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Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin A/C gene

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

View larger version (74K): [in a new window]   Fig. 1. Nuclear shape abnormalities and uneven distribution of A- and B-type lamins in fibroblasts from FPLD patients. (A) Control and FPLD-fixed cells were labelled with DAPI, rabbit anti-lamins B antibodies (LB, green) and mabs directed against lamins A/C (LA/C, red), before analysis by conventional immunofluorescence microscopy. Two control nuclei are shown, a regular ovoid nucleus and a dysmorphic budding nucleus (arrowhead), the latter being observed in a low percentage of cells (2-3%). In the two nuclei, A- and B-type lamins are detected at the periphery, including in the bud. In the nucleus from an FPLD patient, A- and B-type lamins colocalize at the periphery of the main part of the nucleus, whereas A-type lamins predominate in the buds. Note that one bud contains a continuous A-type lamin meshwork (arrowhead), whereas the lattice in the other one is discontinuous (asterisk). Note also the faint DAPI staining of DNA, especially in the buds. (B) Cells from a control individual and from FPLD patients JM (LaA/C R482Q) and LM (LaA/C R482W) were treated as described above and examined by confocal microscopy. As in A, B-type lamin staining was weak or absent in buds, whereas signals for A-type lamins were of a normal intensity. Note the variation in size and shape of the nuclear herniations, some being connected to the nucleus by large necks (arrowheads), others showing a strong lamin staining at their base (arrows). Note also the honeycomb aspect of both lamin types meshworks in some buds or poles (asterisks). Bars, 10 µm.

View larger version (66K): [in a new window]   Fig. 2. Abnormal localization of INM proteins and nucleoporins in the NE of fibroblasts from FPLD patients. Fibroblasts from control individuals (top) and from FPLD patients (bottom) were labeled for double immunofluorescence as indicated, then observed with a confocal microscope. The patterns of nuclear defects were similar in the three patients. Note the systematic colocalization of lamin A/C and emerin in nuclei from controls and patients and, in B-type lamin-depleted buds (arrowheads), the presence of emerin and the partial or total absence of LAP2ß and Nup153. Bars, 5 µm.

View larger version (76K): [in a new window]   Fig. 3. Cell-cycle progression in FPLD cells. (A) FACS analysis of fibroblasts from control individuals and patients JM (LaA/C R482Q) and LM (R482W). Data are the mean±s.e. of four different experiments, each being performed with cells from two control and two patients. Apo, apoptotic cells. (B) A living fibroblast from patient LM containing a dysmorphic nucleus was observed by phase contrast and microcinematography. Note the stability of the nuclear morphology over a 28 hour period and the ability of the cell to divide. Bar, 10 µm.

View larger version (59K): [in a new window]   Fig. 4. Heat shock induces deep alterations in the nuclear shape of cells from FPLD patients. Unheated (Time 0) and heat-shocked (30 min H.S.) fibroblasts from a control individual and patient JM (LaA/C R482Q) were fixed, then immunolabeled for A-type lamins (LA/C, red), and finally analyzed by confocal microscopy. Three sections of each cell were examined (upper, middle, lower). A row containing the superposition of the three sections is shown at the bottom of the figure (Total). Note in the nuclear envelope of heated fibroblasts of JM patient the presence of folds (arrowheads), and ‘holes’ (arrows) that may represent invaginations, evaginations or channels (arrows). The end of one of these channels is visible (double arrow). The honeycomb structure of the lamina in a nucleus from JM patient is indicated by asterisks. Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 5. Lamins, emerin and LAP2ß are more readily extracted from cells of the FPLD patients than from control cells. (A) Fibroblasts from control individuals and FPLD patients were lysed in SDS, and whole-cell extracts corresponding to 1.2, 2.5, 3.7 and 5x105 cells were analyzed by immunoblotting using anti-lamins (LA/C and LB), anti-emerin and anti-LAP2ß antibodies. Note the increase in intensity of the different signals that parallels the increase in cell number, with a saturation of the signals at 5x105 cells. Note also the similar intensity of signals for each protein in both panels. (B) Control (C) and fibroblasts from FPLD patients were sequentially extracted under increasingly stringent conditions (see Materials and Methods). Soluble fractions (S1, S2 and S3), insoluble material (Ins.) and proteins from whole-cell extracts (Total) were analyzed in parallel by immunoblotting. Samples loaded in each lane correspond to an identical cell number. Note that lamins, emerin and LAP2ß are extracted at a lower stringency in cells from the patients than in control cells. Similar data were obtained with cells from patient JM (LaA/C R482Q) or from patients KN and LM (R482W). A representative blot is shown.

View larger version (67K): [in a new window]   Fig. 6. Ectopic expression of LaA R482W in human control fibroblasts generates lamina and chromatin abnormalities similar to that observed in fibroblasts from FPLD patients. Human control fibroblasts were transfected with expression vectors containing either FLAG-wild-type prelamin A (FLAG-WT-LA) or R482W-prelamin A (FLAG-R482W-LA) cDNAs, then maintained in culture for 29 hours. Cells were fixed, then processed for DNA staining with DAPI (A, D, G) and lamin staining with anti-FLAG (red, B, E, H) and anti-lamin B (green, C, F, I) antibodies, before examination by conventional immunofluorescence microscopy. A cell that does not express ectopic lamin A is shown by arrows (A-C). Ectopic FLAG-WT-LA was generally located at the NE where it colocalized with B-type lamins (A-C). By contrast, among cells expressing ectopic FLAG-R482W-LA, some had dysmorphic nuclei (D-I) with nuclear buds containing a disorganized A-type lamin network with a honeycomb aspect (E, H, arrowheads), and with faint B-type lamins (F, I, arrowheads) and DAPI (D, G, arrowheads) stainings. Note in H and I the presence of holes in the A- and B-type lamins networks. Bar, 10 µm.