The Journal of Clinical Investigation| February 2004| Volume 113| Number 3
The molecular mechanisms underlying the varied
phenotypes are unknown, and two alternative hypothe-
ses have been proposed to explain the tissue-specific
effects observed in laminopathies. The “structural
hypothesis” suggests that lamin mutations lead to
increased nuclear fragility and eventual nuclear dis-
ruption in the mechanically stressed tissue, while the
“gene regulation hypothesis” proposes a tissue-specif-
ic role of lamins in DNA transcription. Lmna–/–mice
are indistinguishable from their littermates at birth but
develop severe muscle wasting and contractures simi-
lar to Emery-Dreifuss muscular dystrophy by 3–4
weeks and die by 8 weeks (4). Cells derived from Lmna–/–
mice have misshapen nuclei and obvious ultrastruc-
tural damage (4, 18). Distorted nuclear shape has also
been demonstrated in fibroblasts from lipodystrophic
patients with heterozygous R482Q/W mutations in the
lamin A/C gene and in cells from Caenorhabditis elegans
with reduced lamin levels (1, 19). Here we show that
nuclear mechanics in cells from Lmna–/–mice are defec-
tive, withLmna–/–nuclei displaying increased deforma-
tion and fragility under strain. In addition, we demon-
strate that transcriptional activation in response to
mechanical stimuli is attenuated in Lmna–/–cells,
impairing viability of mechanically strained cells. These
data suggest that the structural and gene regulation
hypotheses of the laminopathies are in fact closely
related, and different mutations may cause specific
phenotypes by differentially affecting these processes.
Lamins are structural components of the nuclear lam-
ina, a protein network underlying the inner nuclear
membrane that determines nuclear shape and size (1).
In addition, lamins play an important role in organiz-
ing nuclear pore complexes (2) and recruiting other
proteins such as emerin to the nuclear envelope (3, 4).
Two types of lamins are found in mammalian cells:
A-type lamins (lamin A, C, A∆10, and C2) are encoded
by a single gene (Lmna) and are developmentally regu-
lated and expressed in differentiated cells. B-type
lamins (B1 and B2/B3) are encoded by two distinct
genes and are constitutively expressed in all cells (1,
5–10). Mutations in the gene encoding A-type lamins
and their binding partners have been associated with a
variety of human diseases, including Emery-Dreifuss
muscular dystrophy, dilated cardiomyopathy, Dunni-
gan-type familial partial lipodystrophy, and Hutchin-
son-Gilford progeria syndrome (11–17).
Received for publication July 30, 2003, and accepted in revised form
November 11, 2003.
Address correspondence to: Richard T. Lee, Partners Research
Facility, Room 280, 65 Landsdowne Street, Cambridge,
Massachusetts 02139, USA. Phone: (617) 768-8282;
Fax: (617) 786-8280; E-mail: firstname.lastname@example.org.
Conflict of interest: The authors have declared that no conflict of
Nonstandard abbreviations used: insulin, transferrin, and
selenium (ITS); nanonewton (nN); hectopascal (hPa).
Lamin A/C deficiency causes
defective nuclear mechanics
Jan Lammerding,1P. Christian Schulze,2Tomosaburo Takahashi,2Serguei Kozlov,3
Teresa Sullivan,3Roger D. Kamm,1Colin L. Stewart,3and Richard T. Lee1,2
1Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
2Cardiovascular Division, Brigham and Women’s Hospital, Cambridge, Massachusetts, USA
3Cancer and Developmental Biology Lab, National Cancer Institute, Frederick, Maryland, USA
Mutations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Drei-
fuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The
tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is
incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C
could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechan-
otransduction, we subjected lamin A/C–deficient mouse embryo fibroblasts to mechanical strain
and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna–/–
cells have increased nuclear deformation, defective mechanotransduction, and impaired viability
under mechanical strain. NF-κB–regulated transcription in response to mechanical or cytokine stim-
ulation was attenuated in Lmna–/–cells despite increased transcription factor binding. Lamin A/C
deficiency is thus associated with both defective nuclear mechanics and impaired mechanically acti-
vated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C muta-
tions observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics
and transcriptional activation.
J. Clin. Invest. 113:370–378 (2004). doi:10.1172/JCI200419670.
See the related Commentary beginning on page 349.
The Journal of Clinical Investigation
| February 2004| Volume 113| Number 3
Cells. Lmna+/+and Lmna–/–mouse embryo fibroblasts
were maintained in DMEM (Invitrogen Corp., Carls-
bad, California, USA) containing 10% FCS (HyClone
Laboratories, Logan, Utah, USA) and penicillin/strep-
tomycin (Invitrogen Corp.).
Nuclear strain experiments. Cells were plated at 900
cells/cm2on fibronectin-coated silicone membranes
in DMEM supplemented with 10% FCS followed by
serum starvation for 48 hours in DMEM containing
insulin, transferrin, and selenium (ITS) supplement
(Sigma-Aldrich, St. Louis, Missouri, USA). Preceding
the strain experiments, cells were incubated with
Hoechst 33342 nuclear stain (1 µg/ml; Molecular
Probes Inc., Eugene, Oregon, USA) in DMEM plus
ITS for 20 minutes. Membranes were placed on a
custom-made strain device mounted on an Olympus
IX-70 microscope (Olympus America Inc., Melville,
New York, USA), and biaxial strain was applied in a
stepwise fashion. Membrane and nuclear strain was
computed on bright field and fluorescence images
using a custom-written image-analysis algorithm.
Normalized nuclear strain was defined as the ratio
of nuclear strain to membrane strain to compensate
for small variations in applied membrane strain
Magnetic bead microrheology. Cells were plated on 35-
mm polystyrene dishes (Corning-Costar Corp., Corn-
ing, New York, USA). The following day, cells were
incubated with fibronectin-coated paramagnetic
beads (Dynal Biotech Inc., Lake Success, New York,
USA) for 30 minutes. To minimize nuclear effects,
only beads attached more than 5 µm from the nucle-
us were selected for analysis. A sinusoidal force
(amplitude 0.6 nanonewtons [nN], frequency 1 Hz,
offset 0.6 nN) was applied through a magnetic trap,
and bead displacement was monitored using a digi-
tal camera (Roper Scientific Inc., San Diego, Califor-
nia, USA). Displacement amplitudes were computed
using custom-written MATLAB (The MathWorks
Inc., Natick, Massachusetts, USA) algorithms. In sep-
arate experiments, smaller (2 µm) fibronectin-coated
polystyrene beads (Bangs Laboratories Inc., Fishers,
Indiana, USA) were incubated together with the mag-
netic beads for 1 hour to adhere to the cell surface.
Cells containing single magnetic beads and several
polystyrene beads were subjected to a brief force
pulse (2.5 nN for 3 seconds). Using custom-written
MATLAB algorithms, maximal induced magnetic
and polystyrene bead displacements were computed
and expressed in cylindrical coordinates (r, θ) with
the magnetic bead at the origin and θ = 0 for the force
direction. The induced strain field can be described
by an analytical cell mechanics model proposed by
Bausch et al. (20) expressing the radial component ur
of the induced bead displacement as a function of the
applied force F, cell stiffness µ∗, the characteristic cut
of radius κ−1, the distance from the magnetic bead
center r, and the polar angle θ:
where Κ0and Κ1are modified Bessel functions of the
second kind (order 0 and 1, respectively) and using
κ1 = [(1 – σ)/2]1/2κ. The parameters µ* and κ were
obtained by fitting equation 1 to the bead displace-
ment data using the GraphPad Prism 4.0 robust curve
fit function (GraphPad Software, San Diego, Califor-
nia, USA) and assuming σ = 0.5 for incompressible
media and a magnetic bead contact radius of 2 µm.
The magnetic trap calibration was performed as
described previously (21). In brief, magnetic beads sus-
pended in viscous solution were monitored while being
attracted to the magnetic trap operated at various cur-
rents. The applied force as a function of current and
distance from the magnetic trap was then computed
based on Stokes’ law.
Microinjection. Cells were plated on fibronectin-coated
glass dishes (WillCo Wells BV, Amsterdam, The Nether-
lands) or silicone dishes and incubated overnight.
Microinjections were performed using an Eppendorf
microinjector with Eppendorf Femtotips (Eppendorf
AG, Hamburg, Germany). In each dish, 20–50 cells were
injected with Texas Red–labeled 70-kDa dextran (Mol-
ecular Probes Inc.) dissolved at 10 mg/ml in PBS (Invit-
rogen Corp.), either into the cytoplasm (injection pres-
sure 500 hectopascals [hPa], injection time 0.6 seconds)
or into the nucleus (injection pressure 10, 100, 500, and
1,500 hPa, injection time 0.6 seconds). Following
microinjection, cells were washed in HBSS (Invitrogen
Corp.), and intracellular localization of dextran–Texas
Red was recorded under a fluorescent microscope.
Selected silicone membranes were subjected to con-
stant biaxial strain (about 32% for 30 minutes) or cyclic
biaxial strain (10% at 1 Hz for 24 hours), and localiza-
tion of Texas Red–labeled dextran in strained and con-
trol cells was analyzed on a fluorescence microscope
(Olympus America Inc.).
Strain experiments. Strain stimulation was carried
out as previously described (22). In brief, cells were
plated on fibronectin-coated silicone membranes
(2,500–3,300 cells/cm2). After 72 hours of serum star-
vation, cells were subjected to biaxial cyclic strain (4%
or 10% at 1 Hz). For chemical stimulation, cells were
incubated with IL-1β (25 ng/ml; R&D Systems Inc.,
Minneapolis, Minnesota, USA) or PMA (200 ng/ml,
Sigma-Aldrich) in DMEM plus ITS.
DsRed/peroxiredoxin-2 localization. Cells were plated on
fibronectin-coated glass dishes or fibronectin-coated
silicone membranes. Following overnight incubation,
cells were transfected with a CMV promoter–driven
DsRed/peroxiredoxin-2 fusion construct (CLONTECH
Inc., Palo Alto, California, USA) using FuGENE 6 (F.
Hoffman–La Roche Ltd., Basel, Switzerland) and incu-
bated for 24 hours. Selected silicone membranes were
subjected to constant (about 19% for 60 minutes) or
cyclic biaxial strain (10% at 1 Hz for 3 hours), and
The Journal of Clinical Investigation|February 2004|Volume 113| Number 3
localization of DsRed-labeled peroxiredoxin-2 in
strained and control cells was analyzed on a fluores-
cence microscope (Olympus America Inc.).
Flow cytometry. For cell viability assays, propidium
iodide (2 µg/ml, Sigma-Aldrich) was added to the dish-
es after 24 hours of strain application. Cells were col-
lected and analyzed using a Cytomics FC 500 flow
cytometer (Beckman Coulter Inc., Fullerton, California,
USA), counting 10,000–30,000 events in each group.
Thresholds for propidium iodide incorporation were
determined based on negative (no propidium iodide
staining) and positive (cells permeabilized by 50%
ethanol) controls. Apoptotic and necrotic cell fractions
were measured in similar experiments using the Vybrant
Apoptosis Assay Kit no. 3 (Molecular Probes Inc.).
Northern and Western analyses. Expression of iex-1 and
egr-1 mRNA was assessed by Northern analysis as
described previously (23). Protein expression was ana-
lyzed by Western analysis of nuclear and cytoplasmic
cell fractions using antibodies against NF-κB p65
(antibody does not recognize p50 or p105), IκBα(both
from Santa Cruz Biotechnology Inc., Santa Cruz, Cal-
ifornia, USA), and actin (Sigma-Aldrich). Additional
immunoblotting was performed on whole cell lysates
using specific antibodies against total ERK1/2 (Santa
Cruz Biotechnology Inc.) and phospho-p44/p42 MAP
kinase (Cell Signaling Technology, Beverly, Massachu-
setts, USA). After incubation with HRP-conjugated
secondary antibody (Bio-Rad Laboratories Inc., Her-
cules, California, USA), specific bands were visualized
by enhanced chemiluminescence (PerkinElmer Inc.,
Boston, Massachusetts, USA).
Luciferase experiments. Cells were transfected with plas-
mids for NF-κB–controlled luciferase expression and
SV40-regulated β-gal (Promega Corp., Madison, Wis-
consin, USA) using FuGENE 6 (F. Hoffman–La Roche
Ltd.). Following transfection, cells were serum starved
in DMEM plus ITS medium for 48 hours, followed by
overnight stimulation with PMA (200 ng/ml) or IL-1β
(25 ng/ml). Luciferase assays were quantified in a Vic-
tor2 Multilabel Counter (Perkin Elmer Inc.). Results
were normalized for β-gal activity and expressed as per-
cent WT control.
Immunohistochemistry. Cells were plated on untreated
or fibronectin-coated chamber slides and serum-starved
for 24–72 hours followed by stimulation with IL-1β.
Cells were fixed in 4% paraformaldehyde or methanol,
washed in PBS, and permeabilized with 0.1% Triton
X-100. After blocking, cells were incubated overnight
with primary rabbit antibody anti–NF-κB (p65; Santa
Cruz Biotechnology Inc.) at 4°C or Alexa Fluor 568
phalloidin (A-12380; Molecular Probes Inc.) for 1 hour
at 25°C, followed by 1 hour of incubation with second-
ary FITC- or TRITC-conjugated antibodies.
Cellular protein fractions and electrophoretic mobility shift
assay. Nuclear extracts were prepared as described pre-
viously (24) with the following modifications. Cells
were washed with ice-cold PBS and lysed in buffer A,
which consisted of 0.1% Triton X-100, 10 mM EDTA,
10 mM EGTA, 10 mM KCl, 10 mM HEPES, 1 mM
DTT, 0.5 mM PMSF, and protease inhibitor cocktail
(P-8340; Sigma-Aldrich). After centrifugation at 1,200
g for 10 minutes, the supernatant was stored as the
cytoplasmic cell fraction, while the nuclear pellet was
washed once in PBS, resuspended in buffer C (1 mM
EDTA, 1 mM EGTA, 0.4 M NaCl, 20 mM HEPES, 5
mM MgCl2, 25% glycerol, 1 mM DTT, 0.5 mM PMSF,
and protease inhibitor cocktail), and incubated at 4°C
for 10 minutes. The nuclear extract was centrifuged for
10 minutes at 4°C at 12,000 g, and the supernatant was
used for Western analysis and electrophoretic mobility
shift assay. NF-κB–specific oligonucleotides (Promega
Corp.) were end-labeled using T4 polynucleotide kinase
and [γ-32P]ATP (DuPont NEN Research Products,
Boston, Massachusetts, USA). Nuclear extracts were
preincubated for 10 minutes in binding buffer followed
by 20 minutes of incubation at room temperature with
labeled oligonucleotide. Samples were separated on a
4% native polyacrylamide gel. For competition studies,
an excess (50 times) of unlabeled oligonucleotide was
used, and for the supershift assay, the nuclear extracts
were incubated with 2 µg of anti-p50 or anti-p65 anti-
body (Santa Cruz Biotechnology Inc.).
Statistical analysis. All experiments were performed at
least three independent times. Data are expressed as
mean ± SEM. Statistical analysis was performed using
Nuclear mechanics is impaired in lamin A/C–deficient cells. (a)
Nucleus of WT fibroblast before strain (red) and at 22% strain (yel-
low). Scale bar: 10 µm. (b) Lmna–/–nucleus before strain (red) and
at 19% strain (yellow). Scale bar: 10 µm. (c) Nuclear strain as a func-
tion of applied membrane strain. Dashed lines represent linear
regression of the data for each cell type forced through the origin
(Lmna+/+: y = 0.299x, Lmna–/–: y = 0.626x). (d) Maximal normalized
nuclear strain was significantly increased in Lmna–/–fibroblasts
(0.306 ± 0.029 vs. 0.626 ± 0.039; P < 0.0001, n = 21).
The Journal of Clinical Investigation
|February 2004| Volume 113| Number 3
PRISM 4.0 and INSTAT software (GraphPad Software
Inc., San Diego, California, USA). The data were ana-
lyzed by unpaired t test (allowing different SDs),one-
way ANOVA, or the Mann-Whitney test in case of non-
parametric distribution. A two-tailed P value below
0.05 was considered significant.
Nuclear mechanics. To explore the role of lamin A/C in
nuclear mechanics, mouse embryo fibroblasts derived
from Lmna–/–and Lmna+/+mice were cultured on trans-
parent membranes and subjected to stepwise increas-
ing biaxial strain (first step: 10.1% ±0.18 %; second step:
18.2% ±0.07%). The cytoskeleton, attached to the mem-
brane through integrin receptors, is exposed to the
same biaxial strain as the membrane, while the stiffer
nucleus exhibits only small deformations in WT cells
(25). The induced nuclear deformations were calculat-
ed by tracking the fluorescently labeled nucleus (Figure
1, a and b) and normalized to membrane strain to com-
pensate for the small variation in applied membrane
strain. For each cell type, nuclear deformation
increased approximately linearly with applied mem-
brane strain (Figure 1c), but Lmna–/–nuclei showed sig-
nificantly larger deformations than did WT cells (Fig-
ure 1, c and d). Fitting a linear regression to the nuclear
deformation data revealed a significantly larger slope
for the Lmna–/–nuclei, and the maximal normalized
nuclear deformation was significantly larger for
Lmna–/–cells, indicating impaired nuclear mechanics in
lamin A/C–deficient nuclei.
Cytoskeletal mechanics. To evaluate the possibility that
the observed increase in nuclear deformation was
caused by more direct force transmission to the nucle-
us, we examined cytoskeletal organization by staining
actin stress fibers with phalloidin. No apparent differ-
ences in cytoskeletal architecture were found between
WT (Figure 2a) and Lmna–/–cells (Figure 2b). Magnetic
bead microrheology (20) was used for quantitative eval-
uation of cytoskeletal stiffness. Small (4.5 µm),
fibronectin-coated paramagnetic beads were attached
to the cell, and the bead displacement in response to an
applied magnetic force was used as an indicator of
cytoskeletal stiffness (Figure 2c). The induced bead dis-
placement amplitude was significantly increased in
Lmna–/–fibroblasts (Figure 2d), indicating decreased
cytoskeletal stiffness. Since magnetic bead displacement
depends not only on cytoskeletal stiffness but also on
the binding characteristics of the cell to the bead, we
applied a second microrheology method that measures
the induced displacement in polystyrene beads located
on the cell surface close to the magnetic bead (Figure 2,
e and f). Lamin A/C–deficient cells exhibited signifi-
cantly larger induced polystyrene bead displacements
Cytoskeletal stiffness is reduced in lamin A/C–deficient cells. (a) Phal-
loidin staining for actin stress fibers in WT (Lmna+/+) fibroblasts. Scale
bar: 20 µm. (b) Phalloidin staining for actin stress fibers in Lmna–/–
cells. Scale bar: 20 µm. (c) Magnetic bead microrheology. Represen-
tative examples of magnetic bead displacement in response to applied
sinusoidal force (thin black line) for WT (thick black line) and Lmna–/–
(thick gray line) fibroblasts. (d) Bead displacement amplitude in
response to applied magnetic forces was significantly increased in
Lmna–/–fibroblasts, indicating reduced cytoskeletal stiffness in lamin
A/C–deficient cells (0.124 ±0.024 µm vs. 0.226 ±0.029 µm; P< 0.01,
n = 60). (e) Fibroblast with magnetic (diameter 4.5 µm) and poly-
styrene beads (diameter 2 µm) attached to the cell membrane. Scale
bar: 10 µm. (f) Graphic representation of the displacement field after
a brief force pulse (2.5 nN for 3 seconds). Bead sizes and positions
are drawn to scale, while bead deflections are enlarged by a factor of
10. (gand h) Distance dependence of the angle-corrected radial bead
displacement component ur/cosθ as defined in equation 1. The dot-
ted line is an optimal fit to equation 1, yielding estimates for cellular
stiffness µ*and dissipation κfor WT (g) and Lmna–/–cells (h), respec-
tively (µ*: 27,537 ±8,458 pN/µm vs. 2,417 ±734.7 pN/µm; P< 0.01,
n= 128 [WT], 153 [Lmna–/–]; κ: 0.020 ±0.017 µm–1vs. 0.201 ±0.072
µm–1; P < 0.05, n = 128 [WT], 153 [Lmna–/–]). pN, piconewton.
The Journal of Clinical Investigation|February 2004| Volume 113| Number 3
that decreased quickly with increasing distance from
the magnetic bead, whereas bead displacements in WT
cells were smaller and hardly exceeded the detection
limit of less than 0.1 µm (Figure 2, g and h). Based on an
analytical model proposed by Bausch et al. (20), the
induced polystyrene bead displacement is a function of
the applied force, the position relative to the magnetic
bead, and two cellular mechanics parameters µ* and κ
that describe the cell stiffness and intracellular strain
dissipation, respectively. By separately fitting the theo-
retical displacement field to the observed bead data for
Lmna–/–and WT fibroblasts, one can estimate the
parameters for cellular stiffness and dissipation (Figure
2, g and h), confirming that Lmna–/–cells exhibit signif-
icantly decreased cytoskeletal stiffness.
Therefore, the observed increased nuclear deforma-
tion in lamin-deficient cells is unlikely to be due to
altered force transmission to the nucleus. In contrast,
the softer cytoskeleton would result in an underestima-
tion of the nuclear stiffness. Based on these findings we
conclude that lamin A/C–deficient cells have decreased
nuclear stiffness and altered nuclear mechanics.
Cellular response to mechanical strain. Strain-induced
damage to the more fragile nucleus could provide one
explanation for tissue-specific effects of lamin A/C
mutations, for example, in mechanically active tissues
like myocardium and skeletal muscle. To examine
nuclear envelope integrity, we monitored the subcellu-
lar location of fluorescently labeled 70-kDa dextran
microinjected into either the cytoplasm or nucleus of
adherent fibroblasts. Cytoplasmic injection revealed
that the high-molecular-weight dextran was excluded
from the nucleus in both WT and lamin-deficient cells
(Figure 3, a and b), indicating that nuclear integrity is
not significantly impaired in Lmna–/–cells under rest-
ing conditions. These results were confirmed by trans-
fecting the cells with a DsRed/peroxiredoxin-2 fusion
protein. The protein lacks a nuclear localization
sequence and is too large (mol wt 57 kDa) to passively
diffuse into the intact nucleus. Consequently, the
fusion protein was excluded from the nucleus of WT
However, when dextran was injected directly into the
nucleus at low and medium injection pressures
(10–500 hPa), nuclear integrity in lamin A/C–deficient
cells was at least temporarily compromised, resulting
in fluorescently labeled dextran escaping into the cyto-
plasm (Figure 3d). In contrast, dextran was confined to
the nucleus in most WT cells when injected at low pres-
sure (Figure 3c), and even at medium pressure signifi-
cantly more WT cells than Lmna–/–cells maintained
their nuclear integrity. Using sufficiently high pressure
(1,500 hPa), both WT and lamin-deficient nuclei could
be ruptured (Figure 3e).
To determine whether Lmna–/–fibroblasts are thus
more susceptible to mechanical strain, cells were sub-
jected to cyclic biaxial strain (10% strain at 1 Hz). After
24 hours of strain, Lmna–/–fibroblasts had a signifi-
cantly increased fraction of dead (i.e., propidium
iodide–positive) cells compared with controls not sub-
jected to strain (Figure 4a). Differences within controls
(WT and Lmna–/–) and between WT control and
strained cells were not significant. Dual labeling with
propidium iodide and an FITC-conjugated annexin V
antibody revealed that the decrease in viability was due
Nuclear fragility is increased in lamin A/C–deficient cells. (a and b)
Fluorescently labeled 70-kDa dextran is excluded from the nucleus
following cytoplasmic injection, indicating an intact nuclear mem-
brane in WT (a) and Lmna–/–(b) cells under resting conditions. Scale
bar: 20 µm. (c) Nuclear injection at low pressure results in fluores-
cently labeled dextran contained in the nucleus of WT cells, indicat-
ing that the nuclear integrity is preserved during injection. Scale bar:
20 µm. (d) In contrast, nuclear integrity is disrupted in nuclei of
Lmna–/–cells even at low pressure, leading to fluorescently labeled
dextran escaping into the cytoplasm during injection. Scale bar: 20
µm. (e) Nuclear rupture as a function of increasing injection pressure
of dextran microinjection into the nucleus. Zero pressure: cytoplas-
mic injection at 500 hPa. Low pressure: nuclear injection at 10–20
hPa (**84.7% ± 4.24% vs. 9.5% ± 4.53% intact nuclei for WT and
Lmna–/–cells, respectively; P < 0.0001, n = 72 [WT], 42 [Lmna–/–]).
Medium pressure: nuclear injection at 100–500 hPa (*40.4% ± 7.16%
vs. 9.5% ± 4.53% intact nuclei for WT and Lmna–/–cells respectively;
P < 0.01, n = 47 [WT], 31 [Lmna–/–]). High pressure: nuclear injec-
tion at 1,500 hPa; all cells showed nuclear rupture.
The Journal of Clinical Investigation
|February 2004| Volume 113| Number 3
to an increase in both necrotic and apoptotic cell frac-
tions compared with WT cells (Figure 4b).
Interestingly, when cells were injected with the fluo-
rescently labeled 70-kDa dextran into the cytoplasm
and subjected to either constant (30% for 30 minutes)
or cyclic (10% at 1 Hz for 24 hours) biaxial strain, strain
application did not significantly increase the number
of dextran-positive nuclei in either WT or Lmna–/–
fibroblasts (data not shown), suggesting that the
extreme event of nuclear rupture under mechanical
strain occurs in only a small fraction of cells that can-
not be detected based on a small number (n= 20–30) of
single cell observations.
The increased fraction of apoptotic cells in mechani-
cally strained Lmna–/–fibroblasts indicates that necro-
sis through nuclear rupture can only partly explain the
increased sensitivity to mechanical stimulation. There-
fore, we investigated the cellular response to mechani-
cal stimulation in more detail. Interestingly, expression
of the mechanosensitive genes egr-1 and iex-1
in response to mechanical stimulation was
impaired in Lmna–/–cells (Figure 4c), whereas
expression of the mechanically unresponsive
genes thioredoxin-1and GAPDHwas unaltered
(data not shown), indicating that transcrip-
tion is not impaired in a nonspecific manner.
To test whether the observed changes in
Lmna–/–cells were specific to mechanical stim-
ulation or represented a more general defi-
ciency in signal transduction, we measured
the expression levels of iex-1 and egr-1 in
response to stimulation with PMA or the
cytokine IL-1β. Cytokine stimulation led to
an attenuated response in iex-1 but not egr-1
expression in Lmna–/–cells, while PMA
responsiveness remained intact for both
genes in Lmna–/–cells (Figure 4d).
NF-κB signaling. Because iex-1is an NF-κB–dependent
survival gene (23), and because NF-κB can be biome-
chanically activated (26), we examined whether biome-
chanical signaling through NF-κB is disturbed in
Lmna–/–cells. The MAPK ERK1/2 is an important reg-
ulator of mechanically induced gene expression and
has been linked to NF-κB activation (27). Analysis of
MAPK phosphorylation after chemical (PMA) or
mechanical stimulation revealed no differences
between Lmna–/–and WT cells (data not shown), indi-
cating that the observed changes are caused by alter-
ations in signal transduction other than impaired cyto-
plasmic MAPK activation.
In resting cells, NF-κB is sequestered in the cytoplasm
by the inhibitor IκB. Upon stimulation, IκB is
ubiquinated and degraded, allowing NF-κB to translo-
cate into the nucleus and activate target genes. Figure
5a shows that cytokine-induced cytoplasmic degrada-
tion of IκBα and translocation of the NF-κB subunit
Impaired mechanotransduction in lamin A/C–deficient
cells. (a) Lmna–/–fibroblasts exhibited a significantly high-
er percentage of propidium iodide–positive cells than did
WT cells (2.88% ±0.49% vs. 1.14% ±0.14%; P< 0.01, n= 7
[WT], 8 [Lmna–/–]) after 24 hours of strain application
(10% at 1 Hz). Differences in unstrained cells were not sig-
nificant (n= 9 [WT], 10 [Lmna–/–]). (b) Dual labeling with
FITC-conjugated annexin V and propidium iodide uptake
indicated that apoptotic (A) and necrotic (N) cell fractions
are increased in Lmna–/–cells following prolonged strain.
Viable cells (V) are propidium iodide–negative and FITC-
negative. Top left: WT unstrained control; top right:
Lmna–/–unstrained control; bottom left: WT cells after
10% strain for 24 hours; bottom right: Lmna–/–cells after
10% strain for 24 hours. PI, propidium iodide. (c) Lmna–/–
(KO) fibroblasts exhibited attenuated mechanical induc-
tion of egr-1 and iex-1 after 2 hours and 4 hours of strain
(4%) compared with Lmna+/+cells (WT). Expression of
GAPDH was not negatively affected. (d) Cytokine–induced
expression of iex-1 was impaired in Lmna–/–cells, while
PMA responsiveness remained intact in Lmna–/–cells.
The Journal of Clinical Investigation|February 2004| Volume 113| Number 3
p65/RelA into the nucleus was not impaired in Lmna–/–
cells. This finding was confirmed in immunofluores-
cence directed against p65/RelA (data not shown).
Total levels of NF-κB subunits p50, p65, and p105 in
whole cell extracts from Lmna–/–cells were indistin-
guishable from those in WT cells (data not shown).
Interestingly, binding of nuclear NF-κB to its tran-
scription factor binding site was increased in lamin
A/C–deficient cells compared with WT cells (Figure
5b). Surprisingly, despite the increased levels of tran-
scription factor binding, IL-1β–induced activity of
NF-κB–dependent luciferase was significantly impaired
in Lmna–/–fibroblasts (Figure 5c). These results indicate
that the deficient response of Lmna–/–cells to mechan-
ical or cytokine stimulation is based on a role for lamin
A/C in transcriptional activation following transcrip-
tion factor binding.
Our results establish the importance of lamin A/C for
nuclear stability and highlight its role in transcrip-
tional regulation in response to mechanical or chemi-
cal stimulation. Measurements of nuclear mechanics
were obtained using a novel technique to measure
nuclear deformation with biaxial strain applied to the
cells. This method yields quantitative measurements of
nuclear stiffness compared with cytoskeletal stiffness
in living cells without having to isolate the nuclei. Our
results for nuclear deformation in strained Lmna–/–
cells are in excellent agreement with measurements
obtained by Caille et al. (25) in endothelial cells under
uniaxial strain and on isolated endothelial cell nuclei
(28). Increased nuclear fragility in Lmna–/–cells was fur-
ther confirmed by nuclear microinjection experiments,
which demonstrated increased nuclear rupture at low
and moderate pressures compared with that in WT
cells. The markedly increased nuclear deformability
and fragility in Lmna–/–cells compared with WT cells
has important implications for the cellular response to
mechanical strain. Impaired nuclear stability can lead
to rupture of the nucleus, resulting directly in cell
death. Evidence of fragmented nuclei has been report-
ed in skeletal muscle fibers from emerin-deficient
Emery-Dreifuss muscular dystrophy patients and in
fibroblasts from Dunnigan-type familial partial lipody-
strophy patients following heat shock treatment (19,
29). This direct effect of impaired nuclear stability is
also consistent with the impaired cell viability after
cyclic strain observed in our experiments.
In addition to the direct effect of nuclear rupture,
altered nuclear mechanics can affect cells through
impaired nuclear mechanotransduction. Recent studies
have reported desmin intermediate filament–mediated
changes in chromatin in response to mechanical strain,
and hypothesized that stretch-induced changes in chro-
matin can lead to activation of hypertrophy-associated
genes (30). Mechanical connections between integrins,
cytoskeletal filaments, and the nucleus have also been
demonstrated by micromanipulation with microbeads
and micropipettes in endothelial cells (31). Our nuclear
strain experiments demonstrate that external strain
application results in increased nuclear strain, indicating
mechanical coupling between the extracellular matrix
and the nucleus mediated through focal adhesion sites
and the cytoskeleton. Mutations in nuclear envelope pro-
teins such as lamin or emerin could interrupt some of
these connections and impair nuclear mechanotrans-
duction pathways. In addition, the impaired nuclear and
cytoskeletal mechanics observed in Lmna–/–cells lead to
significantly increased nuclear strain, which could fur-
ther result in altered nuclear mechanosensing.
The observation of decreased cytoskeletal stiffness in
Lmna–/–cells raises additional questions. Alterations in
cytoskeletal stiffness could arise as a compensatory
mechanism to protect a more fragile nucleus, but could
also play a pivotal role in the pathophysiology of the
disease. Altered cytoskeletal mechanics not only affect
the transmitted force to the nucleus under applied
strain, but can also play an important role in cell shape,
migration, and other critical functions with direct con-
sequences to the affected tissue.
Defective NF-κB signaling in lamin A/C–deficient cells. (a) Western
blot analysis of nuclear and cytoplasmic protein fractions. Cytokine-
induced nuclear translocation of p65/RelA and degradation of cyto-
plasmic IκBα was indistinguishable between WT and Lmna–/–
fibroblasts. Cytoplasmic proteins were loaded at a lower concen-
tration. Con, control. (b) Electrophoretic mobility shift assay for
NF-κB target sequence using protein from the same nuclear frac-
tions as in a. Probe specificity was confirmed using unlabeled com-
petitive and noncompetitive probes. Identity of NF-κB subunits p50
and p65 was confirmed by supershift assay. (c) Cytokine-induced
NF-κB–regulated luciferase activity was significantly impaired in
Lmna–/–cells (percent of baseline: 282% ± 18.5% vs. 185% ± 22.2%;
P < 0.001, n = 9). Baseline activity was not significantly different
between WT and Lmna–/–cells.
The Journal of Clinical Investigation
| February 2004| Volume 113| Number 3
However, increased nuclear fragility can only partial-
ly explain the phenotypes observed in several
laminopathies. Even though nuclear mechanics are
clearly impaired in the vast majority of Lmna–/–cells,
only a very small fraction of cells (about 3–5%) exhibit-
ed nuclear rupture under strain in our experiments,
and only a small number of ruptured nuclei are found
in affected muscle tissue in patients suffering from
Emery-Dreifuss muscular dystrophy (29). In addition,
fibroblasts obtained from lipodystrophic patients with
the lamin R482Q/W mutation also exhibit defective
nuclear mechanics, even though these patients lack a
muscular phenotype (19). Therefore, most cells appear
to be functional despite distorted nuclear shape and
altered nuclear mechanics, and additional events might
be necessary to trigger nuclear failure. Therefore, defec-
tive nuclear mechanics could play a more important
role in muscle tissue that is subjected to large mechan-
ical strain and stress compared with (for example) adi-
pose tissue, with cumulative cellular damage through
both direct nuclear rupture and impaired mechan-
otransduction signaling, eventually leading to the mus-
cular dystrophy observed in several laminopathies.
In addition to their role as nuclear envelope proteins,
lamins form stable structures within the nucleoplasm
as shown by fluorescence recovery after photobleaching
(FRAP) (32). The impaired response of iex-1 and egr-1
expression to mechanical and cytokine stimulation, as
well as the attenuated response of NF-κB–regulated
luciferase activity in Lmna–/–fibroblasts despite
increased transcription factor binding, indicate an
important role of lamin A/C in transcriptional activa-
tion. Interactions between lamin and nuclear transcrip-
tion factors have been previously demonstrated in vivo
and in vitro (33, 34), and lamin A/C speckles can medi-
ate spatial organization of splicing factor compart-
ments and RNA polymerase II transcription (35). Dis-
rupting the normal organization of nuclear lamins by
expression of a dominant-negative lamin mutant has
been shown to inhibit RNA polymerase II–dependent
transcription in mammalian cells and active embryon-
ic nuclei from Xenopus laevis (36). The impaired tran-
scriptional activation observed in our experiments indi-
cates a yet-unknown role of lamin A/C in the assembly
of enhancesomes or as a scaffolding protein for tran-
scription factors and coactivators.
NF-κB is a mechanical stress–responsive transcription
factor that can function as an antiapoptotic signal.
Impaired transcriptional activation can therefore lead
to increased apoptosis in mechanically strained tissue.
Furthermore, direct evidence for the role of lamin in
apoptosis has previously been demonstrated in cultured
cells that expressed an uncleavable mutant form of
lamin (37). In these cells, chromatin failed to condense
and DNA cleavage was delayed despite the activation of
caspases. Kumar et al. demonstrated in ex vivo experi-
ments that mechanical stress activated NF-κB in skele-
tal muscle fibers and that this response is altered in mdx
mice, a model for Duchenne muscular dystrophy, stress-
ing the importance of NF-κB signaling in muscle tissue
that is affected most often in laminopathies (38).
Interestingly, several cells such as lymphoblasts, basal
skin cells, and early embryonic cells do not express
lamin A/C. We speculate that nuclear mechanics and
mechanotransduction in these cells would be normal,
since these cells are known to be capable of normal
function. In these cells, the role of lamin A/C might be
taken over by a different protein or complex of pro-
teins. However, it is important to note that our experi-
ments used only fibroblasts, and future studies of cells
from different tissues are essential.
While all experiments were performed in fibroblasts
that completely lack lamin A and lamin C, most human
diseases arise from heterozygous lamin mutations. In
these situations mutant lamin often appears as stable
as WT lamin and is expressed at similar levels (39). In
many cases, especially in phenotypes involving striated
muscle, the mutation may lead to a structurally
impaired form of lamin A/C that could act as a domi-
nant negative and lead to cellular mechanical deficien-
cies as observed in the lamin A/C–null cells. Other
mutations, such as those resulting in lipodystrophy,
could affect the binding of lamin to other proteins or
chromatin with fewer effects on the structural role of
lamin itself, resulting in a partially functional protein
that might affect only specific signaling pathways. Fur-
thermore, combinations of mechanical and transcrip-
tional regulation defects could result in complex phe-
notypes affecting several tissue types in diseases such as
progeria. By providing independent tests for measuring
structural and gene-regulatory functions of lamin A/C,
our experiments could help distinguish the effects of
individual mutations on the function of lamin.
Tissue-specific effects observed in several lamino-
pathies may thus arise from two mechanisms: the
impaired nuclear stability renders mechanically
strained tissue more susceptible to cellular damage,
and abnormal transcriptional activation impairs adap-
tive and protective pathways. Individual mutations in
the lamin A/C gene could potentially selectively inter-
fere with any of these functions, explaining the diversi-
ty of observed phenotypes.
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