group were mounted in the stretch device and
stretched immediately by screwing the well down against an annular
indenter that pressed on the Silastic membrane on the side opposite
the attached cells, causing equibiaxial stretch of the Silastic membrane
(ⵑ 25% change in surface area) and the adherent cells (11).
Glass micropipettes were pulled to a resistance of 4–6 M⍀, loaded
with a solution of (in mM): 156 KCl, 1.5 CeCl2, 1 MgCl2, 10 HEPES,
1 EGTA, pH 7.3, and mounted over a silver electrode. A saturated
salt agar bridge placed in the extracellular medium served as a reference
electrode. Electrophysiologic measurements were made with an EPC9
amplifier and its companion Pulse software (HEKA Electronik, Lambrecht, Germany). Using a micromanipulator, the micropipette was
brought into contact with the cell membrane, and a seal was formed
using a small, manually applied negative pressure in the micropipette.
Electrode potential was set at ⫺60 mV and pipette capacitance was
compensated automatically in the Pulse software before perforating
the cell membrane by manually applying quick negative pressure pulses
through the patch electrode. Under optimal conditions a successful
patch could be made and perforated, and whole cell capacitance and
series resistance could be measured in 5 min. Thus, in stretched wells,
the earliest measurements were made 5 min after the stretch event. (In
unstretched wells, this lag was not as relevant, as there was no stretch
event.) Generally multiple cells were probed in each well, though no
well was kept on the microscope at room temperature for more than
40 min. Thus all measurements were made within 5–40 min of mounting
the well on the microscope, which coincided with the moment of stretch
for stretched wells. On average, one to six cells were successfully probed
in each of six to eight wells for each experimental group (three to four
stretched wells and three to four unstretched control wells). Exact
numbers of samples are given in the results of individual experiments.
In another set of experiments, seven cells were stretched tonically
for 10 min and then returned to an unstretched state before capacitance
was measured. Again, creating a perforated patch clamp required ⵑ 5
min so that the earliest measurements were made 5 min after the cell
was “unstretched.” In three of the seven cells, capacitance was recorded
for 5 min (i.e., the period from 5 min post-stretch to 10 min poststretch) to determine whether capacitance was still decreasing or if a
steady state had been reached. In the remaining four cells, the patchclamp did not last for an entire 5-min recording period.
After a cell was probed, a photomicrograph was captured using a
Nikon camera and Kodak ISO 100 print film. Micrographs were digitally
scanned and stored as TIFF arrays. Using Adobe Photoshop, cell boundaries were traced with the magnetic lasso tool, and contrast was enhanced between cells and background. Basal surface area was measured
in each contrast-enhanced image using the Scion Image Participle Analysis tool and then used to plot cell capacitance as a function of projected
cell surface area for stretched and unstretched cell groups.
Treating Cells
Previous studies have shown that vesicular trafficking in alveolar epithelial cells is sometimes dependent upon normal functionality and integrity of the actin cytoskeleton (42) and is often stimulated by increased
intracellular calcium, brought about by calcium influx into the cell or
intracellular calcium release (43). In other cell types, lipid insertion
and insertion into the plasma membrane has been shown to be ATPdependent (44, 45). In urothelial cells, which expand via initial plasma
membrane unfolding and subsequent vesicular insertion during bladder
filling, translocation of vesicles into and out of the apical membrane
was dependent on intact microfilaments but not an intact microtubule
system (46). ATP was also required for vesicle insertion during stretch
but not vesicle endocytosis during bladder collapse (47).
To test the dependence of tonic stretch-induced lipid insertion on
integrity of the actin cytoskeleton, additional capacitance experiments
were performed with cells that were pretreated for 1 h with latrunculin
A (100 nM; Sigma), which sequesters G-actin to prevent dynamic
F-actin remodeling. In other cells, ATP levels were depleted for 1 h
before study using the glycolytic metabolic inhibitors 2-deoxy-D-glucose (2 mM; Sigma) and antimycin A (10 ␮M; Sigma), which have been
shown to reduce intracellular ATP levels 45–90% in cultured epithelial
cells (48). Finally the effects of Ca2⫹ were tested first by stretching cells
in a Ca2⫹-free medium to isolate the effect of Ca2⫹ influx and next by
using Ca2⫹-free medium after also sequestering intracellular Ca2⫹ for
1 h using BAPTA-AM (50 ␮M; Molecular Probes, Eugene, OR). Capacitance was measured in 4–12 stretched and 4–12 unstretched cells in
each treatment group (exact numbers for each group given in results).
Images were captured and processed as described above for stretched,
untreated cells.
Data Analysis
In studies comparing changes in projected cell surface area to total cell
surface area, all data were captured from paired image stacks of the
same cells before and after stretch. The image slice that contained the
greatest cellular cross-sectional surface area was designated the basal
slice, and the cross-sectional area of the cell or cells in this slice was
defined as basal surface area. Also in the basal slice, where cell boundaries were most crisp, we selected a lower bound intensity threshold
to eliminate background signal, using the guide that it be as high as
possible without cropping any part of the cell image. This threshold
parameter was used in the Scion Image Particle Analysis tool for all
slices within a cell. Cell volume was calculated using Simpson’s Rule
(34) to calculate the area under the curve of slice area versus slice
height. Total cell surface area was calculated using Simpson’s Rule to
sum surface area sections of slice-by-slice conic sections.
To extract change in plasma membrane surface area from measured
changes in capacitance, we used a parallel plate capacitor relationship
between cell capacitance, C, and plasma membrane surface area, A:
C⫽ εA/d, where ε is the dielectric modulus of the plasma membrane
and d is membrane thickness. Energetic constraints limit change in
membrane thickness to d ⬍ 3.6% (13), and the only possible change
in dielectric modulus ε might be a small decrease resulting from rarefaction of the lipid bilayer, which would decrease measured capacitance.
Thickness and dielectric modulus being relatively constant, observed
increases in capacitance can be attributed primarily to increased plasma
membrane surface area, as established by other investigators (25, 35).
Because micropipette probing to measure capacitance is a destructive technique, we are unable to probe the same cell twice to measure
a change in capacitance with stretch. Hence, we must look for a change
in the capacitance versus size relationship between stretched and unstretched cells (Figure 1). If a group of cells is stretched and their
apparent surface areas increase by membrane unfolding without lipid
insertion (and without the consequent increase in capacitance), the
capacitance versus surface area regression line will shift to the right,
but not upward. In this case, the capacitance-area regression line in
the stretched cells would be distinct from that in unstretched cells.
However, if the surface area of stretched cells increases solely via lipid
insertion into the plasma membrane, the capacitance versus surface
area relationship will shift both to the right and upward, as both surface
area and capacitance increase. In this second limiting case, the capacitance-area regression line would coincide with the unstretched cells, as
if the stretched cell had the same capacitance and plasma membrane
surface area of a large unstretched cell, a state it can achieve after
stretch only by lipid insertion.
The standard statistical approach for detecting differences in two
regression lines is an analysis of covariance (ANCOVA). ANCOVA
Fisher, Levitan, and Margulies: Tonic Stretch Expands Plasma Membrane
Figure 1. Capacitance and surface area in two membrane
expansion models. If plasma
membrane expansion occurs via
membrane unfolding, the capacitance versus area relationship
for a group of cells (top panel,
gray solid line) will shift to the
right as cells grow larger with
stretch (top panel, black dashed
line). However, if the plasma
membrane does not unfold but
expands by adding additional
lipid, the line will shift both to
the right with stretch and upward with increased capacitance
(bottom panel, black dashed
line). If the lines coincide, as
shown here below, the relationship between surface area and
capacitance is conserved, meaning a stretched cell has increased the size
of its plasma membrane in proportion to its size and looks simply like
a larger unstretched cell. The coincidence of the stretched and unstretched cell plots, fit to real data with a certain degree of variability,
is tested by ANCOVA, which essentially measures the parallelism and
distance between two regression lines.
is used for testing a hypothesized difference between two sets of values
while factoring out the effect of a covariate. It is appropriate to use
ANCOVA to test a difference in capacitance between stretched and
unstretched cells while factoring out how capacitance varies with area
(the covariate in this example). Because we hypothesize that tonic
stretch stimulates lipid insertion rather than membrane unfolding, we
expect ANCOVA to detect no difference between stretched and unstretched groups. A null hypothesis can be accepted only after a power
analysis is performed to ensure that ANCOVA and the given data have
sufficient statistical power to detect a difference if a true difference
exists. To perform a power analysis, we simulated separation between
the stretched and unstretched data sets by increasing the gap in capacitance, by increasing stretch without adjusting capacitance, or by increasing capacitance in stretched cells between a pure unfolding model and
a pure lipid insertion model. We created power curves for each simulation. From these curves we determined the smallest change in capacitance, the smallest amount of stretch without capacitance, and the
smallest amount of lipid insertion without unfolding that can be detected
(at 80% power) given the collected data (W. J. Ewens, personal communication to J.L.F., Sept.–Oct. 2003), and thus constructed a confidence
interval for concluding the null hypothesis.
Results
Nonconfluent Cells Stretch Similarly to Confluent Cells
Confocal microscopy experiments in this study observed a cellular ⌬BSA of 25.0% ⫾ 10.6% (mean ⫾ SD) in nonconfluent cells
with a 25% ⌬SA stretch of the substratum Silastic membrane.
These results were statistically indistinguishable in both mean
(P ⬎ 0.5) and variance (P ⬎ 0.05) from results of previous studies
that found that confluent epithelial monolayers on a Silastic
membrane stretched to 25% ⌬SA changed by ⬇ 24% ⫾ ⬇ 7%
(mean ⫾ SD) (11). We did note, however, that larger cells with
greater basal surface area in contact with the underlying membrane stretched closer to 25% ⌬BSA with less variability than
smaller cells.
This study was performed on nonconfluent cells to avoid
artifacts in measurement of cell capacitance that are due to
electrical coupling between cells. To maintain consistent conditions throughout all experiments and to allow for result comparison, nonconfluent cells were used in all experiments. Analyses
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of the studies were paired so that differences between Silastic
membrane and actual cell stretch magnitudes did not affect ultimate results. In confocal microscopy studies correlating ⌬BSA
to ⌬CSA, each stretched cell was compared with its own unstretched image. In capacitance studies, capacitance was recorded in stretched and unstretched cells as a function of the
absolute value of basal surface area, not ⌬BSA. Thus, as long
as stretched cells received a different stimulus from unstretched
cells—and they did stretch on average 25.0% ⌬BSA—variation
around the 25% ⌬SA of the Silastic membrane is not an important detraction.
Increases in Overall Cell Surface Are Strongly Linked
to Changes in Basal Surface Area
In stretched cells we detected a very close correlation between
percent ⌬BSA and the percent ⌬CSA (Figure 2). Volume
changes, however, were more variable; some of the tested cells
(n ⫽ 5) increased their volume in proportion to basal stretch,
whereas other cells flattened (n ⫽ 4) during stretch, leading to
only small increases and, in some cells, even slight decreases in
volume (Figure 3). A geometric analogy, which helps to explain
the close match between ⌬CSA with ⌬BSA and also the mismatch between ⌬BSA and volume, considers alveolar epithelial
cell shape to be similar to a short cone with a broad base. The
surface area of a cone is the area of the base, A ⫽ ␲ r2, plus
the lateral surface, S ⫽ ␲ r (r2 ⫹ h2)1/2. Volume, V, is equal to
Ah/3, where r is the radius of the base and h is the height of
the cone. For a very short, very wide cone (r ⬎ ⬎ h), a change
in total surface area (that is, ⌬CSA in the cells in this study) is
nearly proportional to a change in the area of its base (⌬BSA),
but is affected little by change in height (⌬h). In contrast, volume
is equally dependent on both basal area and height. This general
concept holds for any geometric solid with a base dimension
much greater than its height. Cells in this study generally had
dimensions of r ⬇ 50–60 ␮m and h ⬇ 3–4 ␮m. Hence with
alveolar epithelial stretch, ⌬CSA was predominantly dependent
on imposed ⌬BSA, whereas volume changes were equally dependent on variable changes in cell height or thickness with stretch
and reflect that same randomness. Because the cell did not maintain constant proportions with stretch, it did not fulfill a twothirds power rule between ⌬BSA (or ⌬CSA) and ⌬V. In fact,
all volume increases were less than the two-thirds power rule
predictions.
Plasma Membrane Enlarges with Stretch
In cells stretched and held for 5 min or more (n ⫽ 14), the linear
relationship between capacitance and area remained statistically
indistinguishable from that of unstretched cells (n ⫽ 14) (Figure
4). Power analysis showed that for the given data and standard
criteria of 80% power and P ⬍ 0.05 to determine significance, any
increase in cell size ⬎ 7.27% ⌬BSA without a change in capacitance
would generate a statistically detectable and significant difference
Figure 2. Change in total cell surface
area versus change in basal surface
area. In stretched cells, the percent
⌬CSA was highly correlated with
percent ⌬BSA. This is due primarily
to the broad flat geometry of the cultured alveolar epithelial cell. This is
consistent for a geometric solid with
a base dimension much greater than
its height, where changes in total surface area are closely correlated with
changes in the area of the broad base.
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Figure 3. Change in cell volume
versus change in basal surface area.
Stretch-induced change in cell volume did not correlate with ⌬BSA,
primarily due to a lack of correlation between stretch and change in
cell height. Unlike overall CSA,
which varied directly with the basal
area (Figure 2) but remained virtually independent of fluctuating cell height, cell volume depended upon
both basal area and height changes. Some cells flattened with stretch,
but others did not, and this randomness in height changes is reflected
in volume changes.
between regression lines. Because cells were stretched to a 25%
⌬BSA increase and no difference was detected, we conclude
that stretched cells enlarged their area and also increased in
capacitance, which is possible only with plasma membrane
expansion. Our power analysis demonstrated that the detectable
limit was 74.6% lipid insertion; any combination with less insertion and more unfolding than 74.6% insertion/25.4% unfolding
would be detectable. Hence, although we cannot rule out the
possibility that up to 25.4% of the imposed ⌬CSA might be due
to unfolding, lipid insertion accounts for the clear majority of
⌬CSA after 5 min of tonic stretch. Because capacitance could
not be measured any sooner after stretch than 5 min, this finding
does not reflect the relative contributions of plasma membrane
unfolding and lipid insertion immediately after stretch. Nonetheless these findings do support the concept of lipids inserting into
the plasma membrane during prolonged tonic stretch.
Enlarged Plasma Membrane Contracts with 5 min of Unloading
The relationship between size and capacitance of stretched cells
that were released was similar to that of control cells that had
never been stretched. Recall that stretch increased the capacitance as well as size of the cells. Interestingly, upon release, both
cell area and cell plasma membrane volume decreased, and none
of the increased plasma membrane lipid inserted during tonic
stretch had been retained (Figure 5). These measurements demonstrate that stretched cells that were returned to their initial
size after a period of tonic stretch reabsorbed excess plasma
membrane and achieved a steady, restored initial state within 5
min. Current technique prevented measuring dynamic capacitance change earlier than 5 min after release from stretch. Capacitance measured over a subsequent 5 min in three stretched and
released cells did not change, indicating steady state had been
achieved.
Figure 4. Capacitance versus basal
surface area in stretched and unstretched cells. The relationship between basal surface area and whole
cell capacitance did not change significantly between stretched (⫹) and
unstretched (⫻) cells, as shown by the
statistically indistinguishable slope
and elevation of the regression lines.
This persistent relationship indicates that the plasma membrane expands
with stretch to account for increased cell surface. Hence, the plasma
membrane area of a tonically stretched cell is no different from that of
a larger unstretched cell. In contrast, if plasma membrane expansion
did not occur and the cell surface increased through membrane unfolding,
the stretched data would shift to the right as area increased without
shifting upward in capacitance.
Figure 5. Capacitance versus basal
surface area in stretched, unstretched and stretched and released
cells. Compared with stretched (⫹)
and unstretched (⫻) cells the relationship between capacitance and
surface area did not change in cells
that had been stretched for 10 min
and then unstretched (diamonds).
As shown at left, cells that were stretched for 10 min to allow lipid
insertion and then returned to their original size (diamonds) also returned to a lower capacitance within 5 min, reabsorbing membrane
rather than keeping the enlarged tonic stretch capacitance.
Lipid Insertion Independent of Ca2ⴙ, ATP Resources,
and Cytoskeleton Integrity
Although calcium fluxes, ATP-based energy resources, and an
intact cytoskeleton have been proved necessary for some cellular
insertion functions, perturbing them had no effect on stretchinduced insertion. In cells treated with latrunculin A (n ⫽ 8,
stretched; n ⫽ 4, unstretched) and cells depleted of ATP (n ⫽
9, stretched; n ⫽ 5, unstretched), the regression lines of stretched
and unstretched cells remained statistically indistinguishable
(Figures 6 and 7). As concluded in untreated cells, this consistent
relationship between cell capacitance and cell surface area between stretched and unstretched cells demonstrated that
stretched cells with larger basal surface areas had undergone
proportional plasma membrane growth via lipid insertion, rather
than surface unfolding. Furthermore, neither eliminating extracellular calcium (n ⫽ 12, stretched; n ⫽ 4, unstretched) nor sequestering intracellular calcium and eliminating extracellular calcium together (n ⫽ 6, stretched; n ⫽ 8, unstretched) had any
effect on cell capacitance before stretch or tonic stretch-induced
lipid insertion and plasma membrane expansion (Figures 8A
and 8B).
It was interesting to note, however, that after 1 h of G-actin
sequestration or ATP depletion, capacitances had increased significantly (P ⬍ 0.05 by ANCOVA) in treated cells relative to
untreated cells, whether examining the stretched or unstretched
condition.
Thus, whereas these two treatments promoted plasma membrane expansion before cells were stretched, they did not preclude further lipid insertion and plasma membrane growth with
tonic stretch.
Discussion
Alveolar epithelial cells are resilient to stretch by necessity.
Attached to the inside surface of repeatedly inflating and deflating alveoli, these broad, flat cells need to withstand repeated
deformations, which are generally small in a person breathing
at rest but can reach a 37% ⌬BSA during deep inspiratory
Figure 6. Capacitance versus basal
surface area after latrunculin treatment. In cells treated with latrunculin
to disrupt the actin cytoskeleton, the
plasma membrane expanded even in
unstretched cells (squares), as shown
by increased capacitance. When latrunculin-treated cells were stretched
tonically (triangles), capacitance increased further, indicating that
stretch-induced lipid insertion and plasma membrane expansion do not
depend upon the integrity of the actin cytoskeleton. Stretched (⫹) and
unstretched (⫻) untreated controls (from Figure 4) are shown for
comparison.
Fisher, Levitan, and Margulies: Tonic Stretch Expands Plasma Membrane
Figure 7. Capacitance versus basal
surface area after ATP depletion.
Like latrunculin treatment, ATP depletion brought about plasma membrane expansion even before cells
were stretched, but the treatment had
no effect on additional, tonic stretchinduced membrane expansion. Because ATP is required for actin
polymerization, ATP depletion could be causing the same effect as
latrunculin treatment. This finding also indicates that plasma membrane
expansion does not depend on ATP as an energy source for lipid insertion. Shown are stretched (triangles) and unstretched (squares) ATPdepleted cells, and stretched (⫹) and unstretched (⫻) untreated control
cells from Figure 4.
maneuvers. Mechanical ventilation in diseased or injured lungs
with inhomogeneous parenchymal mechanical properties and
surfactant dysfunction can lead to uneven distribution of inspired
gas with unusually high regional inflation and even greater, supraphysiologic epithelial strain (49). Frequently, such overinflation can trigger what is aptly dubbed ventilator-induced lung
injury (VILI), a syndrome whose symptoms can include pneumothorax, alveolar edema, changes in pulmonary mechanics and
lung cell function and, ultimately, lung cell death (49). VILI is
especially concerning in patients already suffering from acute
respiratory distress syndrome (ARDS), where VILI has an incidence of 5–15% and an associated mortality of 34–60% (50).
But even in normal lungs, VILI can occur with high tidal volume
mechanical ventilation (49). Therefore, how alveolar epithelial
cells and their plasma membranes routinely accommodate physiologic strains and the consequences of exceeding strain tolerances are particularly germane to understanding VILI.
Recently, Vlahakis and Hubmayr proposed four possible
ways in which plasma membrane could respond to stretch: (i )
stress failure, (ii) insertion of additional lipids from intracellular
stores to the plasma membrane, (iii) increased intramolecular
distance in the lipid bilayer, or (iv) plasma membrane unfolding
(51). They have identified the first two mechanisms in their
own studies under particular stretch conditions; stress failure
appeared when A549 alveolar epithelial cells were stretched at
high strain rates (52), and lipid insertion took place in tonically
stretched A549 cells (20). Energy considerations of phospholipid
hydrophobicity rule out increasing molecular distances as a significant factor. Biological membranes can only sustain elastic
expansion of ⬍ 3% before failure. The fourth proposal, plasma
membrane unfolding, remains a likely means of accommodating
Figure 8. Capacitance versus basal surface area in calcium-depleted cells.
Neither depleting calcium in the extracellular medium alone (A ) nor depleting extracellular calcium and sequestering intracellular calcium (B )
affected cell capacitance. Cell capacitance increased with cell size in
tonically stretched cells regardless of
calcium depletion, indicating that tonic
stretch-induced lipid insertion occurs
independent of calcium levels or signaling. Shown are stretched (triangles)
and unstretched (squares) calciumdeprived cells, and, for comparison,
stretched (⫹) and unstretched (⫻) untreated control cells (from Figure 4).
205
membrane stretch at medium strain rates, which are not so explosive as to cause stress failure, but are still faster than lipid insertion alone could accommodate. It is also possible that even
after 5 min of tonic stretch, when we observed lipid insertion,
membrane unfolding still could account for up to 25% of the
total cell surface increase and remain statistically undetectable
in our data. By measuring changes in membrane capacitance,
the present study not only used a new technique to confirm that
lipid insertion occurs during tonic stretch of alveolar epithelial
cells in primary cultures but also showed that stretch stimulates
a net plasma membrane expansion sufficient to account for at
least 75% of the entire increase in cell surface area. Because of
the time required to take a capacitance reading after stretch,
our findings cannot predict relative contributions of membrane
unfolding and lipid insertion in the short term. In fact, because
lipid insertion cannot occur as quickly as unfolding, unfolding
is probably more important in buffering the immediate cell response to stretch. Nevertheless within 5 min of tonic stretch,
membrane expansion via lipid insertion accounts for the majority
of surface area changes, relieving plasma membrane stress and
potentially allowing the cell surface to refold (Figure 9).
Under various physiologic conditions across cell types, plasma
membrane expansion via lipid insertion appears to serve as a
protective buffer against high membrane tension and potential
stress failure. The ultimate strain capacity of biological membranes is ⬍ 3%, or ⬍ 6.1% ⌬SA under equibiaxial stretch. Hence
the plasma membrane must be supplemented with additional
phospholipids to avoid stress failure during any cell deformation
beyond this limit. Neuron, muscle, and epithelial cells have all
been shown to add membrane from intracellular stores when
swollen by osmotic perturbation and to reabsorb membrane
upon reshrinking. Laser tweezers can be used to pull a membrane
tether from a cell and monitor relaxation and recovery of membrane tension during such events. With osmotic shrinking and
swelling, membrane tension and surface area appeared to be
linked in a feedback mechanism that tightly constrains the range
of membrane tension. In similar studies, tethers pulled from
fibroblasts recorded an initial increase in tension as the tether
formed, but then tension remained constant as the tether was
pulled further at a constant rate. Only at very high strains did
tension begin to increase again (14). This plateau in which strain
increased with no increase in tension is attributed to lipid insertion into the plasma membrane from a tension-buffering reservoir. In attached alveolar epithelial cells, stretching the basement
membrane stimulated lipid insertion to the cell surface, detected
Figure 9. Lipids are inserted
during tonic stretch and account for the greatest part
of surface area increases. In
summary, our results confirmed that tonic stretch results in lipid insertion and
plasma membrane expansion. In this series of cartoons, the cell begins unstretched. Because apparent
surface area increases faster
than lipid insertion is known
to occur, surface unfolding is presumed, though we have not demonstrated
that in this study. Then, according to our results, additional lipid is added
to the plasma membrane, increasing the cell surface area. Within 5 min
the plasma membrane has expanded and refolded such that the membrane
capacitance of a stretched cell appears little different from that of a large
unstretched cell. This phenomenon persists even after actin derangement,
intracellular ATP depletion, or calcium sequestration.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 31 2004
by dye dilution in the plasma membrane (20). Although this
prior study did not discriminate between net lipid insertion and
lipid recycling, which could possibly produce no net plasma membrane expansion, the results of the present study show that the
plasma membrane does expand and that this expansion accounts
for at least 75% of the total stretch-induced change in cell surface
area.
Our finding that alveolar epithelial plasma membrane expansion takes place within 5 min of tonic stretch also sheds light on
the reported discrepancy between functional sensitivity to cyclic
and tonic stretch stimuli. Previously we found that Na⫹-K⫹ATPase activity is stimulated by 1 h of cyclic stretch, and that
this stimulus was significantly dependent upon SAC function
(3). One hour of tonic stretch, in contrast, had no effect. The
present study clarifies that tonically stretched plasma membrane
expands via lipid insertion within the first 5 min of stretch. In
cyclic stretch, the opening of SACs is made energetically favorable by cyclically repeating increases in membrane tension. But
as lipids insert and the plasma membrane expands to relieve
membrane tension, SAC opening would become energetically
unfavorable. Stretch-induced cell death, and other cell functions
that exhibit a similar contrast between cyclic and tonic stretch
responses, may also be related to SACs or other MMAPs, which
are no longer stimulated once lipid insertion occurs in tonic
stretch. Meanwhile, cell functions such as surfactant secretion,
which are stimulated by either cyclic or tonic stretch, are probably not signaled through plasma membrane tension and MMAP
stimulation, but rather through other potential stretch transducers such as the cytoskeleton.
Unlike many cellular insertion mechanisms, our results demonstrate that stretch-induced lipid insertion is not dependent
on calcium, ATP availability, or cytoskeletal integrity. Similar
calcium-independent, tension-regulated lipid insertion is found
in plant cells (53) and osmotically swollen neurons (37, 54).
Morris and colleagues argue from cellular energy economy that
a tension-regulated lipid insertion mechanism is not only experimentally evident but also theoretically appealing (35). Rather
than depend upon dissipating ion gradients and complex signaling pathways, high membrane tensions could act as both signal
and potential energy source for lipid insertion. This could also
explain the null effect of ATP depletion on stretch-induced capacitance increases; if tension provides the necessary well of
potential energy, insertion would not have to depend upon another cellular energy source, like ATP.
Although cytoskeleton disruption with latrunculin and ATP
depletion did not affect plasma membrane expansion with
stretch, they did bring about a capacitance increase even in
unstretched cells. Disassembly of the cytoskeleton has previously
been shown to increase cell capacitance (55–57) and decrease
membrane tension (58, 59). Two mechanisms have been proposed for this. On one hand, breakdown of the cortical cytoskeleton is thought to enhance exocytosis by allowing freer movement
of vesicles below the cell surface (55–57). On the other hand,
disassembly of the actin cytoskeleton prevents the structural and
mechanical foundation for bulk membrane reabsorption through
vesicular endocytosis (39, 40, 54). Regardless of latrunculin treatment leading to inhibition of bulk retrieval, endocytosis and
exocytosis rates were found to remain normal (40). Because
ATP hydrolysis is required for actin polymerization (60), ATP
depletion could produce the same results. Capacitance increases
recorded in this study once cells were treated with latrunculin
or a combination of 2-deoxy-D-glucose and antimycin A can
presumably be attributed to the same mechanism. It is also worth
noting that after treatment and a resultant capacitance increase,
cells still responded to stretch with additional lipid insertion.
Thus it appears that although the potential of stretch increasing
tensions and stimulating insertion might be buffered by previous
expansion, lowered resistance to lipid insertion caused by cytoskeletal breakdown might have permitted response to lower
tension stimuli.
One topic for further consideration is the interplay between
plasma membrane unfolding and lipid insertion over time. Along
with lipid insertion, unfolding of a convoluted cell membrane
could be a crucial protection against high membrane tension.
Mast cells inflated with hydrostatic pressure via micropipette
quadrupled their volume without rupturing and with only slight
changes in cell capacitance, indicating a substantial capacity of
membrane unfolding to buffer lytic tensions (61). Viewed with
an electron microscope, alveolar epithelial cells are convoluted
and ruffled, suggesting a similar buffering capacity. Yet, unlike
inflated mast cells, alveolar epithelial cells also insert lipids and
increase their capacitance to match their size. Unfortunately,
it is presently impossible to record capacitance changes in an
attached cell as it stretches without tearing the cell membrane
or losing the patch. The movement of a cell during stretch is
mesoscopic relative to the microscopic size of the cell and the
patch electrode, so that during stretch a cell might move tens
of cell diameters away from the micropipette. Thus cells must
be first stretched and then patched. In this study, the fastest
patching and capacitance recordings were performed ⵑ 5 min
after stretch, at which time, we observed, full expansion had
already taken place. Using fluorescent dyes, Vlahakis and colleagues also reported that lipid trafficking had reached a steady
state in alveolar epithelial cells within 60–90 s after stretch (20).
Thus it appears that lipid trafficking occurs and possibly relieves
tensions in stretched cells within a window of ⬍ 1 min.
To date, it is known that stretch stimulates a variety of responses in alveolar epithelial cells. Some are beneficial, so we
seek to enhance them. Others are injurious, so we seek to avoid
them. Less clear is how these stretch-induced responses, good
or bad, are affected by the rates at which epithelial cells are
stretched and the consequent fluctuations in membrane tension.
Some studies addressed cell injury and plasma membrane rupture when cells are stretched a very high strain rates, and how
they recover afterward (53, 62). It has also previously been
reported that cells stretched at 60 cycles/min sustain greater
injury than cells stretched more slowly at 15 cycles/min (63).
But less is known about even lower stretch frequencies between
15 cycles/min and the 0 cycles/min of tonic stretch. Understanding the specific stimuli and responses during the tension-bearing,
membrane unfolding, and lipid insertion phases of stretch could
elucidate how the alveolar epithelial cell plasma membrane responds to various stretch frequencies. It is currently clear that
stretch at a rate of 15 cycles/min stimulates a number of cell
functions, and at least some of these functions are decreased or
abolished by blocking SACs. But perhaps cells could also be
stretched cyclically but so slowly that lipid insertion can take
place during stretch, avoiding membrane tension increases. Although this rate may well be below what is needed to supply
adequate ventilation to a mechanically ventilated patient, it may
be possible to superpose low-volume, high-frequency waves with
low-frequency recruitment maneuvers. Ultimately, such research
might enable the identification of ventilation strategies that could
target desired plasma membrane tensions, which might be associated with healthy alveolar epithelial cell responses to stretch,
while reducing harmful tension levels that might overstimulate
or even rupture the cell’s plasma membrane.
As a first step toward such strategies, this study has found
that alveolar epithelial cells undergo changes in shape and size
during stretch, and, when that stretch is held tonically, cells
expand their plasma membrane through lipid insertion to accommodate at least 75% of stretch-induced changes in cell surface
Fisher, Levitan, and Margulies: Tonic Stretch Expands Plasma Membrane
area within 5 min. This suggests that stretching cells over a period
of 5 min or more might allow lipid insertion that could prevent
high membrane tensions and possibly temper the cellular perception of stretch. However, we have also found that when released
from tonic stretch, cells also reabsorb excess membrane within
5 min, meaning that tonic stretch induced relaxation is transient
if stretch is not maintained. We have also determined that
stretching the alveolar epithelial by stretching its adherent basement membrane to a certain percent change in surface area
enlarges the entire cell surface by the same percent increase. This
valuable one-to-one relationship between basal surface increase
and whole cell surface area increase allows one to use traditional
techniques of stretching an alveolar epithelial cell’s basal surface,
using a variety of custom-made or commercially available devices, and know that entire cell surface area is increasing in the
same proportion. Conveniently, as a result of this finding, in
making capacitance measurements, it was only necessary to take
two-dimensional photographs to record change in total cell surface area. Finally, we report that lipid insertion and plasma
membrane expansion appear to function at a low level linked
to plasma membrane tension and are independent of actin cytoskeleton integrity, ATP availability, or calcium signaling.
These findings provide an additional step toward better understanding how alveolar epithelial cells respond to stretch, and
may ultimately provide insight for the development of safer,
more effective ventilation strategies.
Acknowledgments: The authors thank biostatistician Dr. Warren Ewens at the
University of Pennsylvania for his expert assistance with the sophisticated statistics
needed to analyze and interpret the data in this study. Support for this study
was provided by National Heart, Lung, and Blood Institute Grant NIH-RO1 2HL-527204. J.L.F. was supported by a Graduate Fellowship from the Whitaker
Foundation.
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