Alterations in mitosis and cell cycle progression caused by a mutant lamin A known to accelerate human aging

Abstract

Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LADelta50/progerin). These modifications cause an abnormal association of LADelta50/progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G(1) is impaired in cells expressing LADelta50/progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.

Fig. 1.

Cells from HGPS patients expressing LAΔ50/progerin contain abnormal cytoplasmic structures enriched in nuclear lamina/envelope components in early G₁. (A) Mid- to late-passage (p) (5) HGPS fibroblasts (HGADFN003, p18) were fixed in 3% paraformaldehyde and processed for immunofluorescence with anti-LA/C (a, d, and g), anti-LA (b), anti-LB (e), and anti-emerin (h). Early G₁ cells displayed abnormal cytoplasmic structures containing LA/C (a, c, d, f, g, and i), LA (b and c), LB (e and f), and emerin (h and i). These structures are also seen in HGPS fibroblasts from another patient (HGADFN127, p16) but not in control fibroblasts [AG08470 (p17) and AG09309 (p18)] (data not shown). Confocal images are shown. (Scale bars, 10 μm.) (B) Late passage HGPS fibroblasts (HGADFN003, p22) were methanol fixed and processed for double immunofluorescence by using anti-LA/C (a–d, red) and either anti-phosphoRb(Ser-807/811) (a and d, green), anti-Rb (b, green), or anti-cyclin D1 (c, green). Confocal images are shown. (Scale bars, 5 μm.)

Fig. 2.

LAΔ50/progerin is farnesylated and carboxymethylated. (A) HeLa cells expressing GFP-LAΔ50/progerin were incubated with the farnesol analogue, AG, and with (+FTI) or without (−FTI) 3 μM FTI-277. GFP-LAΔ50/progerin was immunoprecipitated with anti-GFP and analyzed by immunoblotting, using either anti-AG (α-AG) or anti-LA/C (α-LA/C). The results of two experiments (±FTI) are shown. (B) HeLa cells expressing GFP-LAΔ50/progerin or GFP-LA were incubated with ([methyl-³H])methionine for 16–20 h. After incubation, proteins were immunoprecipitated consecutively with antibodies against LB, LA, and EGFP, separated on SDS/PAGE, and identified by immunoblotting. Protein bands (n = 3) were excised, and carboxymethylation was determined as described in Materials and Methods.

Fig. 3.

LAΔ50/progerin is membrane associated during mitosis. (A) HeLa Tet-on cells expressing either GFP-LA (a–c) or GFP-LAΔ50/progerin (d–l) were fixed with 3.7% formaldehyde and processed for immunofluorescence by using anti-emerin (b, c, e, and f), anti-LA (k and l), and anti-LB (h and i). DNA was stained with Hoechst dye (c, f, i, and l). Confocal images of metaphase cells are shown. (Scale bars, 5 μm.) (B) HeLa Tet-on cells expressing GFP-LAΔ50/progerin were arrested in mitosis and extracted with (+T) or without (−T) detergent (see Materials and Methods). Supernatant (Sn) and pellet (P) fractions were collected. Immunoblots using anti-LA/C (α-LA/C), anti-LB (α-LB), or anti-emerin (α-emerin) are shown. (C and D) Fluorescence loss in photobleaching analyses of metaphase HeLa Tet-on cells expressing either GFP-LA or GFP-LAΔ50/progerin. GFP-fluorescence was repeatedly photobleached in the area indicated by the closed white circle at ≈4-sec intervals. An image was captured immediately after each time interval. (C) Images of representative time points are shown (0 sec represents prephotobleach). The fluorescence intensity was measured at each time point in an area opposite to the photobleached area (dotted white circle). (Scale bars, 5 μm.) (D) The images obtained from 10 GFP-LA (red dots) and from 11 GFP-LAΔ50/progerin (green dots) expressing metaphase cells were analyzed (see SI Materials and Methods). For each time point, the normalized fluorescence intensity measurements were averaged and plotted as a function of time. Error bars are indicated for each time point.

Fig. 4.

LAΔ50/progerin re-localizes exclusively to the nuclear periphery at the end of mitosis and delays cytokinesis and nuclear envelope reassembly. (A) HeLa Tet-on cells expressing either GFP-LA (a) or GFP-LAΔ50/progerin (b) were followed by time-lapse microscopy from the metaphase/anaphase transition (0 sec) into G₁. Confocal images were acquired at ≈79-sec intervals (see SI Materials and Methods). Representative time points for the onset of cytokinesis (arrows) and for the initial localization of the GFP-fusion proteins to the newly forming sister nuclei (arrowheads) are shown (394−631 sec), as well as the time when most of the GFP-LA was located in the nucleus (1,025 sec). (Scale bars, 10 μm.) (B) GFP-LA- (a) and GFP-LAΔ50/progerin- (b) expressing HeLa Tet-on cells were fixed with 3.7% formaldehyde, and DNA was stained with Hoechst dye. In addition, GFP-LAΔ50/progerin expressing cells were incubated with FTI-277 before fixation (c). Confocal images of telophase cells are shown. (Scale bars, 5 μm.)

Fig. 5.

LA and LB are retained in the cytoplasm in early G₁ cells expressing GFP-LAΔ50/progerin. HeLa Tet-on cells expressing either GFP-LAΔ50/progerin (A–I) or GFP-LA (M–O) were fixed with 3.7% formaldehyde and processed for immunofluorescence by using anti-emerin (B and C), anti-LA (E and F) and anti-LB (H, I, K, L, N, and O). In addition, GFP-LAΔ50/progerin expressing cells were incubated with FTI-277 before fixation (J–L). Confocal images of late telophase/early G₁ cells are shown. (Scale bars, 5 μm.)