The truncated prelamin A in Hutchinson–Gilford
progeria syndrome alters segregation of A-type
and B-type lamin homopolymers
Erwan Delbarre1, Marc Tramier1, Maı ¨te ´ Coppey-Moisan1, Claire Gaillard2,
Jean-Claude Courvalin1and Brigitte Buendia1,*
1De ´partement de Biologie Cellulaire, Institut Jacques Monod, CNRS, Universite ´ Paris 6 and 7, 2 Place Jussieu Tour
43, 75251 Paris Cedex 05, France and2Centre de Recherches Biome ´dicales des Cordeliers, 15 rue de l’E´cole de
Me ´decine, 75006 Paris, France
Received December 22, 2005; Revised January 27, 2006; Accepted February 9, 2006
Hutchinson–Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused
by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are inter-
mediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear
membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently
or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in
cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is con-
sidered important for the development of therapeutic strategies. Here, we investigated the alterations in the
lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the
polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and
mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer
(FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluor-
escence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-
type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast,
expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type
and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the
primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such
as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.
dimerization domain or rod (Fig. 1A) that polymerize to form
the lamina network underlying the inner nuclear membrane
(1). Lamina forms a highly stable structure (2) and anchors
nuclear pore complexes, heterochromatin and regulatory pro-
teins at the nuclear periphery (3). B-type lamins are constitu-
tive, whereas A-type lamins (lamins A and C) which arise
from the LMNA gene by alternative splicing (1,4) are expressed
only in differentiated cells (5). Lamin A is synthesized as a pre-
cursor, prelamin A, that terminates with a CAAX motif (4).
This motif triggers sequential post-translational modifications:
farnesylation of the cysteine, removal of the AAX amino
acids by the endoprotease Zmpste24 and carboxymethylation
of cysteine (6–8). After completion of these modifications,
the last 15 amino acids of prelamin A, including the modified
cysteine, are also cleaved by the Zmpste24 endoprotease, gen-
erating mature lamin A (6–10). The interactions of A- and
B-type lamins have been investigated by biochemical
methods (11–14) and two-hybrid analyses (15) and homotypic
and heterotypic interactions were detected. Whether in vivo
lamins co-assemble or assemble independently at the mem-
brane–chromatin interface of somatic cell nuclei is unknown.
Mutations in the LMNA gene cause a wide array of inherited
diseases, including myopathies, a partial lipodystrophy, a
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Human Molecular Genetics, 2006, Vol. 15, No. 7
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peripheral neuropathy and premature aging syndromes (16).
Hutchinson–Gilford progeria syndrome (HGPS) is a dominant
autosomal premature aging disease due to the expression of a
truncated prelamin A designated Pgn. HGPS is most commonly
caused by a point mutation in exon 11 of LMNA that activates a
cryptic donor splice site (17,18). This mutation leads to the del-
motif intact, but removes the final endoproteolytic cleavage site.
Hence, the mutant prelamin A is predicted to undergo post-
translational modification of the terminal cysteine that will be
retained in Pgn (19–23). Expression of Pgn in cells of subjects
with HGPS triggers an increase in perimeter length of nuclei
with an extensive folding of the nuclear envelope, accompanied
and chromatin (24). Misshapen nuclei in HGPS have been con-
as a test for therapeutic efficiency. This nuclear phenotype is
reversed upon splicing correction of prelamin A pre-mRNA
(25), or by blocking the farnesyltransferase (19–23).
Here, we analyzed the alterations in the lamina structure
that may underlie the disorganization caused in the nuclear
envelope by Pgn expression. We studied the interactions
between enhanced green fluorescent protein (EGFP)- and red
fluorescent protein (DsRed)-tagged wild-type and mutated
lamins in the nuclear envelope of living cells by measuring
fluorescence resonance energy transfer (FRET), using a quan-
titative method, time domain fluorescence lifetime imaging
microscopy (tdFLIM). We first studied the interactions of
wild-type A- and B-type lamins, in an attempt to establish a
putative model of their assembly in the lamina. We then
used the same approach to analyze how tagged Pgn integrates
and/or disturbs the lamina network.
tdFLIM is an appropriate method to study lamin–lamin
interactions in living cells
We used tdFLIM to detect energy transfer between lamins
tagged with EGFP and DsRed fluorescent molecules, as this
method possesses features that are most appropriate for the
Briefly, when an EGFP-tagged protein (donor) interacts with
a DsRed-tagged protein (acceptor) in a co-transfected cell,
FRET can occur if the distance between the two fluorophores
is less than 10 nm, leading to a faster fluorescence decay of
involved in FRET (tFRET) is shorter than that associated with
the EGFP molecules that do not interact with DsRed (tEGFP).
These two lifetimes allow the calculation of real FRET effi-
ciency (E) through a simple equation (E ¼ 1 2 tFRET/tEGFP;
see Materials and Methods). The measurement of this E-value
is the basis of the tdFLIM method, as differences in E-values
will only reflect variations in the distance/orientation between
the fluorescent molecules, but neither the variations in the cel-
relative concentrations of green and red species. This property
of tdFLIM was essential for the present study because the
dilution of the exogenous fluorescent lamins by endogenous
lamins may vary from cell to cell, as well as the relative
Figure 1. Over-expressed tagged lamins, wild-type or mutated (Pgn) are inte-
grated within the lamina. (A) Schematic diagram of the lamins expressed by
the plasmid constructs. Numbers refer to the amino acids of lamin proteins.
The large box represents the dimerization rod domain. The small squares
and triangles correspond to the EGFP and DsRed fluorophores, respectively.
La A, La B1 and Pgn refer to lamin A, lamin B1 and Pgn, respectively. (B)
Endogenous and exogenous lamins are similarly resistant to extraction.
Control C2C12 cells (upper panels) and C2C12 cells co-expressing tagged
lamin A (DsRed–La A) and lamin B1 (EGFP–La B1) (middle panels), or
expressing tagged Pgn (EGFP–Pgn) (lower panels), were sequentially
extracted with TX-100, TX–NaCl and finally digested with DNase I and
RNase A followed by salt extraction (TX–N-DNase). The three supernatants
(TX, TX–NaCl, TX–N-DNase) and the final insoluble fraction (Ins) were
analyzed by immunoblotting. In left panels, endogenous lamins A and C
(La A and La C) and DsRed–lamin A (DsRed–La A) were revealed by
rabbit antibodies directed against lamins A and C, and EGFP–progerin
(EGFP–Pgn) was revealed by a monoclonal antibody directed against GFP.
In right panels, endogenous lamin B1 (La B1) and exogenous lamin B1
(EGFP–La B1) were revealed by rabbit antibodies directed against lamin
B1. Each lane corresponds to material extracted from 5?104cells. The
amount of extracted proteins in each fraction (supernatants and pellet), as
judged by Ponceau red staining (not shown), was similar in all experiments.
1114Human Molecular Genetics, 2006, Vol. 15, No. 7
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content in green and red fluorescent lamins. In most of the
experiments presented here, lamins are tagged at their N-
porated in the lamina, the orientation of their fluorophores is
expected to be the same. We therefore interpret the differences
in E-values found for various lamin combinations as mostly
reflecting differences in the distances separating the tagged
lamins in the lamina (see Materials and Methods). The second
basic feature of tdFLIM is that it makes it possible to measure
the proportion of EGFP-tagged molecules involved in FRET
in a given cell. This proportion varies depending on two para-
meters: (i) the ratio of exogenous versus endogenous lamins
(unknown at the level of individual cells); and (ii) the ratio of
acceptor (DsRed) versus donor (EGFP) molecules, that is
measured through the R/G ratio (see Materials and Methods).
In a fraction of cells with an unfavorable balance between
these two parameters, the probability of a close proximity of
green and red fluorescent markers is low and FRET is undetect-
able. The measurement at a low R/G value of the fraction
of cells with detectable FRET signals allowed us to compare
the ability of different lamins to interact with each other
Over-expressed lamins are integrated within the lamina
Lamins tagged at their N-terminal end (Fig. 1A) were
transientlyexpressedinC2C12 myoblasts.A major
characteristic of the nuclear lamina is its resistance to
extraction by non-ionic detergents and salt. We analyzed
the resistance to extraction of exogenous and endogenous
lamins in control C2C12 cells (Fig. 1B, upper panel), and
in C2C12 cells over-expressing either DsRed–lamin A
and EGFP–lamin B1 (Fig. 1B, middle panel) or EGFP–
Pgn (Fig. 1B,lower panel).
extracted with Triton X-100 (TX-100), Triton and salt and
finally digested with DNase I and RNase A followed by
salt extraction. Supernatants and the final insoluble fraction
were analyzed by immunoblotting using antibodies directed
against lamins A and C, lamin B1 and EGFP. Exogenous
lamins were as resistant to extraction as endogenous
lamins (Fig. 1B), showing that they were integrated within
The localization of the exogenous lamins at the nuclear
envelope was confirmed by fluorescent microscopy data. In
C2C12 cells that contain A- and B-type lamins and in P19
embryonic cells with only B-type lamins (26), exogenous
wild-type and mutated lamins were targeted to the nuclear
envelope with a pattern indistinguishable from that of
endogenous lamins (Fig. 2). Over-expression of lamin B1
induced in some cells an enlargement of the nucleus with
nuclear envelope folding (Fig. 2A, arrowheads). A similar
increase in size of the nucleus with occasional formation
of herniations and blebs was observed in some cells over-
expressing Pgn (Fig. 2A, arrows).
Figure 2. Over-expressed tagged lamins localize at the nuclear envelope in C2C12 (A) and P19 (B) cells. Endogenous lamins A and C (La A/C) and B1 (La B1)
are revealed by indirect immunofluorescence in C2C12 and in P19 cells, using antibodies against lamins A and C (anti-LaA/C) and antibodies against lamin B1
(anti-La B1). DNA in (B) is labelled with DAPI. Note that A-type lamins are not expressed in P19 cells. Exogenous EGFP- or DsRed-tagged wild-type lamins
and Pgn, detected by direct fluorescence, localize at the nuclear periphery. The nuclei in some C2C12 cells expressing progerin are larger than usual and dys-
morphic (arrows) with extensive nuclear envelope folding (arrowheads). A similar folding is also observed in some cells expressing exogenous lamin B1 (arrow-
heads). Bars ¼ 10mm.
Human Molecular Genetics, 2006, Vol. 15, No. 7 1115
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Homotypic and heterotypic lamin interactions
are detected within the lamina
We first investigated the occurrence of heterotypic interactions
at the nuclear envelope of C2C12 cells transfected with
plasmids encoding EGFP–lamin A and DsRed–lamin B1.
Figure 3A shows that the fluorescence decay of EGFP–
lamin A at the nuclear envelope was more rapid in cotrans-
fected cells (blue curve) than in monotransfected cells
(green curve), signaling FRET occurrence. In monotransfected
cells, the EGFP fluorescence decay was monoexponential with
a lifetime (tEGFP) of 2.27 + 0.07 ns (Table 1). In cells
co-expressing tagged lamins in which FRET was detectable,
fluorescence decay became faster because of the appearance
of a shorter lifetime. As discussed earlier, this shorter lifetime
(tFRET) corresponds to EGFP–lamin A molecules that interact
with DsRed–lamin B1 (see also Materials and Methods).
Whatever the lamin combination, DsRed–Lamin B1/EGFP–
lamin A or DsRed–La A/EGFP–lamin B1, similar values
for tFRET and real FRET efficiency (E) were obtained
(tFRET¼ 0.76 + 0.09 ns
tFRET¼ 0.80 + 0.09 ns and E ¼ 0.65 + 0.04, respectively)
(Table 1). As negative control, we analyzed the interactions
of lamins with Pom 121, a transmembrane nucleoporin that
cannot interact with lamins for topological reasons (27). No
FRET was detected when EGFP–Pom 121 was co-expressed
with either DsRed–lamin B1 (Fig. 3B) or DsRed–lamin A
(data not shown), whatever the levels of expression of
E ¼ 0.66 + 0.04versus
lamins and Pom 121. These data that assess the existence of
heterotypic interactions in the lamina of living cells are in
agreement with the data of Moir et al. (28) who made
similar observations in post-mitotic cells using a qualitative
In cells co-expressing homologous lamins, FRET was also
detected (Fig. 3C, Table 1), but the EGFP fluorescence
lifetime and the corresponding real FRET efficiency of
lamin B1 homotypic interactions (tFRET¼ 1.00 + 0.08 ns;
E ¼ 0.57 + 0.04) were distinct from those of lamin A homo-
typic interactions (tFRET¼ 0.83 + 0.09 ns; E ¼ 0.64 + 0.04)
(P , 0.0001) and from those of heterotypic interactions (see
above; Table 1) (P , 0.0001). This difference means that
the range of distances between lamin B1 molecules in
lamina is different from that between lamin A molecules or
between lamin A and lamin B1 molecules.
Interactions between exogenous lamins A and B1 were also
tested in P19 cells that express B-type lamins but not A-type
lamins (26). Heterotypic interactions were detected with a
mean real FRET efficiency value (E ¼ 0.65 + 0.05) close to
the value obtainedin C2C12
(Table 1). The similar FRET efficiency in two cell lines, one
of which is devoid of endogenous A-type lamins, supports
the scaffolding role postulated for B-type lamins in the assem-
bly of lamin A (29). It is worth noting that the real FRET effi-
ciency for homotypic lamin B1 interactions (E ¼ 0.65+ 0.05)
was higher in P19 cells than in C2C12 cells (E ¼ 0.57 + 0.04;
P , 0.0001) (Table 1), indicating that the range of distances
cells(E ¼ 0.65 + 0.04)
Figure 3. Heterotypic and homotypic interactions between tagged lamins are detected by FRET. C2C12 cells were mono- and cotransfected as indicated.
Steady-state fluorescence images were acquired with a CCD camera (CCD panels). Time integrated images obtained for each cell with the quadrant anode detec-
tor are presented in the quadrant panels. Nuclear envelope domains (highlighted in yellow in quadrant panels) were selected, and corresponding fluorescent
decays are normalized and plotted in the graphs. Green and blue curves refer to fluorescence decays in mono- and cotransfected cells, respectively.
(A) FRET occurs in cells co-expressing tagged lamins A (La A) and B1 (La B1). (B) FRET is not detected in cells co-expressing tagged lamin B1 and
Pom121. (C) FRET occurs in cells co-expressing EGFP- and DsRed-tagged lamin B1. ns refers to nanosecond. Bars ¼ 10 mm.
1116Human Molecular Genetics, 2006, Vol. 15, No. 7
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between lamin B1 molecules in the lamina is different in the
two cell lines. This suggests that the structure of lamin B1
polymers varies with the composition of the nuclear envelope,
and in particular with the level of expressed lamin A.
The conclusion from this series of experiments is that
FRET occurred between homologous- and heterologous-
tagged lamins in the lamina of living cells.
Lamin–lamin interactions detected by FRET
occur within high order polymers
As the first step in lamin polymerization is homodimerization
(1,30), heterotypic lamin A–lamin B1 interactions detected by
FRET were only occurring between homodimers of both types
in high order polymers. However, a plausible explanation for
the homotypic interactions would be their occurrence at the
very first stage of lamin assembly, within the homodimers.
To check this possibility, we co-expressed homologous
lamins tagged with the two fluorophores and analyzed
mitotic cells in which the lamina is disassembled into distinct
populations of A-type and B-type homodimers (30). No FRET
was detected indicating that energy transfer did not occur
within homodimers (data not shown). This suggests that in
interphasic cells, FRET occurs between dimers within higher
order polymers. To strengthen this hypothesis, we analyzed
the signals generated in the nuclear envelope of interphasic
C2C12 cells by co-expression of lamin A molecules tagged
with EGFP or DsRed at different sites. One lamin A molecule
was fused at its N-terminal end to DsRed and another
one with EGFP immediately after the rod domain (Fig. 1A,
intra-EGFP–La A). In a putative homodimer containing
both fluorophores, FRET should not occur because the two
tags would be separated by a distance roughly the length of
the rod domain (?50 nm; see Fig. 1A), i.e. greater than the
maximum distance compatible with FRET (10 nm). The data
showed that FRET was detected between intra-EGFP–lamin
A and DsRed–lamin A with an associated lifetime of
0.94 + 0.14 ns (n ¼ 25). Real FRET efficiency (E ¼ 0.59 +
0.06) was lower than that obtained when both tags were
present at the N-terminal end of lamin A (E ¼ 0.64 + 0.04;
P , 0.001). This difference in E-values is probably because
of different distances between the fluorophores in the lamina
and to possible distinct orientations related to their locations
in lamin A. These data demonstrate that FRET signals did
not occur between two lamin molecules within homodimers,
but rather between lamins present in distinct dimers within
higher order polymers.
Homotypic interactions are favored over heterotypic
In cotransfected cells, it was checked if among the different
lamin combinations used here some were more favorable
than others in triggering FRET. The acceptor (DsRed) to
donor (EGFP) ratio (R/G) was measured in individual cells
and found to vary between 2 and 25 (Table 2; see Materials
and Methods). In cells containing a large excess of acceptor
over donor (R/G . 15), FRET was detected in all cells, what-
ever the lamin combination (Table 2). With a lower excess of
acceptor over donor (R/G , 15), homotypic combinations
generated a higher proportion of cells positive for FRET
than heterotypic combinations (Table 2, 83 and 93% versus
20%), meaning that the threshold for FRET triggering was
lower for homotypic interactions. In the lamina, homotypic
interactions are therefore clearly favored when compared
with heterotypic interactions.
Integration of Pgn within the lamina
We then assessed how truncated prelamin A (Pgn) integrates
into the lamina. Figures 1B and 2A show that both EGFP-
and DsRed-tagged Pgn molecules are targeted to the nuclear
Table 1. FRET lifetimes and real FRET efficiencies in cells expressing EGFP and DsRed tagged lamins
Co-expressed tagged proteins FRET lifetime
Real FRET efficiency
E ¼ 1 2 tFRET/tEGFP
Interphasic C2C12 cells: tEGFP¼ 2.27+0.07 ns
DsRed–La B1/EGFP–La A
DsRed–La A/EGFP–La B1
DsRed–La A/EGFP–La A
DsRed–La B1/EGFP–La B1
Interphasic P19-EC cells: tEGFP¼ 2.26+0.02 ns
DsRed–La B1/EGFP–La B1
DsRed–La A/EGFP–La B1
FRET lifetimes (tFRET) and real FRET efficiencies (E) in transfected C2C12 and P19 cells. Mean tFRETvalues are expressed in nanoseconds. In each
series of experiments, tEGFPis the mean of the lifetimes of EGFP in monotransfected cells. For C2C12 and P19 cells, a total of 136 and 27 nuclei in
monotransfected cells were analyzed, as indicated. For each cotransfection, monotransfection with the corresponding EGFP-tagged protein was
performed. Mean tFRETand E-values include standard errors.
?P , 0.0001 versus DsRed–La B1/EGFP–La B1 in C2C12 cells.
??P , 0.002 versus DsRed–La A/EGFP–La A; P , 0.0004 versus DsRed–La A/EGFP-La B1 in C2C12 cells.
???P , 0.0001 versus DsRed–La B1/EGFP–La B1 in C2C12 cells.
Human Molecular Genetics, 2006, Vol. 15, No. 71117
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envelope in C2C12 cells and incorporated into an extraction-
resistant structure. As the toxic effect of Pgn in cells is dose-
dependent (24), FRET analyses were performed at a high
level of Pgn expression with Pgn as the acceptor molecule
(DsRed–Pgn). When the tagged Pgns were co-expressed, or
when DsRed–Pgn was expressed with EGFP-tagged lamins
B1 or A, FRET occurred in all cases, with nearly identical
real FRET efficiencies of 0.58 + 0.07, 0.56 + 0.07 and
0.59 + 0.05, respectively (Table 1). Thus, the range of dis-
tances in the lamina separating Pgn molecules from each
other, from lamin A and from lamin B1 is identical, supporting
an even distribution of these three lamin proteins in the lamina.
These similar real FRET efficiency values found for Pgn inter-
actions were lower than those previously found for lamin A
interactions with either lamin A (E ¼ 0.64+0.04; P , 0.002)
or lamin B1 (E ¼ 0.65 +0.04; P , 0.0004) (Table 1). In con-
trast, the E-values found for Pgn interactions were similar to
those previously measured for lamin B1 homotypic interactions
(E ¼ 0.57 +0.04), indicating that the common range of dis-
tances separating lamins A, B1 and Pgn in the lamina is
similar to that previously found between lamin B1 molecules.
These data suggest that Pgn behaves similar to lamin B1
during lamina assembly. The fact that lamin B1 co-assembles
preferentially with Pgn rather than with wild-type lamin A is
supported by the high proportion of cells in which FRET
was detectable when DsRed–Pgn and EGFP–lamin B1 were
co-expressed, compared with the low proportion when
DsRed–lamin A and EGFP–lamin B1 were co-expressed (62
versus 20%; P , 0.05) (Table 2; R/G , 15). Taken together,
our data support a model in which Pgn would displace a pool
of lamin A from homopolymers to a heterotypic structure
containing also lamin B1 and Pgn.
We used tdFLIM, a quantitative FRET analysis method, to
study alterations in lamin polymerization in living cells
provoked by the expression of Pgn. As the in vivo homo- or
heteropolymerization of A- and B-type lamins had not been
elucidated, the interactions of wild-type lamins of both types
tagged with EGFP and DsRed were first studied, in an
attempt to establish a putative model of their assembly in
the lamina. Three possible models of lamin polymer organiz-
ation were considered (Fig. 4A). In model 1, A-type and
B-type lamins form homopolymers that are not in contact. In
model 2, A-type and B-type lamins interact within heteropoly-
mers where they are evenly distributed. In model 3, A-type
and B-type lamins interact within polymers where they are
not evenly distributed. We then checked with which model
the FRET data fit the best. Since, as shown here, lamin A
and lamin B1 interact, model 1 was discarded. An even
distribution of lamins of both types into polymers, as in
model 2, would have provided homogeneous real FRET effi-
ciencies values and similar proportions of FRET positive
cells at all R/G ratios, whatever may be the lamin combi-
nation. Instead, we obtained different values of real FRET
efficiencies for the homotypic interactions of lamin A and
lamin B1, and a low sensitivity for FRET triggered by heter-
ologous combinations of lamins when compared with both
homologous combinations of lamins. These data eliminate
model 2 and support model 3 that postulates an uneven
distribution of lamins in polymers. Model 3 is compatible
Table 2. Percentage of cells with detectable FRET as a function of the acceptor
(DsRed) to donor (EGFP) expression ratio (R/G)
Co-expressed tagged proteins R/G , 15
DsRed–La A/EGFP–La B1
DsRed–La A/EGFP–La A
DsRed–La B1/EGFP–La B1
The relative quantity of DsRed- and EGFP-tagged proteins in transfected
cells were calculated from fluorescence intensity measurements as
described in Materials and Methods. Data were obtained from about 20
cells in each case and expressed as a percentage (%) of cells in which
FRET was detectable. nd, not determined. Statistical data concerning
this Table are presented in the text.
?P , 0.05 versus DsRed–La A/EGFP–La B1.
Figure 4. Hypothetical models of lamina structure represented in a transverse
section of the nuclear envelope. (A) Schematic cross-sectional representation
of possible polymer assembly of A- and B-type lamins in the lamina. In model
1, lamins are integrated into homopolymers that are not in contact. In model 2,
both types of lamins are integrated into mixed polymers where they are evenly
distributed. In model 3, lamins of both types interact but they are not evenly
distributed. They are integrated either in a heteropolymeric structure or in two
homopolymers that are in contact. (B) In a somatic cell, wild-type A- and
B-type lamins form homopolymers that interact. Because of the insertion of
the farnesyl group (squiggly line) into the inner nuclear membrane, B-type
polymers are more closely associated with the membrane than A-type
polymers. As lamina is a fenestrated structure, a fraction of the chromatin is
represented in contact with the membrane. (C) In a cell expressing Pgn, farne-
sylated Pgn assembles with B-type lamins to form heteropolymers, whereas
homopolymerization of B-type lamins is abolished. As Pgn still associates
with a fraction of wild-type lamin A, the new mixed polymer of lamin B
and Pgn, also contains lamin A (B-P-A).
1118 Human Molecular Genetics, 2006, Vol. 15, No. 7
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with either an heteropolymeric structure with an uneven distri-
bution of both lamins or the juxtaposition of homopolymers of
lamin A and lamin B1 in close contact. FRET analysis did not
allow us to distinguish between these two possibilities, but the
existence of homotypic structures of both lamin types in living
cells supports the latter possibility. In the nuclei of embryonic
and stem cells, the lamina is exclusively composed of B-type
lamins (5). In contrast, nuclei in cultured fibroblasts of
individuals expressing lamin A bearing missense mutations
(31,32) develop membrane herniations that contain a lamina
structure exclusively composed of A-type lamins. As homo-
polymerization of A- and B-type lamins were shown by
these previous studies to occur in living cells and heterotypic
interactions of lamins are demonstrated in the present study,
we favor the model of lamina in which interaction occurs
between juxtaposed A-type and B-type lamin homopolymers.
We further propose an additional feature of the lamina
structure linked to the difference in post-translational modifi-
cations present at the C-terminal end of mature lamin B1
and lamin A. B-type lamins are permanently farnesylated at
their C-terminal end (7,8), whereas this modification occurs
only transiently in the process of prelamin A maturation,
because of the action of the specific membranous endopro-
tease Zmpste24 (6,8–10). We suggest that polymers of farne-
sylated B-type lamins associate more closely with the
membrane than polymers of A-type lamins (Fig. 4B),
because of the integration of the hydrophobic farnesyl group
within the inner nuclear membrane and/or association with a
putative isoprenyl-carboxymethyl-lamin receptor (33).
The same approach was used to analyze how tagged-Pgn
integrates and/or disrupts the lamina network. We showed
that Pgn can interact with both lamin A and lamin B1, as
well as with Pgn. The observation that real FRET efficiencies
are the same for these lamin combinations (Table 1) supports
an even distribution of the three lamin proteins in the lamina.
The shared real FRET efficiency value was lower than that
previously found for wild-type lamin A homotypic and hetero-
typic interactions, but similar to that measured for lamin B1
homotypic interactions. Thus, the range of distances separat-
ing Pgn and lamins A and B1 in the lamina is identical to
the range of distances separating lamin B1 molecules in homo-
typic polymers. These data suggest that Pgn behaves like
lamin B1 during lamina assembly. This conclusion was
strengthened by the fact that lamin B1 preferentially
co-assembles with Pgn rather than with wild-type lamin A
(Table 2). Taken together, our data support a model in
which Pgn would displace a pool of lamin A from homopoly-
mers to form a heterotypic structure containing evenly distri-
buted lamin B1, Pgn and lamin A (Fig. 4C). Expression of
Pgn would thus switch lamina structure from model 3 to
model 2 (Fig. 4A).
This change in lamina assembly may explain how endo-
genous as well as exogenous Pgn generate dysmorphic
nuclei with extensive nuclear membranes (24,25) (Fig. 2A).
In normal cells, the cysteine residue post-translationally modi-
fied at the C-terminus of B-type lamins is responsible for the
growth of the nuclear membrane (34,35). This may result from
the insertion of this hydrophobic moiety into the membrane
lipid bilayer or by interaction with a putative receptor (33).
The terminal cysteine modifications are abnormally conserved
in Pgn (20) and may allow this truncated prelamin A to prefer-
entially associate with lamin B1 and induce abnormal mem-
brane biogenesis via the same mechanisms. In support with
our model, similar altered nuclear phenotypes occur in
mouse cells that accumulate full-length prelamin A because
of a deficiency in the expression of the Zmpste24 endopro-
tease (6,9), a reversal of the phenotype being achieved by
blocking farnesyltransferase (19–23).
The assembly of Pgn with B-type and A-type lamins in an
abnormal network may also modify the interactions of these
lamins with their respective partners in adjacent structures
(16,33,36). Ultrastructural modifications of the lamina and
peripheral heterochromatin are observed in nuclei of subjects
with HGPS, together with an alteration in the distribution of
nuclear pore complexes in the nuclear envelope (24). Ultra-
structural chromatin abnormalities are also present in nuclei
from Zmpste24-deficient mice (37). Finally, the loss of
A-type and B-type polymer segregation may also disturb the
interactions of the lamina with various partners, generating
pleiotropic downstream cellular events such as upregulation
of p53 target genes (38), defective DNA repair (37) and
impairment of DNA replication and transcriptional activity
(24), finally leading to increased apoptosis and abnormal
The tdFLIM technique is an appropriate method to analyze
the changes of lamina structure in living cells expressing
either Pgn or various mutated lamins A responsible for num-
erous severe disorders (16). It also represents a promising
quantitative approach to evaluate reversal to a normal pheno-
type in the development of therapeutic strategies.
MATERIALS AND METHODS
pEGFP–lamin B1 and POM121–EGFP3 were gifts of
pEGFP–prelamin A construct has been described (40).
The pDsRed–lamin A and pDsRed–lamin B1 constructs
were produced in pDsRed–C1 vectors (Clontech Laboratories
Inc., CA, USA) with DsRed in frame at the N-terminus of the
full-length cDNAs of lamins. Intra-EGFP–prelamin A con-
struction was made following a three-step cloning strategy.
From the pEGFP–prelamin A plasmid, a cDNA encoding
the first 414 residues of prelamin A [prelamin A (1–414)]
was amplified by PCR, with an EcoRI restriction site engin-
eered at the 50-end of the sense primer (50GCG AAT TCT
ATG GAG ACC CCG TCC CAG GGG 30) and a KpnI site
engineered at the 50-end of the anti-sense primer (50GC GGT
ACC CCC ACC CTG TGT CTG GGA TGA 30) (restriction
sites underlined). The reaction products were digested with
EcoRI and KpnI and ligated into the EcoRI and KpnI sites
of pSVK3 (Amersham Pharmacia Biotech Inc., Uppsala,
Sweden) similarly digested. From the pEGFP–C1 plasmid,
the EGFP cDNA was amplified by PCR, with a KpnI site
engineered at the 50-end of the sense primer (50GC GGT
ACC ATG GTG AGC AAG GGC GAG GAG 30) and a
BamH1 site engineered at the 50-end of the anti-sense primer
(50GC GGA TCC CTT GTA CAG CTC GTC CAT
GCC 30). The reaction products were digested with BamH1
Human Molecular Genetics, 2006, Vol. 15, No. 7 1119
by guest on June 5, 2013
and KpnI and ligated into the BamH1 and KpnI sites of
pSVK3–prelamin A (1–414) similarly
pEGFP–prelamin A, a cDNA encoding the last 220 residues
of prelamin A was amplified by PCR, with a BamH1 site
engineered at the 50-end of the sense primer (50GC GGA
TCC AGC GTC ACC AAA AAG CGC AAA 30) and an
XhoI site engineered at the 50-end of the anti-sense primer
(50GC CTC GAG TTA CAT GAT GCT GCA GTT CTG 30).
The reaction products were digested with BamH1 and XhoI
and ligated into the BamH1 and XhoI sites of pSVK3–prelamin
A (1–414)–EGFP similarly digested.
From pEGFP–prelamin A and pDsRed–prelamin A, the
sequence corresponding to a region in the C-terminal
domain of prelamin A was removed by digestion with
SanDI and Acc65I and replaced by the DNA sequence specific
of Pgn (internal deletion of bp 1819–1968; residues
607–656). The dsDNA sequence corresponding to the C-
terminal fragment of Pgn was obtained from oligonucleotides
synthesized by Proligo Primers and Probes (Proligo, Paris,
France). The first couple of single strand (ss) oligonucleotides
was designed to correspond to the LMNA gene from bp 1723
to 1772 with a SanDI site in the 50-end of the sense oligonu-
cleotide (sense oligonucleotide: 50GAC CCC GCT GAG
TAC AAC CTG CGC TCG CGC ACC GTG CTG TGC
GGG ACC TG 30; anti-sense oligonucleotide 50TG CCC
GCA GGT CCC GCA CAG CAC GGT GCG CGA GCG
CAG GTT GTA CTC AGC GGG 30). The second couple of
ss oligonucleotides was designed to correspond to bp 1773–
1818 of the LMNA gene followed by bp 1969–1992, extended
with CTTA and an Acc651 site at the 50-end of the anti-sense
oligonucleotide (sense oligonucleotide 50C GGG CAG CCT
GCC GAC AAG GCA TCT GCC AGC GGC TCA GGA
GCC CAG AGC CCC CAG AAC TGC AGC ATC ATG
TAA G 30; anti-sense oligonucleotide 50GT ACC TTA CAT
GAT GCT GCA GTT CTG GGG GCT CTG GGC TCC
TGA GCC GCT GGC AGA TGC CTT GTC GGC AGG C
30). After duplex formation, the two double strand (ds) oligo-
nucleotides were annealed and the purified dsDNA sequence
(154 bp) was inserted either into the pEGFP–prelamin A or
into pDsRed1–prelamin A vector previously digested with
SanDI and Acc651.
The apparatus used for FRET determination performs tdFLIM
by the time- and space-correlated single photon counting
method (41). This method directly gives the picosecond (ps)
time-resolved fluorescence decay for every pixel by counting
and sampling single emitted photons according to: (i) the
time delay between photon arrival and laser pulse (ps time
(256 ? 256 pixel image). A titanium sapphire laser (Millennia
5W/Tsunami 3960-M3BB-UPG kit, Spectra-Physics, France)
that delivers ps pulses was tuned at 960 nm to obtain an exci-
tation wavelength at480 nmafter frequencydoubling. Therep-
etition rate was 4 MHz after pulse-picker (Spectra-Physics
3980-35, France). The laser beam was expanded and inserted
into an inverted epifluorescence microscope (Leica DMIRBE,
France) for wide-field illumination (a few mW/cm2). The
microscope stage was equipped with an incubator system
orescence decayimages were
Plan-Apochromat 100X 1.3NA oil objective, a dichroic beam
France), an emission filter (535DF35; Omega; Optophotonics),
and the quadrant-anode TSCSPC detector (QA, Europhoton
(515 nm , lem, 560 nm) was chosen to select the donor
fluorescence (EGFP) and to reject the acceptor fluorescence
(DsRed). The count rate was up to 50 kHz. Acquisition of
fluorescence decay images was done after accumulation of
sufficient single photon events, usually 3–6 min. A temporal
resolution of less than 100 ps and a spatial resolution of
500 nm were determined previously for this system (41).
taken usinga Leica
tdFLIM data analysis using a three-exponential model
For qualitative determination of FRET, the fluorescence
decays of EGFP within the regions of interest were extracted
from the acquisition matrix and the decays of EGFP-tagged
lamins (donor) in the presence of DsRed-tagged lamins
(acceptor) were compared with the control decays of the
EGFP-tagged proteins measured in the absence of acceptor.
To perform the quantitative analysis of FRET, the experimen-
tal fluorescence decays were further deconvoluted with the
instrument response function and fitted by a Marquardt non-
linear least-square algorithm using Globals Unlimited soft-
ware (University of Illinois at Urbana-Champaign, IL, USA)
with discrete lifetimes as theoretical model.
For each tdFLIM experiment, the fluorescence decays of
EGFP in cells expressing EGFP-tagged proteins alone (mono-
transfected cells) or with DsRed-tagged proteins (cotrans-
fected cells) were analyzed. The fluorescence decay of
EGFP-tagged proteins in monotransfected cells was mono-
exponential with a lifetime of 2.27 + 0.07 ns in C2C12 cells
(n ¼ 136). As the DsRed protein emits a weak green fluor-
escence before maturation in vitro (42) as well as in vivo
(43), the decays of the green species of DsRed (Supplementary
Material, Fig. S1) in the 515 and 560 nm interval were ana-
lyzed and found to fit with a two-exponential model, with life-
times of 2.30 + 0.50 ns and 0.24 + 0.07 ns in C2C12 cells
(n ¼ 51), and 2.54 + 0.26 ns and 0.25 + 0.02 ns in P19
cells (n ¼ 17), respectively. These values were consistent
with those previously found in vitro and in vivo (44,45). The
component with the shorter lifetime was 89–96%. In cotrans-
fected cells in which the fluorescence decay was more rapid
than in monotransfected cells, FRET analyses were carried
out by fitting the fluorescence decays of EGFP using a
three-exponential model. For C2C12 and P19 cells, the fluor-
escence lifetime of free EGFP–lamins (tEGFP) was fixed at
2.27 and 2.26 ns, and that of the major component of the
green species of DsRed–lamins (tDsRed) at 0.24 and 0.25 ns,
respectively. The third lifetime corresponding to FRET
(tFRET) remained free. In cotransfected cells, the time-
dependent fluorescence decay I(t) in the selected area is
given by the sum:
IðtÞ ¼ aEGFP:e?t=t EGFPþ aDsRed:e?t=t DsRed
1120Human Molecular Genetics, 2006, Vol. 15, No. 7
by guest on June 5, 2013
in which aEGFP, aDsRed and aFRET are the corresponding
Calculation of FRET features
The proportion of EGFP-tagged proteins involved in FRET
corresponds to the ratio
The value of this ratio varied as a function of the intensity of
the acceptor (DsRed) fluorescence, similarly for all couples of
lamins analyzed in the present study (Supplementary Material,
The real FRET efficiency (E) can be calculated by the
E ¼ 1 ? tFRET=tEGFP
E depends on the distance separating the donor and acceptor
fluorophores and on their orientations. In most of the experi-
ments presented here, lamins are tagged at their N-terminal
end (Fig. 1A). Once lamins tagged at this site are incorporated
in the lamina, the orientation of their fluorophores is expected
to be the same. We therefore interpret the differences in
E-values found for various lamin combinations as mostly
reflecting differences in the distances separating the tagged
lamins in the lamina. To a lower tFRETcorresponds a higher
transfer efficiency, indicative of a shorter distance (R)
between the chromophores engaged in FRET, following the
E ¼ R6
where the Fo ¨rster distance R0corresponds to the distance at
which 50% efficiency of energy transfer takes place. In the
case of fluorophores that move freely, R0is 4.7 nm for the
couple EGFP and DsRed (46).
The percentage of cells with detectable FRET varies as a
function of the acceptor to donor expression ratio. The relative
quantity of EGFP- and DsRed-tagged proteins in transfected
cells was calculated from additional green and red fluores-
cence intensity images acquired using appropriate filter
cubes, a mercury lamp and a conventional CCD camera
(Silar, St Petersburg, Russia). The intensities of EGFP (IGFP)
and DsRed (IRed) signals were estimated in arbitrary units
separately for green and red images. The mean gray level of
regions of the nuclear envelope was calculated by taking
into account the exposure time and excitation level, allowing
for a comparison of the signals between different cells.
FRET was measured in cells in which the acceptor was in
excess over the donor, with a ratio IRed/IGFP(R/G) between
2 and 25. IGFPwas above 2 (maximum 50) and IRedabove
10 (maximum 300). Global analysis of the data shows that
the value 15 for the R/G ratio divides the data into two
classes. Above this value, nearly 100% of cells generate
FRET signals whatever may be the lamin combination
(Table 2). Below this saturation value, the percentage of
cells generating FRET signals was highly dependent upon
the lamin combination (Table 2). Therefore, the latter con-
dition was used to determine if some lamin conditions were
more favorable than others to trigger FRET.
Cell culture and transfections
The original mouse myoblast C2C12 cell line was grown in
DMEM medium containing 15% fetal calf serum (FCS),
1 mM glutamine, and 1% antibiotics (100 units/ml penicillin
and 100 mg/ml streptomycin). Mitotic C2C12 cells accumu-
lated by overnight culture in the presence of 40 ng/ml noco-
dazole. Mouse embryonic P19 cells were grown in a-MEM
medium containing 7.5% FCS and identical additives. Cells
grown to 50% confluency in chamber slides were transfected
using FuGene6 (Roche Diagnostic Co., IN, USA) following
the manufacturer’s instructions. Cells were used 40–48 h
Immunoblotting and fluorescence microscopy
Both methods were performed as previously described
In Table 1, real FRET efficiencies were compared using the
Student t-test. Percentages of cells positive for FRET
(Table 2) were compared using the x2test.
Supplementary Material is available at HMG Online.
We thank J. Ellenberg for providing the EGFPC3–lamin B1
and Pom121–EGFP3plasmids, H. Herrmann and F. Strauss
for helpful discussions and A.-L. Haenni for careful reading
of the manuscript. This work was supported by the Centre
National de la Recherche Scientifique and by the Institut
National de la Sante ´ et de la Recherche Me ´dicale, and grants
from the Association Franc ¸aise de Lutte contre les Myopathies
(A.F.M. to B.B.), and from the Groupement des Entreprises
Franc ¸aises dans la Lutte contre le Cancer (to M.C.-M. and
J.-C.C.), and fellowships from Ministe `re de l’E´ducation
Nationale, de la Recherche et de la Technologie (MENRT)
and from A.F.M. (to E.D.).
Conflict of Interest statement. None declared.
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