A progeria mutation reveals functions for lamin A in nuclear assembly, architecture, and chromosome organization

Abstract

Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the alpha-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LADelta50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization.

Fig. 1.

Lamin alterations in E145K cells. (A) E145K (p11) and control cells (p19) were stained with anti-LA/C, anti-LB1, and Hoechst. Mid-plane confocal sections show extensive lobulation of the NE in E145K cells. LA/C is associated mainly with the outer region of the lobules (bracket), which stain weakly for LB1 and surround a euchromatic region as indicated by weak staining with Hoechst. Arrows indicate small blebs containing LA/C, but no detectable LB1. LB1 is associated with the heterochromatic proximal regions of the lobules, which contain little LA/C (arrowheads). (Scale bars, 5 μm.) (B) Immunoblotting analysis of E145K and control cells at p14. The amount of LB1 and to a lesser extent of LB2 is reduced in E145K cells, while LA and LC are expressed at levels comparable to controls.

Fig. 2.

Mislocalization of heterochromatin, centromeres, and telomeres in E145K cells. (A) E145K (p11) and control cells (p11) were stained with either anti-H3K9me3 or anti-H3K27me3 and Hoechst. Mid-plane confocal sections show enrichment of H3K9me3 in the central region of the nucleus in E145K cells (arrowhead), while it is enriched in the nuclear periphery and in perinucleolar regions in controls (arrowheads). H3K27me3 is distributed throughout the nucleus in both cells and enriched in the Xi in female control cells (arrowhead). (Scale bars, 5 μm.) (B) E145K (p13) and control cells (p15) were stained with anti-LA, CREST antiserum, and Hoechst. Maximum projections of series of z-sections spanning the entire nucleus and side projections are shown. Centromeres are clustered in the central region of the nucleus in E145K cells, while they are either closely associated with the peripheral lamina region or elsewhere in the nucleoplasm in control cells. Inset shows an example of the close association of one centromere with the lamina in a single confocal section. (Scale bars, 5 μm.) (C) E145K and control cells were stained with anti-LA/C, CREST antiserum, and Hoechst. A FISH probe was used to visualize telomeres. Maximum projections of series of z-sections spanning the entire nucleus and side projections from merged images are shown. Centromeres and telomeres are distributed throughout the nuclei in control cells. In E145K cells centromeres are clustered in the middle of one region of the nucleus, while telomeres are more frequently associated with the peripheral lamina region. (Scale bars, 5 μm.)

Fig. 3.

Expression of FLAG-E145K-LA in HeLa cells induces nuclear lobulations and mislocalization of centromeres and telomeres. (A) HeLa cells were transfected with either FLAG-WT-LA or FLAG-E145K-LA for 72 h and stained with anti-FLAG and CREST antiserum. Maximum projections of series of z-sections spanning the entire daughter cell nuclei are shown. Note the extensive lobulation of the lamina and clustering of centromeres in FLAG-E145K-LA-expressing cells. (Scale bars, 5 μm.) (B) HeLa cells were transfected with either FLAG-WT-LA or FLAG-E145K-LA for 72 h, fixed, and stained with anti-FLAG, CREST antiserum, Hoechst, and a FISH probe to visualize telomeres. Maximum projections of z-sections and side projections are shown as above. Centromeres and telomeres are distributed throughout the nuclei in FLAG-WT-LA-expressing cells. In FLAG-E145K-LA-expressing cells centromeres and telomeres are less interspersed, and telomeres are more likely to be associated with the peripheral lamina region. (Scale bars, 5 μm.)

Fig. 4.

Multilobulated E145K patient cells are capable of replicating their DNA despite the abnormal chromatin configuration. Double replication labeling assays (first pulse: IdU, 4 h Chase, second pulse: CldU) were carried out on E145K and control cells and subsequently stained with CREST antiserum and Hoechst. Single confocal sections showing both early and mid-to-late replication patterns are presented. In control cells, early replication foci are distributed throughout the nucleus, while mid-to-late replication foci are either found in association with the lamina region or deeper in the nucleoplasm. Some of the mid-to-late foci colocalize with CREST (arrows). In E145K cells early replication foci are enriched in lobules while many of the mid-to-late replication foci are concentrated in the central region of the nucleus with the clustered centromeres (arrows). (Scale bars, 5 μm.)

Fig. 5.

FLAG-E145K-LA interferes with postmitotic nuclear assembly and centromere positioning in daughter cells. HeLa cells stably expressing YFP-CENP A (green) were transfected with FLAG-E145K-LA and mCherry-WT-LA (red) for 48-72 h. Cells with normally shaped nuclei were followed through mitosis and nuclear reassembly by using time-lapse imaging. A single confocal section is shown for each time point. mCherry-LA is localized normally during metaphase, anaphase, and telophase (0.5 to 1.5 h), but the daughter cells eventually show lobulated NE and clustered centromeres for up to 10 h. (Scale bar, 10 μm.)

Fig. 6.

Structural analysis of filaments and PCs assembled from WT-LA, E145K-LA, and LAΔ50/progerin. Bacterially expressed and purified WT-LA, E145K-LA, and LAΔ50/progerin were allowed to self-assemble into filaments and PCs in vitro and analyzed by TEM. Negatively stained images show that filaments of varying diameters assembled from WT-LA and LAΔ50/progerin are well ordered after 10 min, while those assembled from E145K-LA are disorganized. For cryo-ET, 60-nm-thick x-y slices along the z-axis through tomograms of PC assembled from WT-LA, E145K-LA, and LAΔ50/progerin are shown. Pseudo-colored higher magnification images show volume rendering views of 30-nm-thick tomographic slices of the marked regions. Both WT-LA and LAΔ50/progerin PCs show similar repeat patterns at low magnification. In contrast the E145K-LA PC is significantly less well ordered. This is emphasized at higher magnification, which shows that the protofilaments in PCs assembled from WT-LA and LAΔ50/progerin are well ordered, while the lateral interactions of protofilaments in PC assembled from E145K-LA are disordered. [Scale bars, 0.2 μm (Left) and 50 nm (Middle).]