1 INSERM U. 402, Faculté de Médecine Saint-Antoine, 75012 Paris, France
2 Cinémicro INSERM, 78110 Le Vesinet, France
3 Département de Biologie Cellulaire, Institut Jacques Monod, CNRS, Université Paris 7, 75005 Paris, France
*Author for correspondence (e-mail: buendia{at}ijm.jussieu.fr)
Accepted September 6, 2001
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SUMMARY |
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Key words: Lamin A/C gene, Lipodystrophy, Mutation, Nuclear dysmorphy, Nuclear envelope disorganization
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INTRODUCTION |
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Lamins A and C are major components of the nuclear lamina. They are members of the intermediate filaments protein family, with a similar primary and secondary structure (McKeon et al., 1986; Fisher et al., 1986). The LMNA gene generates lamin A and lamin C by alternative RNA splicing (Lin and Worman, 1993). These proteins are identical for their first 566 amino acids, which encompass the N-terminal head, the central rod helicoidal domain, and most of the tail domain. Thus the R482Q/W mutations, which are the most frequent mutations in FPLD, affect both A and C lamins. Lamins B1 and B2 are the other major components of the lamin family of proteins and are coded by different genes (Stuurman et al., 1998; Worman and Courvalin, 2000). A- and B-type lamins polymerize in various ratios to form the nuclear lamina, a protein network that is located between inner nuclear membrane and chromatin. Lamin genes are differentially expressed during development and cell maturation; B-type lamins are constitutive and A-type lamins are preferentially expressed in differentiated nonproliferating cells (Stewart and Burke, 1987; Guilly et al., 1987; Guilly et al., 1990).
A specific set of nuclear integral proteins interacts with lamina and could mediate its attachment to the inner nuclear membrane (INM) (Worman and Courvalin, 2000). Among these proteins, the lamin B receptor (LBR) and lamina associated protein 2 ß (LAP2ß) interact more specifically with B-type lamins (Furukawa et al., 1995; Worman et al., 1988), whereas emerin preferentially binds to A-type lamins (Fairley et al., 1999; Sullivan et al., 1999; Clements et al., 2000). The lamina is tightly associated with nuclear pore complexes (NPCs), possibly involving a direct interaction between lamins and Nup153, a peripheral component of the NPCs (Smythe et al., 2000). Finally, the nuclear lamina also interacts with chromatin, through lamin sequences located in their carboxyl-terminal end, downstream from the rod domain (Taniura et al., 1995; Goldberg et al., 1999).
Several roles for the nuclear lamina have been proposed, including its involvement in nuclear structure, gene expression, cell-cycle progression, and DNA replication (Lourim and Lin, 1992; Liu et al., 2000; Moir et al., 2000a). To investigate the cellular alterations involved in FPLD, we have performed a morphological and biochemical analysis of cultured skin fibroblasts from patients with the LaA/C R482Q/W heterozygous mutations. We show here that a population of cells from FPLD patients and from control individuals expressing ectopic LaA R482W present dysmorphic nuclei or a disorganization of the lamina, or both. In addition, cells in FPLD patients can have an altered protein composition of the INM, a discontinuous repartition of the nuclear pore complexes, accompanied by an increased sensitivity to heat shock and to extraction by nonionic detergents.
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MATERIALS AND METHODS |
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Fluorescence cytometric analyses
For FACS analysis, 105 trypsinized fibroblasts from control individuals and patients JM and LM were collected by centrifugation, resuspended in PBS and finally fixed in ethanol (70%) at 4°C. After RNase A (500 µg/ml) digestion and propidium iodide (50 µg/ml) staining, cells were analyzed on a FACS Calibur (Becton Dickinson) equipped with an argon laser emitting at 488 nm. Cells sorted according to the different phases of the cell cycle were observed by fluorescence microscopy and the percentage of abnormal nuclei in each fraction was calculated. These experiments were repeated four times. For cytofluorometric analysis of adherent cells, cells grown on coverslips were fixed in methanol, stained by propidium iodide as indicated above, and finally analyzed individually by DNA fluorescence using Meridian ACAS 570 interactive laser. Fluorescence of 100 cells was measured for each of the three patients and the two control individuals.
Time-lapse cinemicrography
After a one day culture, cells from control individuals and patients JM and LM, seeded onto glass slides in Petri dishes, were mounted in observation microchambers known as the Rose chamber (Rose, 1954). A time-lapse unit generated one impulse per minute that controlled a 16 mm Arriflex camera. Phase contrast recordings were made over periods of 2 days. Between two successive frames, the light was turned off to avoid cellular damage due to light energy. After development, films were analyzed on a frame by frame projector and selected frames were enlarged and printed.
Antibodies and immunological methods
For conventional and confocal immunofluorescence microscopy, human fibroblasts grown on glass coverslips were fixed in methanol at 20°C, then processed as described (Buendia and Courvalin, 1997; Buendia et al., 1999). Monoclonal antibodies (mab) directed against A-type lamins, QE5 mab directed against nucleoporins p62, Nup153 and Nup214, and a mab specific for Nup153 were generous gifts from B. Burke (University of Calgary, Alberta, Canada) and R. Bastos (Institut Jacques Monod, CNRS, Paris). Mab anti-emerin (NCL-emerin clone 4G5) was purchased from Novocastra laboratories (Newcastle upon Tyne, UK), and mab anti-FLAG (M2) from Sigma (St Louis, MO). Rabbit antibodies directed against B-type lamins and LAP2ß and human anti-lamin B antibodies have been previously described (Chaudhary and Courvalin, 1993; Buendia et al., 1999). Affinity-purified FITC-conjugated anti-rabbit antibodies and Texas Red-conjugated anti-mouse antibodies were purchased from Jackson ImmunoResearch laboratories (West Grove, PE). Given that the mean thickness of nuclei was statistically not different in the different populations of fibroblasts (z values estimated by observation at the confocal microscope), surface measurements were performed on two-dimensional images of DAPI-stained nuclei (58 control nuclei, 53 and 74 dysmorphic nuclei from patients JM and NK, respectively, and 59 eumorphic nuclei from patient NK) using manual outlining in Canvas 5.0. Intensity of the DAPI-staining was evaluated in 83 dysmorphic nuclei from patients JM and NK, in parallel in the bud and in the rest of each nucleus, using Metaview software. Immunoblotting analysis of cell extracts was performed as previously described (Buendia and Courvalin, 1997).
Heat-shock treatment
Heat-shock experiments were performed by transferring cells from 37°C to 45°C for 30 minutes. Cells were then either used immediately or transferred back to 37°C. For morphological examination, fibroblasts from the two control individuals and the three patients were grown on glass coverslips, washed for one second in PBS after heat shock, then immediately fixed in cold methanol (20°C) for 10 minutes, and finally processed for immunofluorescence studies. After staining with anti-lamin A/C antibodies, 200-300 nuclei from patient and control fibroblasts were examined. Cellular viability after heat shock was evaluated at 0, 24 and 48 hours. Attached cultured cells, heated or unheated, from control individuals and patients LM and KN were trypsinized, collected by centrifugation, and counted. This experiment was repeated four times.
Cell fractionation
Monolayers of fibroblasts grown on Petri dishes were sequentially extracted as described (Fey et al., 1984). The first extraction was performed with 0.5% Triton-X100 to release soluble proteins (S1). This extraction was then repeated in the presence of 0.25 M (NH4)2SO4 to release cytoskeletal proteins (S2). Finally, cells were digested with DNase 1 and RNase A, then extracted again with 0.25 M (NH4)2SO4 to release chromatin material (S3). Insoluble final material (Ins.) contained nuclear matrix and intermediate filament proteins, including lamins as checked by immunofluorescence microscopy (data not shown). Proteins from the different soluble fractions were collected by precipitation with 10% TCA, then resuspended in one volume of SDS sample buffer (Laemmli, 1970). The insoluble material (Ins.) was directly resuspended in the same volume of SDS sample buffer. Whole-cell extracts were prepared by direct solubilization of fibroblasts in SDS sample buffer. Electrophoresis was performed on a 7.5% polyacrylamide gel, according to Laemmli (Laemmli, 1970).
Transfection of control human fibroblasts
Primary cultures of control human fibroblasts were grown in DMEM medium containing 15% fetal calf serum. cDNAs encoding wild-type or R482W-prelamin A were inserted into pSVK3 plasmids. For detection of expressed proteins, FLAG-epitopes were fused to the amino-termini of the constructs (Östlund et al., 2001). Cells grown at a 60% confluency were transfected in chamber slides using Lipofectamine PLUSTM (Life Technologies), following the manufacturers instructions. The cells were overlaid with the lipid-DNA complexes for 5 hours in serum-free medium, then grown in fresh complete medium with 15% serum for 24 hours. Cells were then fixed in methanol for 10 minutes at 20°C and processed for immunofluorescence studies. In these cells, the efficiency of transfection was low (2-4%).
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RESULTS |
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DNA staining appeared homogeneous in control nuclei but heterogeneous in dysmorphic nuclei of FPLD patients, with a weaker labeling in buds (Fig. 1A). DAPI staining intensity was quantified in the buds versus the rest of the nuclei in a series of fibroblasts (80) from patients JM (LaA/C R482Q) and KN (LaA/C R482W). Results were similar in the two patients and showed that DAPI staining was significantly lower in the buds versus the rest of the nuclei (26±16%). These data suggested that, in the buds of FPLD nuclei, the chromatin was more decondensed.
Variability in the nuclear abnormalities observed in cells from FPLD patients
Although the type of nuclear abnormalities present in the fibroblasts from the three patients was similar, large variations in the frequency of these alterations were observed between patients and between cells from the same patient. First, only a subset of the cells had dysmorphic nuclei, its proportion increasing with cell passages. Patient JM (LaA/C R482Q) had the more prevalent dysmorphic nuclei, their percentage increasing from 10 to 22% between cell passages 4 and 10. Only 5-13% of nuclei were dysmorphic in fibroblasts from patients LM and KN (LaA/C R482W), and 2-4% in nuclei from control fibroblasts. JM dysmorphic nuclei were also larger (367±126 µm2) than control nuclei (192±79 µm2) and than the nuclei from the two other patients (203±79 µm2). Second, the frequency of the honeycomb aspect of lamin A/C staining was different among patients, varying from 1% (JM) to 15% or 50% (LM and KN, respectively) of fibroblasts with dysmorphic nuclei. This lamin A/C lattice disorganization was also observed in 3% of eumorphic nuclei from patients LM and KN (Fig. 1B, asterisks). Third, the loss in B-type lamins was more pronounced in nuclear buds or poles of fibroblasts from patient KN (80%) than in those from patient JM (30%).
Abnormal localization of INM proteins and nucleoporins in the NE of cells from FPLD patients
The consequences of lamina disorganization on the localization of emerin, LAP2ß and nucleoporin Nup153 was checked by double immunofluorescence and confocal microscopy. In control nuclei, a similar nuclear peripheral localization was observed for both types of lamins, emerin, LAP2ß and NPC markers (Fig. 2, top). Nuclear envelope abnormalities were identical in patients with the R482Q and W mutations in lamin A/C, with a conserved colocalization of these lamins and emerin even in areas where the lamin network was disorganized (Fig. 2, bottom, left column). In nuclear buds depleted of B-type lamins, (1) emerin was still present but with a honeycomb aspect, likely reflecting the structure of the underlying A-type lamin lattice (Fig. 2, bottom), (2) the signals for LAP2ß and Nup153 were variable, ranging from normal to reduced to abolished (Fig. 2, bottom). The reduction of Nup153 signal was not specific for this nucleoporin, as similar data were obtained with mab QE5 which, in addition to Nup153, also recognizes nucleoporins p62 and Nup214 (data not shown).
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DISCUSSION |
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Abnormalities of lamin networks in fibroblasts from FPLD patients
Immunofluorescence analysis revealed the presence in fibroblasts from FPLD patients of a subpopulation of cells with dysmorphic budding nuclei surrounded by a layer of lamins A and C. No aggregation of A-type lamins was observed in the cytoplasm or in the nucleoplasm. However, defects in the lamin A/C meshwork were often present, mainly in the buds, but also sometimes at poles of normally shaped nuclei. These alterations in lamin A/C distribution generated either a honeycomb structure or a few large holes. A striking additional modification of the lamina structure in this subpopulation of cells was the partial or total loss of B-type lamins around nuclear buds or at some nuclear poles, contrasting with their normal localization and density in the rest of the nuclei. Although nuclear abnormalities were qualitatively similar in the patients with the different mutations, some quantitative differences were apparent. The patient with the LaA/C R482Q mutation had numerous (10-25%) large dysmorphic nuclei, whereas nuclei from the patients bearing the R482W substitution had a more frequent disorganization of the A-type lamin network (5-50%). These differences in cellular disorders were of no prognostic value because the severity of the disease was similar in the three patients of our study. Owing to our lack of knowledge concerning A- and B-type lamin polymerization in vivo (Herrmann and Aebi, 2000), it was difficult to assess if the loss of B-type lamins in some membrane domains of FPLD nuclei was due to a decrease in the concentration of B-type lamins in lamin heteropolymers, or to a low density or disappearance of B-type homopolymers.
Inner nuclear membrane proteins, NPCs and chromatin abnormalities
In addition to lamina disorganization, other components of the NE were altered in this subset of fibroblasts from FPLD patients, including the INM, nuclear pore complexes and adjacent chromatin. Emerin strictly colocalized with A-type lamins, even when the lamina network was severely altered, suggesting that both molecules were remaining tightly associated. This observation is in agreement with (1) the lamin A-dependent localization of emerin in the nuclear envelope of LMNA(/) mice fibroblasts expressing either wild-type or R482W mutated lamin A (Sullivan et al., 1999; Raharjo et al., 2001), and (2) the ability of LaA R482Q to interact with emerin in vitro (Holt et al., 2001). LAP2ß, which, like emerin is a member of the LEM family of INM proteins (Lin et al., 2000), has been reported to specifically bind lamin B1 (Foisner and Gerace, 1993). Therefore, one would have expected a parallel loss of both proteins in the nuclear envelope areas lacking B-type lamins. However, the loss of LAP2ß in these membrane domains was less frequently severe than that of lamin B1. This result suggested that component(s) other than lamin B1 may also play a role in targeting LAP2ß to the INM, or more trivially, that the antibody against LAP2ß had a higher affinity for its antigen than the antibody against LB1.
Nuclear pore complexes are tightly associated with the lamina (Dwyer and Blobel, 1976), thus we were expecting an alteration in their distribution in dysmorphic nuclei from FPLD patients. As for LAP2ß, the presence of nucleoporins Nup153, p62 and Nup214 in blebbing membranes was either normal, diminished or abolished, suggesting that some NE domains may be impoverished or depleted in NPCs. The requirement of an intact nuclear lamina for a normal NPC localization in the NE has been emphasized by other experimental data including, (1) the polar defects in NPCs observed in nuclei from LMNA(/) mice (Sullivan et al., 1999) and from mouse cells overexpressing lamin A mutated in the rod domain (Östlund et al., 2001), (2) the NE clustering of NPCs in Drosophila and C. elegans cells depleted of B-type lamins (Lenz-Bohme et al., 1997; Liu et al., 2000), or in cultured cells undergoing apoptotic cleavage of lamins (Lazebnik et al., 1993; Buendia et al., 1999).
The intensity of DAPI staining in nuclear buds was significantly weaker than that measured in other parts of the nuclei, suggesting that chromatin was decondensed in areas with disorganized NE domains. Despite these nuclear pleiotropic disorders, cell-cycle analysis by FACS and other methods showed that fibroblasts from FPLD patients, were euploid, normally cycling and not apoptotic, as control fibroblasts.
Ectopic expression of lamin A R482W provokes nuclear abnormalities similar to that observed in FPLD fibroblasts
By expressing LaA R482W in control human fibroblasts, we were able to generate alterations in nuclear shape and in A- and B-type lamin networks similar to that observed in fibroblasts from FPLD patients. This is a strong argument in favor of the involvement of mutated lamin A at the origin of the nuclear abnormalities observed in FPLD. Lamina disorganization can also be induced by expressing ectopic LaA R482W in mouse cells (C. Favreau et al., unpublished). Furthermore, the prevalence of aberrant nuclei in response to expression of mutated LaA/C appeared cell-type specific (Raharjo et al., 2001) (C. Favreau et al., unpublished), consistent with the tissue-sensitivity of human diseases linked to LMNA mutations.
The nuclear abnormalities in cells from FPLD patients reported here are reminiscent of those present in fibroblasts from LMNA(/) mice. Similarities include polar losses in B-type lamins, in LAP2ß and in NPCs, as well as chromatin disorganization (Sullivan et al., 1999). Our finding that a subset (5-22%) of FPLD cells had nuclear structural defects is also similar to findings by Ognibene et al. (Ognibene et al., 1999), who found an abnormal distribution of lamins and chromatin in nuclear poles of a subset of fibroblasts from a patient with an emerin-null mutation.
Mechanical properties of NEs are altered in fibroblasts from FPLD patients
Putative functional consequences of the altered nuclear lamina structure were investigated by checking the resistance to heat shock of fibroblasts from FPLD patients. Compared with control fibroblasts, extensive modifications were observed in nuclei from patients, with the appearance in the NEs of extensive folds, invaginations, evaginations and transnuclear channels. Nuclear channels are common features present in a variety of living cells in culture (Fricker et al., 1997); however, those observed by immunofluorescence in heat-shocked cells from patients were of an unusual large size. Similar large channels were also observed in cells expressing ectopic lamin A or LBR (Broers et al., 1999; Ellenberg et al., 1997), showing that these particular NE alterations may occur in response to different modifications in NE composition. Finally, heat-shocked cells from FPLD patients had a twofold reduction in their survival rate compared with control cells. Therefore, the mechanical properties of the NEs, which play a crucial role under stress conditions (Krachmarov and Traub, 1993; Zhu et al., 1999), may be impaired by missense mutations in lamins A and C. In that respect, lamins would be similar to other members of the intermediate filament proteins family (Morley et al., 1995).
The modifications of the mechanical properties of the NEs in fibroblasts from FPLD patients were also attested by their low resistance to extraction by nonionic detergents and salt (Ellis et al., 1998; Fey et al., 1984; Gerace and Blobel, 1980). A significant fraction of A- and B-type lamins, emerin and LAP2ß were solubilized from patients nuclei under conditions that do not remove these components from control nuclei. If this diminished resistance to extraction was restricted to dysmorphic nuclei or also affected nuclei with a normal shape is presently unknown. Finally, the parallel extraction of A-type lamins and emerin, together with their strict colocalization in immunofluorescent studies, further confirmed that the R482Q/W mutation does not affect the interaction of these two NE components.
In summary, our initial attempts to unravel the cellular pathophysiology of FPLD in cultured skin fibroblasts of three patients bearing the R482Q and R482W mutations in lamins A and C, has revealed abnormalities in the shape of a subset of nuclei, associated with an unusual NE organization, composition and fragility. Similar nuclear alterations probably occur in adipocytes from FPLD patients, impairing essential functions of adipose tissue and leading to lipodystrophy and its metabolic consequences. Further work on adipocytes will be necessary to understand the cellular specificity of this lamina disorganization. The evidence for mechanical interactions between nuclear scaffolding proteins, cytoskeletal filaments and integrins (Maniotis et al., 1997) may provide a clue to understanding the amplification at the tissue level of this intranuclear structural disorganization.
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ACKNOWLEDGMENTS |
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