Decreased mechanical stiffness in LMNA2/2
cells is caused by defective nucleo-cytoskeletal
integrity: implications for the development
Jos L.V. Broers1,2,*, Emiel A.G. Peeters2, Helma J.H. Kuijpers1, Jorike Endert1,
Carlijn V.C. Bouten2, Cees W.J. Oomens2, Frank P.T. Baaijens2and Frans C.S. Ramaekers1
1Department of Molecular Cell Biology, Cardiovascular Research Institute Maastricht, University Maastricht, PO Box
616, 6200 MD Maastricht, The Netherlands and2Department of Biomedical Engineering, Biomechanics and Tissue
Engineering, Eindhoven University of Technology, The Netherlands
Received June 22, 2004; Revised and Accepted September 6, 2004
Laminopathies comprise a group of inherited diseases with variable clinical phenotypes, caused by
mutations in the lamin A/C gene(LMNA). A prominent feature in several of these diseases is muscle wasting,
as seen in Emery–Dreifuss muscle dystrophy, dilated cardiomyopathy and limb-girdle muscular dystrophy.
Although the mechanisms underlying this phenotype remain largely obscure, two major working hypotheses
are currently being investigated, namely, defects in gene regulation and/or abnormalities in nuclear architec-
ture causing cellular fragility. In this study, using a newly developed cell compression device we have tested
the latter hypothesis. The device allows controlled application of mechanical load onto single living cells,
with simultaneous visualization of cellular deformation and quantitation of resistance. With the device, we
have compared wild-type (MEF1/1) and LMNA knockout (MEF2/2) mouse embryonic fibroblasts (MEFs),
and found that MEF2/2 cells show a significantly decreased mechanical stiffness and a significantly
lower bursting force. Partial rescue of the phenotype by transfection with either lamin A or lamin C prevented
gross nuclear disruption, as seen in MEF2/2 cells, but was unable to fully restore mechanical stiffness in
these cells. Our studies show a direct correlation between absence of LMNA proteins and nuclear fragility
in living cells. Simultaneous recordings by confocal microscopy revealed that the nuclei in MEF2/2 cells,
in contrast to MEF1/1 cells, exhibited an isotropic deformation upon indentation, despite an anisotropic
deformation of the cell as a whole. This nuclear behaviour is indicative for a loss of interaction of the dis-
turbed nucleus with the surrounding cytoskeleton. In addition, careful investigation of the three-dimensional
organization of actin-, vimentin- and tubulin-based filaments showed a disturbed interaction of these struc-
tures in MEF2/2 cells. Therefore, we suggest that in addition to the loss of nuclear stiffness, the loss of a
physical interaction between nuclear structures (i.e. lamins) and the cytoskeleton is causing more general
cellular weakness and emphasizes a potential key function for lamins in maintaining cellular tensegrity.
Nuclear lamins are karyoskeletal proteins located at the inner
nuclear membrane and belonging to the family of intermediate
filament proteins. Two main classes can be distinguished, i.e.
A- and B-type lamins. B-Type lamins are either products of
the LMNB1 gene, encoding lamin B1 (1), or the LMNB2
(LMN2) gene (2), encoding lamins B2 and B3 (3). Until
now at least four different proteins are known to arise from
the A-type lamin gene (LMNA) (4), i.e. lamin A, lamin
AD10, lamin C (5) and lamin C2 in (mouse) spermatocytes
(6). Unlike B-type lamins, which are expressed in most
eukaryotic cells (7,8), A-type lamins are differentially
expressed, i.e. A-type lamins are reduced or absent in early
Human Molecular Genetics, Vol. 13, No. 21 # Oxford University Press 2004; all rights reserved
*To whom correspondence should be addressed at: Department of Molecular Cell Biology, Cardiovascular Research Institute, University Maastricht,
PO Box 616, NL-6200 MD Maastricht, The Netherlands. Tel: þ31 433881366; Fax: þ31 433884151; Email: firstname.lastname@example.org
Human Molecular Genetics, 2004, Vol. 13, No. 21
Advance Access published on September 14, 2004
embryonic cells, in cells of a low degree of differentiation, in
cells of the cellular immune and hematopoietic system, and
in proliferating cells (7–9).
Although some functional aspects of the nuclear lamins
have been established, several other proposed functions
remain to be confirmed. For B-type lamins, it has become
clear that the absence of these proteins is not compatible
with life. Silencing of lamin B1 or lamin B2 by siRNA pro-
duces apoptotic features both in mammalian cells and in
cells from Caenorhabditis elegans (10,11). Apparently, the
presence of B-type lamins is critical for vital cellular pro-
cesses, such as the elongation phase during DNA synthesis
(12). As mutations in either LMNB1 or LMNB2 can cause
cell death, this can explain why no mutations in these genes
have been found in human heritable diseases.
In contrast to B-type lamins, the loss of A-type lamins leads
to specific loss of function. This can be seen in human herita-
ble diseases resulting from LMNA mutations and in LMNA
knockout mice, where the loss of A-type lamins leads even-
tually to death (13,14). However, the exact impact of the
impairment of LMNA expression on the functioning of cells
is not yet well understood.
A variety of inherited diseases are associated with
mutations in the LMNA gene. These diseases are collectively
called laminopathies, and include Emery–Dreifuss muscular
dystrophy (EDMD) (15), dilated cardiomyopathy type 1A
(16), limb-girdle muscular dystrophy type 1B (17), familial
partial lipodystrophy (18,19), Charcot–Marie–Tooth disease
type 2 (20) mandibuloacral dysplasia (21) and rare childhood
syndromes of premature ageing, the Hutchinson–Gilford pro-
geria (22–24) and atypical Werner’s progeria (25). To explain
this relatively heterogeneous spectrum of diseases, two main
working hypotheses are currently exploited. First, it is
suggested that patients with laminopathies experience mech-
anical weakness in cells, caused by a disturbed nuclear archi-
tecture. This mechanical stress hypothesis could explain the
loss of muscle tissues seen in the group of muscle failure-
related diseases. The second hypothesis trying to explain
development of symptoms deals with the loss of gene regulatory
functions of the A-type lamins. Studies have suggested that
A-type lamins can play a role in gene regulation by interaction
with the retinoblastoma protein (26,27), possibly mediated
by the Lamina Associated Protein 2 alpha (LAP2alpha) (28)
or by the prevention of transcriptional complex formation
by binding to transcription factors such as SREBP1 (29) and
In this article, we have examined the role of A-type lamins
in providing structural support to the nucleus and to cells in
general, by performing compression experiments on mouse
embryonic fibroblasts (MEFs) with normal expression of the
LMNA gene (MEFþ/þ cells), and on cells lacking expression
of the LMNA gene (MEF2/2 cells). As the nucleus seems to
account for most of the mechanical stiffness of a cell (31,32), a
disturbance in the nuclear architecture should be reflected by a
decreased mechanical stiffness of these cells.
To test this hypothesis, unconfined compression exper-
iments were performed on single cells, using a recently devel-
quantification of the mechanical resistance to compression,
and during the experiments cell deformation and cell
. The deviceenables
damage can be observed using confocal microscopy (33).
Our results show that, using this device, we can indeed
measure a significantly reduced mechanical stiffness in cells
lacking expression of the LMNA gene. In contrast to nuclei
of normal cells, the integrity of MEF2/2 nuclei was lost
after a compression experiment. Moreover, the preferred
deformation direction as seen in the nuclei of MEFþ/þ
cells was not observed in MEF2/2 nuclei. This behaviour
is explained by the absence of a link between the nucleus
and the cytoskeleton scaffolds in MEF2/2 cells. Rescue of
MEF2/2 cells with lamin A or lamin C restored some but
not all mechanical characteristics of the MEF2/2 cells.
Decreased resistance to compression in LMNA null cells
Cell cultures from MEFþ/þ and MEF2/2 cells were sub-
jected to compression at different passages, at variable den-
sities, and at different time points after seeding.
The low variation between measurements of the individual
cell types under these different conditions allowed us to group
them. Cellular stiffness of 36 MEFþ/þ cells and 31 MEF2/2
cells was evaluated. Figure 2 shows confocal recordings of
two representative cells from a MEFþ/þ and an MEF2/2
cell culture during compression. Full recordings of the beha-
viour of these cells during indentation are available as Sup-
plementary Material. During compression, the cell flattens
and enlarges dramatically in lateral directions (Fig. 2A and B).
Immediately after cellular disruption (seen in all cells during
compression) propidium iodide (PI) enters the cytoplasm
followed by an almost immediate concentration in the
nucleus, probably via the nuclear pores complexes. Binding
of PI to the chromatin shows a morphologically intact
nucleus after compression in the MEFþ/þ cell, and large
strands of DNA protruding into the cytoplasm in the case of
the MEF2/2 cell (compare Fig. 2A and B). Comparison of
the mean force needed to induce indentation of a cell in
z-direction (axial deformation) shows that, as expected, the
force needed to cause an axial deformation increased with
increasing indentation of the cells (Fig. 2C). Furthermore,
these mean force versus axial strain curves show that the
forces needed to compress a cell were larger for MEF þ þ,
than for MEF2 cells. These differences became statistically
significant from the point where the indentor caused more
than 27.5% axial deformation (Student’s t-test, P , 0.05).
Beyond 75% of axial deformation, the resistance of both
cell types increased rapidly, probably owing to the fact that
the tip of the probe reached the surface of the slide and/or
that the presence of cellular debris (?20% of the initial cellu-
lar height) prevented further compression.
In 50% of the cells, a typical decrease in the force could be
observed during compression, caused by rupture of the cellular
membrane. The maximum force at cell membrane bursting is
referred to as the bursting force. Two typical curves of individual
MEFþ/þ and MEF2/2 cells exhibiting this characteristic
decrease in force are shown in Figure 2D (arrows). The mean
bursting force for MEFþ/þ cells (8.7 + 3.0 mN) was signifi-
cantly higher than that for MEF2/2 cells (2.9 + 1.9 mN,
Student’s t-test, P , 0.0001). The accompanying axial
2Human Molecular Genetics, 2004, Vol. 13, No. 21
deformation at cellular rupture is significantly different for
both cell types, i.e. 68.8 + 7.5% for MEFþ/þ cells and
62.0 + 5.8% for MEF2/2 (Student’s t-test, P , 0.05). For
rescued cells, no clear cellular membrane rupture point could
be calculated from the force versus deformation plots. Poss-
ibly, transfection influences the cellular membrane properties.
Lamin A and/or lamin C rescue does not restore
mechanical resistance to deformation
In order to investigate whether lamin A or lamin C tagged with
the green fluorescent protein (GFP) are capable of rescuing the
MEF2/2 phenotype, we transfected lamin A or lamin C into
MEF2/2 cells. In order to examine whether a correct nuclear
structure was restored, we studied the (re)distribution of other
nuclear markers next to lamins. The localization of emerin,
which is known to be largely dependent on the presence of
lamin A and/or lamin C (13), shows the expected staining
pattern at the nuclear membrane in unaffected MEFþ/þ
cells (Fig. 3A), whereas in MEF2/2 cells, emerin is distrib-
uted throughout the endoplasmic reticulum with only a minor
portion of emerin present in the nuclear periphery (Fig. 3B).
Another marker for verification of a correct nuclear membrane
organization is the nuclear pore complex protein nucleoporin
p62. In normal cells, nuclear pores are evenly distributed
over the nuclear membrane surface. In lamin null cells,
nucleoporin can be absent from large areas of the nuclear
membrane, as seen in Figure 3C (arrow). Transient restoration
of lamin A or lamin C expression or both could not fully
restore the normal architecture of affected nuclei. First, the
typical honeycomb appearance of lamins at the distal poles
of the nuclei, seen in many cases of mutated lamins, was
still apparent after cotransfection with (GFP-tagged) lamin A
and lamin C (Fig. 3D), with a relatively heavy decoration of
lamin A– and lamin C–GFP in these areas. These findings
indicate that distorted MEF2/2 nuclei did not revert to
their normal phenotype after rescue. Indeed, the percentage
of abnormal nuclei on the basis of nuclear DNA staining
remained unaltered (?5% of the cells). In addition, in the
transfected cells, emerin was not concentrated at the nuclear
membrane but was still mainly dispersed throughout the endo-
plasmic reticulum (Fig. 3E), similar to cells lacking A-type
lamins (compare Fig. 3C and E).
Further, co-localization studies of cells transfected with
lamin A and stained for p62 expression showed that nuclear
pore complex distribution remained depleted in some areas
of the nuclear envelope (Fig. 3F, arrow). Similarly, lamin B
remained absent in the same regions (data not shown).
Measuring resistance to indentation of MEF2/2 cells
transfected with A-type lamins did not show significant differ-
ences when compared with untransfected MEF2/2 cells
(Fig. 3G). Strikingly, no significant differences in cellular
stiffness were observed, when comparing rescued cells with
morphologically normal nuclei to cells with aberrantly loca-
lized A-type lamins. Apparently, restoring A-type lamin
expression and incorporation into the nuclear lamina is not
sufficient to restore cellular stiffness.
Differences in nuclear damage resulting from
On the basis of the abnormalities in nuclear morphology, pre-
viously seen in MEF2/2 cells (13), we anticipated that
lamina and nuclear membrane aberrations would cause prema-
ture disruption of nuclei in affected cells during compression.
Indeed, most nuclei of MEF2/2 cells could not endure the
complete compression regimen without bursting or showing
otherwise irreversible nuclear damage. Figure 4 shows the
nuclear morphology of MEFþ/þ and MEF2/2 cells after
compression, and compares the number of affected nuclei
in the two types of cell culture. Although the majority of
MEFþ/þ nuclei remained largely intact throughout the
Figure 1. Schematic representation (A) and picture (B) of the cellular compression device. Cells grown on cover slips are placed in the incubation chamber, and
individual cells are tracked by confocal imaging using the 40? oil objective. Next, the indentor is placed on top of a single cell, and the cell is compressed.
Human Molecular Genetics, 2004, Vol. 13, No. 213
Figure 2. Confocal image and force recordings of cellular deformation during the compression routine. (A) Confocal images of a characteristic MEFþ/þ cell,
stained with Cell Tracker green and propidium iodide (PI) during compression and subsequent relaxation. The influx of PI into the cell starts immediately after
cellular rupture and rapidly concentrates at the nuclear DNA. (B) Idem for a MEF2/2 cell. Note the presence of large strands of DNA protruding from the
damaged nucleus into the cytoplasm after compression. (C) Plot of compression force versus axial deformation of MEFþ/þ and MEF2/2 cells. Asterisks indi-
cate the statistically significant differences between both cell types. Average of n ¼ 36 (MEFþ/þ) and n ¼ 31 (MEF2/2). (D) Force plots of MEFþ/þ cell
from (A) and of MEF2/2 cell from (B), showing the difference in bursting force for both cell types (arrows). t1–t4 correspond to time points t1–t4 in (A) and
(B). (E) Bursting force plots showing average force needed to induce cellular bursting and corresponding axial deformation at which cellular bursting took place.
Statistically significant differences were noted between MEFþ/þ and MEF2/2 cells, both for force needed (# denotes P , 0.001, Student’s t-test) and axial
deformation (?denotes P , 0.05, Student’s t-test). Bar represents 20 mm in all figures.
4Human Molecular Genetics, 2004, Vol. 13, No. 21
complete compression regimen (Fig. 4A and B), most
MEF2/2 nuclei became damaged, resulting in chromatin
protruding into the cytoplasm as seen by PI staining.
Figure 4C shows a massive eruption of DNA outside of the
nucleus, whereas in Figure 4D more subtle DNA protrusions
into the cytoplasm are observed (arrows in Fig. 4D).
A-Type lamin rescue reduces nuclear damage
Rescue with either lamin A(–GFP) or lamin C(–GFP) had a
positive impact on maintaining the nuclear shape during com-
pression. After transfection of A-type lamin–GFP into
MEF2/2 cells, a morphologically intact nuclear surface
was observed in many cells, highlighted by the lamin GFP
signal lining the nuclear lamina (Fig. 4H). These nuclei in
general remained intact after compression, as deduced from
the PI staining. In cells with an irregular lamina after rescue,
nuclear areas with a relatively strong A-type lamin–GFP
expression would not burst, whereas simultaneously the
nuclear areas with less A-type lamin expression did burst
upon indentation, revealed by PI labelling protruding into
the cytoplasm after compression (Fig. 4E and F). Strikingly,
a cell with a predictable weak spot in the nucleus before com-
pression (Fig. 4G, inset, arrow), did burst in this area before
any noticeable damage was seen in the remaining parts of
the nucleus (Fig. 4G; Supplementary Material). Quantitative
comparisons of visual nuclear damage among MEF2/2,
MEFþ/þ and transfected MEF2/2 cells (Fig. 4I) show
that, indeed, nuclear coherence is increased after rescue,
resulting in fewer cells with prominent nuclear damage after
MEF1/1 nuclei deform anisotropically, MEF2/2
To explain the apparent discrepancy between the restoration
of nuclear coherence and simultaneous lack of restoration of
cellular stiffness, we more closely examined the deformation
behaviour of cells and nuclei during compression. In
MEFþ/þ cells, nuclear deformation was mainly anisotropic,
showing nuclear enlargement perpendicular to the long axis
in cells with a prevalent growth orientation (Fig. 5A).
However, careful inspection of nuclear deformation direction
of MEF2/2 cells did not reveal this correlation in most
cells (Fig. 5B). The nuclei deformed equally into all directions
(isotropic), with no correlation with the initial cellular orien-
tation. Comparing the width to length ratio in MEFþ/þ
cells before indentation and at the time of maximum indenta-
tion, an average relative increase of the width to length ratio of
40% was observed (Fig. 5C). The initial shape of the nucleus
did not predict the anisotropic behaviour, as both round and
oval nuclei deformed with similar changes in width to
length ratios. MEF2/2 cells or A-type lamin-rescued cells
showed only an average increase in the length to width ratio
of 8–11%, indicating an almost isotropic deformation in
most of these cells.
The anisotropic deformation of nuclei is not caused by the
shape or structure of the nuclei in itself. When isolated
Figure 3. Rescue experiment of MEF2/2 cells usinglaminA–GFPand lamin
C cDNA in pcDNA3. (A and B) Immunofluorescence patterns of untransfected
MEFþ/þ (A) and MEF2/2 cells (B) showing the pronounced difference in
emerin localization. Note that a MEF2/2 cell shows mainly endoplasmic reti-
culum staining, next to some fluorescent signal at the nuclear membrane (B),
whereas in the MEFþ/þ cell, emerin is exclusively localized at the nuclear
membrane (A). (C) p62 is unevenly distributed in a small percentage (3–5%)
of untransfected MEF2/2 cells, being absent at the lower part of the nucleus
(arrow). (D) Fluorescence of a MEF2/2 cell co-transfected with lamin A–
GFP (left) and lamin C cDNA (right). Although both lamins are present in
of MEF2/2 cells, even 6 days after transfection. (E) lamin A–GFP fluor-
escence (left) and emerin immunostaining (right) showing that in the lamin A
rescued cell, emerin is not relocalized to the nuclear membrane but is still dis-
tributed throughout the endoplasmic reticulum. (F) lamin A–GFP fluorescence
(left) and p62 immunostaining (right). In this cell, transfected with lamin A–
GFP (left), nuclear pore complexes visualized by the p62 antibody (right) are
still confined to the lamin-containing part of the nuclear membrane, and
absent from areas devoid of lamin A (arrow). (G) Force versus axial defor-
mation curves of MEF2/2 cells and cells rescued with either lamin A–GFP
or lamin C-GFP. No significant differences between different MEF2/2 cells
and rescued cells are seen. Bar represents 10 mm in all figures.
Human Molecular Genetics, 2004, Vol. 13, No. 215
nuclei, obtained by treatment of MEFþ/þ or MEF2/2 cells
with acetic acid, were subjected to compression, both round
and oval nuclei of both cell lines deformed strictly
isotropically (data not shown).
Cytoskeletal disorganization in MEF2/2 cells
The direction of nuclear deformation is most likely determined
by extranuclear factors involved in maintaining cellular shape
Figure 4. Nuclear damage during compression, visualized with Cell Tracker Green combined with PI (A–D) or with Cell Tracker Orange combined with PI
(E–H). In all samples, the prominent fluorescent signal in the central region of the cell results from PI fluorescence. Note that especially Cell Tracker Orange
forms cytoplasmic aggregates in these cells. (A and B) Absence of visual nuclear damage in two MEFþ/þ cells after compression. The nuclear shape of these
nuclei remains intact, with chromatin still located in a sharply delineated nuclear compartment. (C and D) Damaged nuclei of MEF2/2 cells that did burst
during compression, with chromatin protruding into the cytoplasm as large (C) or small irregular lobules (arrows in D). (E–H) Depending on the amount
of transfected lamins at the nuclear membrane, rescued nuclei transfected with lamin A(–EGFP) showed no disruption, or partial disruption at areas with
so-called honeycomb lamin patterns (E), and less disruption in areas with high levels of lamin A (F, arrow). Similar observations were made for lamin
C(–EGFP) rescued cells with preservation of nuclear shape in cells, with high levels of lamin C (H) and disruption of areas lacking lamin C (G, arrow).
Inset in (G) shows lamin C fluorescence before compression and the predicted weak spot in the nucleus, which indeed did burst during compression
(Supplementary Material, Movie S3). (I) Quantitative comparison of visual nuclear damage between MEFþ/þ and MEF2/2 cells and A-type lamin-
rescued cells. The number of analysed cells for each category is shown in parentheses. Undamaged nuclei: all DNA remains in nucleus after compression.
Partial damage: one or more areas of the nucleus show protrusions of DNA into the cytoplasm, whereas the remaining nucleus remains intact. Damage:
DNA protrudes into the cytoplasm into all directions. Note that nuclear damage as seen in MEF2/2 is largely prevented after rescue with either lamin
A or lamin C. Bar represents 10 mm in all figures.
6 Human Molecular Genetics, 2004, Vol. 13, No. 21
and orientation. As cytoskeletal structures are the likely candi-
dates in this respect, we examined their organization in
MEF2/2 and MEFþ/þ cells. Figure 6 shows representative
cells from these populations. At the peripheral regions of these
cells, no obvious differences in the organization of stress fibres
were observed. However, in areas close to the nucleus, the
actin stress fibres seemed to be disrupted in MEF2/2 cells
and not in MEFþ/þ cells (compare Fig. 6A and B). In
these areas, numerous dots were observed consisting of actin
not correctly organized into fibres (Fig. 6B). We observed
actin abnormalities in 5–10% of MEF2/2 cells, with
considerable regional variations within the same sample and
even larger variations between samples. Careful inspection
of the microtubule network (Fig. 6C and D) in perinuclear
areas showed that the characteristic concentration of microtu-
bules around the nucleus starting at the centrosomes, as seen in
MEFþ/þ cells (Fig. 6C), is disturbed in MEF2/2 cells
(Fig. 6D). From the centrosomes, microtubules were diverted
into all directions, showing little or no orientation at the
nuclear periphery. Although in most normal cells the nuclear
membrane seemed to be completely covered by microtubules,
large nuclear membrane areas in MEF2/2 cells were devoid
Figure 5. Determination of direction of deformation of nuclei during compression. (A) MEFþ/þ cell showing anisotropic deformation (hatched ovals) compared
to the long axis of the cell (straight line). (B) MEF2/2 cell showing isotropic deformation during compression, i.e. an even expansion in lateral directions. (C)
Comparison of the type of nuclear deformation in MEFþ/þ cells, MEF2/2 cells and A-type lamin-rescued cells. The number of analysed cells is shown in
parentheses. The degree of (an-)isotropic nuclear deformation during compression is expressed in width to length ratio in cells with a well-defined growth direc-
tion. On the basis of this length axis, the width to length ratio before compression and at the moment of maximal expansion are compared. This resulted in an
average increase of 40% of the width compared with the length in MEFþ/þ cells, and 8–11% in other cell types. Asterisk indicates significant differences in
ratio before and after full indentation (P , 0.05, Student’s t-test). Before, before compression; After, after full indentation, i.e. at maximum compression. Bar
represents 10 mm in (A) and (B).
Human Molecular Genetics, 2004, Vol. 13, No. 217
Figure 6. (A and B) Actin staining in MEFþ/þ (A) and MEF2/2 (B) cells with Phalloidin-Texas Red (left) and counterstained with Syto-13 (right). In both the
cases, a single slice from a confocal z-series is shown. Note the apparently truncated actin filaments and granular actin staining patterns in the perinuclear region
of a MEF2/2 cell, whereas intact actin fibres are seen in the MEFþ/þ cell. (C and D) Microtubular organization in a MEFþ/þ (C) and a MEF2/2 cell (D). In
both the cases, a single slice from a confocal z-series is shown. Microtubule staining with the E7 antibody shows a close association of microtubule endings with
the nucleus in the MEFþ/þ cell (C, left panel), whereas the perinuclear area of the MEF2/2 cell lacks microtubules in part of the perinuclear area (D, left
panel). Nuclei are counterstained with PI (C and D, right panels). (E–H) Vimentin organization of MEFþ/þ (E and F) and MEF2/2 cells (G and H). Vimentin
immunofluorescence with antibody BV118 shows disturbed vimentin organization in MEF2/2 cells (G and H, left panels), when compared with vimentin lab-
elling in MEFþ/þ cells (E and F, left panels). Note the absence of vimentin in large areas at the nuclear periphery and the patched staining in other areas. In (E)
and (G) projections of confocal z-series are shown, stressing the dramatic differences in vimentin distribution between MEFþ/þ and MEF2/2 cells, whereas in
(F) and (H) a single slice from a confocal z-series is shown. Bar represents 10 mm in all figures.
8 Human Molecular Genetics, 2004, Vol. 13, No. 21
of microtubule attachments. The number of cells with abnor-
mal tubulin organization was similar to the number of cells
with actin disturbances. However, as actin and tubulin proteins
abnormalities only became fully apparent after confocal
microscope recording at high magnification of individual
cells, the number of abnormal cells was most likely underesti-
mated. An even more disturbed vimentin network was seen
(Fig. 6E–H). MEFþ/þ cells show a characteristic perinuclear
filament organization (Fig. 6E and F), whereas in MEF2/2
cells several disturbances were noted (Fig. 6G and H). A strik-
ing loss of vimentin organization in the cytoplasm was seen as
regions with very bright patches of vimentin, whereas other
areas were almost completely devoid of vimentin fibres. The
typical wrapping of vimentin around the nucleus was almost
absent in these cells. This vimentin disorganization was
much more obvious, and could be readily observed even
with standard fluorescence microscopy. The percentage of
MEF2/2 cells with these vimentin abnormalities was
between 20 and 30%.
The phenotype of several of the diseases associated with
A-type lamin mutations, points to a correlation between cellu-
lar and/or nuclear weakness, and the loss in mechano-sensitive
tissues, such as heart and skeletal muscle. EDMD, limb-girdle
dystrophy and dilated cardiomyopathy are all characterized by
the loss of muscle tissue (reviewed in 34). Next to stretching
forces, muscle cell nuclei also encounter heavy local com-
pression forces during muscle activity. As muscle strength
increases with age, nuclear strain will increase with age as
well, possibly explaining the gradual increase of symptoms
in most patients (34). B-Type lamins are crucial for cellular
survival, and cells depleted of B-type lamins will die via
apoptosis (10). Also, in cell lines with A-type lamin mutations,
increased apoptosis can be observed, especially after trigger-
ing of differentiation (35,36). Organisms lacking A-type
lamin expression, including LMNA null mice (13) or
humans expressing no functional lamins (14), die shortly
after birth. The occurrence of apoptotic cells in cardiac
muscle cells of LMNA deficient mice is probably a late
effect, which would imply that these cells die as a result
from ‘wear and tear’ effects of repeated contractions (37).
A possible explanation would be the loss of cellular stiff-
ness due to A-type lamin mutations. On the basis of the locali-
zation of the nuclear lamina, it has already been postulated
long ago that lamins play a prominent role in maintaining
nuclear integrity (38). Indeed, cellular extraction studies in
cell lines harbouring A-type lamin mutations indicate that
cells with mutations display increased nuclear fragility upon
More direct evidence for nuclear weakness as a result
of A-type lamins abnormalities was recently supplied by
Lammerding et al. (40), who showed that, indeed, defective
nuclear mechanics are observed in LMNA null fibroblasts
after stretching of cells. These authors concluded that the
absence of lamins causes loss of nuclear stability, resulting
in cellular weakness. In order to examine directly the effects
of strain onto the nucleus, we chose an alternative method to
mechanically challenge cells, i.e. by using cellular com-
pression rather than mechanical stretch. Our set-up enables
the direct visualization of the response of the nucleus during
compression, with simultaneous recording of the force
needed to exert this indentation. We measured the mechanical
stiffness in normal fibroblasts and fibroblasts completely
lacking LMNA gene expression. We chose fibroblasts, as
these cells are easy to obtain from (future) patient samples
and these cells should reflect general mechanical weakness in
cells, if caused by structural abnormalities. Moreover, loss of
fibroblasts themselves could induce weakening of connective
tissues at several sites in the body. Using a newly developed
cell loading device (33), we found a significantly higher resist-
ance against indentation force in MEFþ/þ cells than in
MEF2/2 cells. Moreover, the force needed to cause cellular
bursting was significantly higher in MEFþ/þ cells.
Simultaneous confocal microscopic imaging allowed us to
more closely examine the type and direction of deformation
of individual nuclei and cells. Although most wild-type
nuclei exhibited an intact morphology after compression,
90% of the LMNA null nuclei burst completely after com-
pression, leaving a disrupted nuclear mass spreading into the
cytoplasm. This percentage is considerably higher than the
rupture observed in 3–5% of the cells after cell strain using
cellular stretching (40), and implies that the mechanical
weakness of these cells could be an important factor in the
development of laminopathies.
This nuclear damage due to compression could be largely
overcome by transfection of A-type lamins into these cells.
However, in these rescue experiments, mechanical stiffness
of the affected cells was not restored. In order to find an expla-
nation for this phenomenon, we investigated the protein
organization of these rescued cells more closely. It became
clear that the presence of lamin A– and/or lamin C–GFP in
the lamina did not result in the localization of emerin to the
nuclear membrane. Moreover, part of the cells transfected
with A-type lamins still showed an abnormal nuclear mor-
phology characteristic for LMNA2/2 cells (13). A possible
explanation for not restoring the normal phenotype in these
cells was the inability to form stable transfectants. Although
we did get an efficiency of transfection of up to 10%, these
numbers are significantly lower than the efficiency obtained
by electroporation (up to 58%) (41). As a result, the
expression levels of A-type lamins in transfected cells might
be too low for obtaining restoration of nuclear architec-
ture (41). Alternatively, the manifestation of changes in the
nuclear structure phenotype might require one or more
cycles of nuclear disassembly and reformation as occurs
during mitosis (42). In our transfected cell, we never saw
mitosis. Some cells would remain single fluorescent cells for
up to 1 month, before disappearing from the culture.
This partial rescue indicated that the diminished cellular
strength in the LMNA2/2 cells results from factors other
than the absence of A-type lamins in the nucleus. Although
nuclear strength is a major component of total cellular stiffness
(31,32), the interaction of cytoskeletal and nucleoskeletal
elements is important for maintenance of the full cellular stiff-
ness. According to a previously postulated tensegrity model
(43,44) for cellular and tissue strength, this can only be
Human Molecular Genetics, 2004, Vol. 13, No. 219
achieved if all cyto- and nucleoskeletal components are cor-
rectly assembled. A strong indication that, indeed, the partially
rescued cells still lacked tensegrity came from our obser-
vations on the direction of deformation of compressed
nuclei. As observed in other cell lines (data not shown),
normal nuclei exhibit anisotropic deformation during com-
pression; if cells have a prevalent direction of orientation
(the length axis), the nuclei will deform in a direction perpen-
dicular to this axis. Indeed, most MEFþ/þ showed this type
of anisotropic deformation during compression. However,
MEF2/2 cells as well as A-type lamin-rescued cells show
an isotropic deformation. Apparently, the interconnection
between nuclear components such as lamins and the cyto-
plasm was lacking in these cells. Anisotropic deformation is
most likely caused by the attachment of the cytoskeletal com-
ponents to the nucleus, including microfilaments, intermediate
filaments and possibly microtubules. Indeed, when cyto-
skeletal connections are disrupted by acetic acid and detergent
extraction, isolated intact nuclei deform isotropically during
compression. These findings are in line with the viscoelastic
behaviour of isolated nuclei (31). The observation of
decreased cytoskeletal stiffness in MEF2/2 cells is in this
respect very intriguing (40).
There is growing evidence that, indeed, the cytoskeleton
is closely connected to the nucleus. Microfilaments are
indirectly connected to the nucleus, most likely via nesprins
(recentlydiscovered giant proteins spanning the completecyto-
plasm of the cell) (45). Of these, nesprin1a is localized in the
intranuclear compartment of the cell, and interacts with
A-type lamins as well as emerin at the nuclear envelope (46).
Binding of nesprin1a to these molecules occurs at the most
C-terminal part of nesprin to A-type lamins and at a their
N-terminal part to emerin. A more recent study indicates that
nesprin 1a is the C-terminal part of a much larger protein,
nesprin 1, which is not present in the nucleus but spans large
areas of the cytoplasm (45). Interestingly, the N-terminal
part of this large protein binds prominently to actin (45). A
second member of the nesprin family, called NUANCE (47)
or nesprin-2 (45,48), shows a similar topology, i.e. binding to
actin in the N-terminal region of the molecule and anchoring
to the nuclear membrane via the common KLS motif (45). In
cells lacking A-type lamin expression, the proper binding of
nesprin 1a is disturbed (14). Re-localization of nesprin 1a
from the nucleus to the cytoplasm occurs in human fibroblasts
lacking expression of the LMNA gene. The correct localization
of this protein is restored if these cells are rescued with either
lamin A or lamin C (14). In these experiments, in contrast to
our study, the correct localization of other nuclear proteins
such as emerin is restored as well.
Although it is not yet shown that nesprin contributes to
mechanical stiffness, it is well known that actin is a major
component in maintaining cellular stiffness (49,50). Moreover,
disruption of the actin cytoskeleton by cytochalasin-D causes
nuclei to respond differently to compression (51), suggesting a
connection of actin to the nucleus. Careful examination of
actin architecture in LMNA2/2 cells did reveal abnormalities
in actin filament formation not seen in control cells. These
abnormalities in actin molecules, especially present in peri-
nuclear areas of affected cells, could have been easily over-
looked in previous studies, since only confocal microscopy
allows the detailed inspection of the cellular interior covered
with a dense layer of brightly fluorescent actin fibres. Next
to an indirect binding of actin to the nucleus via nesprins,
other studies showed direct interaction between cytoplasmic
actin and emerin (52), thought to be localized exclusively at
the inner nuclear membrane after transport from the endoplas-
mic reticulum to the nucleus. However, the presence of emerin
(or similar transmembrane proteins containing a LEM domain)
at the outer nuclear membrane could be the missing link in
connecting the inner and the outer nuclear membranes. Such
a link is necessary if direct mechanical strain is to be trans-
duced from the cellular membrane to the nucleus. This hypoth-
esis is supported by the finding that similar muscle dystrophic
diseases are caused by mutations in the actin gene itself
(myopathies; reviewed in 53), or by inappropriate attachment
of actin to the periphery of the cell. For instance, limb-girdle
muscular dystrophy type 1A is associated with the actin-
binding protein myotilin (54); sporadic dilated cardiomyopa-
thies can be associated with dystrophin gene mutations (55)
and actinin mutations can be associated with congenital
muscular dystrophy (56).
The importance of the other two main cytoskeletal
components, microtubules and vimentin intermediate fila-
ments, for cellular stiffness has also to be considered. Both
components are important for resisting mechanical load (43),
and are clearly disorganized in MEF2/2 cells. Direct
binding of microtubules to the nuclear surface has recently
been suggested (57). In addition, Patterson et al. (58)
showed in Drosophila the close interaction between lamins,
Klarsicht and the microtubule organization centre. The
proper localization of Klarsicht appeared to be dependent on
the presence of lamins. Similarly, the correct targeting of
nesprins 1a, containing a Klarsicht-like signal, to the
nuclear membrane is dependent on the presence of A-type
lamins in the nuclear lamina (14). Disruption of the desmin
intermediate filament in muscle cells of LMNA null mice,
seen as the loss of attachment sites for desmin at the nuclear
envelope (37) suggests a close correlation between A-type
lamin expression and a proper desmin organization. Although
for intermediate filaments, similar to the other cytoskeletal
components, no direct connection with the nuclear membrane
is shown, these EM studies as well as other cellular extraction
studies (59) do indicate the existence of such a connection.
The disorganization of vimentin intermediate filaments, as
seen in our study, confirms this correlation. Moreover,
mutations in intermediate filaments themselves, in this case
in the neurofilament light subunit, can cause Charcot–
Marie–Tooth neuropathy type 2 (60), similar to the disease
caused by a LMNA mutation (20).
We observed the most dramatic cytoskeletal changes in the
vimentin network. Clearly, more research should solve the
exact (loss of) interaction between these cellular components.
In summary, we have demonstrated that fibroblasts lacking
A-type lamin expression have a significantly reduced mechan-
ical stiffness and reduced bursting force, when compared with
normal fibroblasts. Next to a loss in nuclear stiffness, we also
observed aberrant deformation behaviour during compression.
This points towards a loss of cytoskeletal–nucleoskeletal
interactions (via emerin and nesprins), which could explain
the general cellular weakness in laminopathies.
10 Human Molecular Genetics, 2004, Vol. 13, No. 21
MATERIALS AND METHODS
Wild-type mouse embryonic fibroblasts (MEFþ/þ) as well
as LMNA knockout mouse embryonic fibroblasts (MEF2/2)
were obtained as described previously (13). Cells were
grown at 378C in a humidified incubator containing 5% CO2
in DMEM (ICN Biomedicals
Netherlands) containing 10% fetal bovine serum. Cells were
passaged by splitting at 1:3 to 1:5 ratios using a 0.125%
trypsin/0.02 M EDTA/0.02% glucose solution in PBS.
In order to restore LMNA protein expression in LMNA null
cells, MEF2/2 cells were transfected using Genejammer
(Invitrogen) according to the manufacturer’s instructions.
Typically, 10–20% of the MEF2/2 cells were transfected.
Cells were transfected either with lamin A–EGFP or with
lamin C–GFP for mechanical stiffness studies. The GFP-tag
was used to identify transfected cells in living cell cultures.
Both lamin A–EGFP and lamin C–EGFP were obtained by
cloning the lamin A or lamin C cDNA fragment from the
PS65T-C1 vector (61) into the EGFP-vector (Clontech
Laboratories Inc., Palo Alto, CA, USA). In order to study
morphological nuclear changes by immunofluorescence,
cells were transfected with lamin A–EGFP, lamin C–EGFP
alone, or were co-transfected with lamin A–EGFP and
lamin C cDNA cloned into pcDNA3 (Invitrogen Life Technol-
ogies, Breda, The Netherlands). No stable transfectants could
be generated from this cell line.
After 2–5 days of transfection, cells were plated onto glass
slides, grown overnight and fixed with 4% formaldehyde in
PBS for 15 min, followed by permeabilization in 0.1%
Triton X-100 for 10 min at room temperature (RT). Alterna-
tively, cells were fixed in methanol (2208C) for 5 min.
Primary antibodies were applied onto the cells for 1 h.
The following primary antibodies were used:
. Mouse monoclonal antibody (MoAb) 133A2 (IgG3, 3 mg/ml
purified immunoglobulin) (62,63) was a kind gift from
Dr Y. Raymond (Montre ´al, Canada). This antibody recog-
nizes lamin A and is reacting with the epitope consisting
of amino acids 598–611.
. Affinity-purified rabbit polyclonal antiserum RaLC (1:50
VSGSRR (position 567–572) of human lamin C, and
exclusively reactive with lamin C and not lamin A (64).
. Affinity-purified rabbit polyclonal antiserum to lamin B1,
kindly provided by Dr J.C. Courvalin (INSERM, Paris,
. Mouse MoAb to nucleoporin p62 (IgG2b, dilution 1:300;
Transduction Laboratories, Lexington, KY, USA) (65).
. Mouse MoAb NCL–emerin to emerin (IgG1, dilution 1:60;
Novocastra, Newcastle upon Tyne, UK).
. Mouse MoAb RV202 to vimentin (IgG1, undiluted; Mubio
Products B.V., Maastricht, The Netherlands).
. Mouse MoAb BV1118 to vimentin (IgG1, diluted 1:5;
kindly supplied by C. Viebahn, Bonn, Germany).
. Mouse MoAb E7 against tubulin (IgG1, diluted 1:10;
Developmental Studies Hybridoma Bank, Iowa City, IA,
After washing in PBS, secondary antibodies were applied for
1 h at RT. Secondary antibodies used are FITC-conjugated
rabbit anti-mouse Ig (1:100, DAKO, Glostrup, DK), FITC-
conjugated goat anti-rabbit Ig (1:50, SBA/ITK, Birmingham,
AL, USA), Texas Red conjugated rabbit anti-mouse Ig
(SBA/ITK) and Texas Red conjugated goat anti-rabbit Ig
(1:50, SBA/ITK). After final washings in PBS slides were
mounted in 90% glycerol, 0.02 M Tris–HCl pH 8.0, 0.8%
Merck, Darmstadt, Germany) containing 0.5 mg/ml diami-
dino-2-phenylindole (DAPI, Sigma) or 1 mg/ml PI and
0.1 mg/ml RNase for DNA staining.
Cells were cultured on cover slips, fixed and permeabilized
as described earlier, and incubated with Texas Red-
conjugated Phalloidin (dilution 1:50; Molecular Probes,
Leiden, The Netherlands). Nuclei were counterstained for
15 min with Syto-13 (1 mM; Molecular Probes), recognizing
Confocal laser scanning microscopy
Fluorescent samples were imaged using a Bio-Rad MRC600
confocal microscope (Bio-Rad Laboratories Ltd, Hemel
Hempstead, UK) equipped with an air-cooled Argon–
Krypton mixed gas laser and mounted onto an Axiophote
microscope (Zeiss), using oil-immersion objectives (40?,
NA ¼ 3D1.3 or 63?, NA ¼ 3D1.4). The laser-scanning
microscope was used in the dual parameter set-up, according
to the manufacturer’s specifications, using dual wavelength
excitation at 488 and 568 nm. Emission spectra were separated
by the standard sets of dichroic mirrors and barrier filters.
Optical sections were recorded in the Kalman filtering mode
using 4–8 scans for each picture. Z-Series were generated
by collecting a stack consisting of optical sections using a
step size of 0.18 or 0.36 mm in the z-direction.
The Huygens System image restoration software (Scientific
Volume Imaging B.V., Hilversum, The Netherlands) was used
to improve the effective resolution of some of the confocal
images and to reduce background noise. Because of the
photon limited character of the data a maximum likelihood
estimation algorithm (66) was used.
Mechanical compression studies using a single cell
After 24 or 48 h of transfection, cells were seeded onto glass
cover slips and allowed to adhere to the glass surface for
18 h. Cells were seeded at a density low enough to allow
compression of single cells. Cellular viability was examined
by the addition of 5 mg/ml PI to the culture medium. In
order to visualize cellular deformation during the experiments,
Human Molecular Genetics, 2004, Vol. 13, No. 2111
living cells were labelled with 7.5 mM Cell Tracker Green
(CTG, Molecular Probes). This green fluorescent dye (exci-
tation 488 nm, emission 520 nm) shows a diffuse staining in
all cellular compartments, with often an increased intensity
in the nuclei of cells. In cells, transfected with EGFP-tagged
lamins, contours of the complete cells were visualized by
addition of 10 mM Cell Tracker Orange (Molecular Probes)
to the culture medium.
Next to cultured cells, isolated nuclei were subjected to
compression. For this purpose nuclei were extracted from
cell cultures using an extraction solution containing 1%
acetic acid and 0.01% NP40 (Fluka) in water. After extraction
for 5 min at RT, nuclei were washed and transferred to the
The set-up used to study the response of cells to indentation
forces has been described previously (33). Briefly, this set-up
consists of a cell-loading device, a cell culture chamber in
which cells can be grown at optimal cell culturing conditions
(378C, 5% CO2in an humidified atmosphere), an inverted
confocal microscope (Zeiss LSM510; Zeiss, Jena, Germany)
and computerized electronic control of microscope and
compression device. The complete set-up is shown in
Figure 1. Full characteristics and technical details about
the newly developed cell loading device have been published
elsewhere (33). Summarizing these data, we can state that
the device is capable of controlled compression of a single
cell while simultaneously recording the force needed to
induce cellular deformation. A combination of three microma-
nipulators and a piezoelectric system allows a controlled
displacement of the glass indentor (diameter 60 mm) in
three dimensions with a displacement range of 5–15 mm
and a frequency range of 0–25 Hz. The resolution of
the force transducer is 10 nN. The device enables very rapid
compression and relaxation, as well as slow or repetitive
compression of cells.
Best reproducible results were obtained with a continuous
indentation regimen applied to cells. The indenter was
moved with a constant velocity, such that the cell was com-
pressed completely within 15 s. Force and position of the
piezoelectric system were recorded continuously with a
sampling frequency of 100 Hz. Before deformation, a com-
plete stack of confocal images of a cell was recorded with a
lateral sampling size of 0.3 ? 0.3 mm2and a slice thickness
of 0.4 mm. During the experiment, a time series of 2D confo-
cal images of the cell was made with a lateral sampling size of
0.22 ? 0.22 mm2and a temporal sampling time of 1.7 s per
image. The CTG stain was excited using an Argon ion laser
at 488 nm, with emission recorded between 515 and 540 nm.
PI or Cell Tracker Orange were excited using a helium neon
laser at 543 nm, with emission recorded above 580 nm (33).
The percentage of indentation in z-direction owing to com-
pression, defined as axial deformation of the cell, was calcu-
lated indirectly in the following way:
1z¼1 2 ðzindentor2 zdebrisþ HdebrisÞ
where H0is the initial height of the cell and Hdebristhe height
of the layer of cell debris at position zdebris. zdebrisis defined
as the position of the indentor at a force of 20 mN, where
only a thin layer of cell debris was left over of the cell. In a
separate experiment, the height of the cell debris layer was
measured for 10 cells using a 63?, 1.4 numerical aperture,
oil-immersion objective (Plan-Apochromat, Zeiss, Germany)
at a force of ?20 mN. Hdebrisappeared to be 1.0 + 0.1 mm.
Supplementary Material is available at HMG Online.
The authors wish to thank Dr Brian B. Burke (Department of
Anatomy and Cell Biology, University of Florida, Gainesville,
FL, USA) and Dr Colin L. Stewart (Laboratory of Cancer and
Developmental Biology, NCI-FCRDC, Frederick, MD, USA)
for providing the embryonic fibroblast cells from LMNAþ/þ
and LMNA2/2 mice. The authors thank Dr Jean-Claude
Courvalin (INSERM, Institut Jacques Monod, Paris) and Dr
Christoph Viebahn (Department of Anatomy, University of
Bonn) for kindly providing antibodies to lamin B1 and vimen-
tin, respectively. We thank Dr Christopher J. Hutchison
(School of Biological and Biomedical Sciences, University
of Durham, UK) for helpful suggestions and critical reviewing
of the manuscript. The vital imaging studies were financially
supported by a grant from the Netherlands Organization for
Scientific Research (NWO Grant 901-28-134).
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