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The cell cycle dependent mislocalisation of emerin may contribute to the Emery-Dreifuss muscular dystrophy phenotype

Elizabeth A. L. Fairley^1,*, Andrew Riddell², Juliet A. Ellis^2, and John Kendrick-Jones¹

¹ MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK
² Wellcome Trust Centre for the Molecular Mechanism in Disease, Addenbrooke’s Hospital, Cambridge, CB2 2XY, UK
Present address: Randall Centre for the Molecular Mechanism of Cell Function, Kings College, New Hunts House, Guy’s Campus, London, SE1 1UL, UK

View larger version (25K): [in a new window]   Fig. 1. (A) Cell cycle profiles of asynchronised and synchronised COS-7 cells. Propidium Iodide (PI) was added to COS-7 cells so that their DNA content could be measured by flow cytometry, and thus the stages of the cell-cycle identified. The black line shows the cell-cycle profile for asynchronised COS-7 cells. The cells are in G1, S and G2M phase, although the majority of the cells are in G1. Cells were synchronised by nocodazole treatment; the blue line shows that now at least 50% of cells are arrested in G2M. (B) Flow cytometry can be used to study EGFP-tagged proteins during the cell cycle. COS-7 cells were transfected with EGFP-emerin constructs and examined by flow cytometry. The untransfected population (dotted black line box) and transfected population (solid black line box) could be gated to allow each population to be analysed separately. The gates also allowed doublet cells or cells expressing a high amount of EGFP to be excluded from the analysis. (C) Cell-cycle profiles of untransfected and EGFP-emerin-transfected cell populations. The PI area (DNA content) and EGFP intensity were both monitored so that cell-cycle profiles show the relative number of cells in G1, S or G2M for each population (untransfected and transfected). In each profile, the orange lines represent the untransfected populations and the green and red lines represent cells expressing EGFP-emerin 12 and 24 hours after being released from G2M. The untransfected population acts as an internal control to compare the length of the cell cycle with that for cells transfected with either wild-type or mutant forms of emerin. The cells expressing the emerin mutant Del236-241 are shown to have a prolonged cell cycle length.

View larger version (25K): [in a new window]   Fig. 2. Tracking untransfected and transfected cells over a number of cell divisions. To establish the extent of the lag caused by EGFP-emerin mutant (Del236-241), COS-7 cells transfected with EGFP-emerin constructs were monitored over four cell divisions using a fluorescent cell-tracking dye (PKH26). The fluorescent cell-tracking dye was incorporated into cell membranes at time 0, and its intensity examined at three time points 0, 56 and 104 hours. For the untransfected populations (wt, black dotted line; D236 (D236-241), blue dotted line; S54F, light-grey dotted line; 1-220, dark-grey dotted line), the fluorescence dye intensity decreases exponentially as the dye is dispersed between the daughter cells during cell division. Thus, the medium fluorescent intensity of PKH26 over time allows the cell-cycle timing to be monitored. Cells transfected with wild-type EGFP-emerin (black solid line) or mutants; missense S54F (light-grey solid line) and 1-220 (dark-grey solid line) showed the normal exponential decrease in dye intensity, whereas cells transfected with EGFP-emerin mutant Del236-241 (D236, blue solid line) show a steady decrease in dye intensity, meaning that these cells took longer to cycle.

View larger version (33K): [in a new window]   Fig. 3. Cell-cycle profiles showing cells expressing wild-type and Del236-241 EGFP-emerin at the same stage in the cell cycle. The PI area (DNA content) and EGFP intensity were both monitored so that cell-cycle profiles show the relative number of cells in G1, S or G2M for each population (untransfected and transfected). In each profile, the orange lines represent the untransfected populations and the black, purple and blue lines represent cells expressing EGFP-emerin 28, 32 and 35 hours after being released from G2M. Cells expressing wild-type EGFP-emerin 28 hours after synchronisation were in G1-S, whereas cells expressing Del236-241 EGFP-emerin 35 hours after synchronisation were in G1-S. Thus cells expressing Del236-241 EGFP-emerin lag behind those cells expressing wild-type EGFP-emerin by six to seven hours per cycle.

View larger version (81K): [in a new window]   Fig. 4. The localisation of emerin mutants through mitosis by immunofluorescence microscopy. To show simultaneously the location of exogenously expressed mutant forms of emerin and endogenous lamin A/C during mitosis, COS-7 cells were transfected with wild-type (A), missense S54F (B), Del236-241 (C) and 1-220 (D) EGFP-emerins and immunolabelled for lamin A/C. The chromatin was stained with DAPI to identify the stages in the cell cycle. The cells expressing wild-type and mutant EGFP-emerins, except for Del236-241, show a similar mitotic distribution to that of endogenous lamin A/C. The cells expressing the emerin mutant Del236-241 show an altered localisation pattern for both the EGFP-emerin and endogenously expressed lamin A/C. Cells transfected with EGFP-emerin 1-220 were similar to those transfected with EGFP-vector alone. Nikon type 108 microscope; bar, 10 µm.

View larger version (104K): [in a new window]   Fig. 5. The localisation of endogenous lamin A/C and exogenously expressed EGFP-emerin in the nucleus after cell division. A series of z-sections (0.2 µm apart) were taken through the nuclei of the transfected COS-7 cells in interphase 28 hours after being released from nocodazole treatment. EGFP-emerin is shown in green and endogenous lamin A/C shown in red. The lamin A/C localisation was unaffected in cells expressing wild-type EGFP-emerin (A; 18 z-sections taken in total); however, in the cells expressing the mutant forms of emerin, endogenous lamin A/C was redistributed. The nuclear morphology was altered in the cells transfected with Del95-99 (B; 23 z-sections taken in total) and those cells with irregularly shaped nuclei consistently had exogenously expressed Del95-99 EGFP-emerin and endogenously expressed lamin A/C lost from one of the poles. The nuclear morphology was also altered in cells that expressed Del236-241 (D; 31 z-sections taken in total) EGFP-emerin, and the number of z-sections show that the cells expressing this mutant were smaller and rounder. Intranuclear tubules of emerin and lamin A/C are seen to a greater extent in cells transfected with either wild-type (A) or missense mutations (C; S54F). Radiance Confocal Laser Scanning System; bar, 5 µm.

View larger version (21K): [in a new window]   Fig. 6. A number of COS-7 and HeLa cells transfected with either wild-type or mutant EGFP-emerins show endogenous lamin A/C redistribution and altered nuclear morphology. The endogenous lamin A/C and nuclear architecture appeared unaffected (100%) in both COS-7 or HeLa cells transfected with wild-type (wt) EGFP-emerin (COS-7; n=3, an average of 857 cells per count, HeLa; n=3, an average of 691 cells per count). The number of cells transfected with either Del236-241 (COS-7; n=3, an average of 595 cells per count, HeLa; n=3, an average of 124 cells per count) or Del95-99 (COS-7; n=3, an average of 1169 cells per count, HeLa; n=3, an average of 852 cells per count) EGFP-emerin that had irregularly shaped nuclei with lamin A/C mislocalised was greater than the number of cells that had round nuclei with lamin A/C localised. The percentage of cells expressing S54F EGFP-emerin (COS-7; n=3, an average of 1204 cells per count; HeLa; n=3, an average of 713 cells per count) that had either irregularly shaped nuclei with lamin A/C mislocalised or round-shaped nuclei with lamin A/C localised was similar. The quantification data show that, after cell division, the redistribution of lamin A/C depends upon the localisation of EGFP-emerin.

View larger version (55K): [in a new window]   Fig. 7. Further localisation of emerin and lamin A/C by representing z-sections as x-z slices through cells. A series of z-sections (0.2 µm apart) were taken through the nuclei of COS-7 cells transfected with either wild-type or Del236-241 EGFP-emerin. The z-sections shown are for: (A) a cell transfected with wild-type EGFP-emerin (18 z-sections taken in total) 28 hours after being released from nocodazole; (B) a cell transfected with Del236-241 EGFP-emerin (31 z-sections taken in total) 28 hours after being released from nocodazole; and (C) a cell transfected with Del236-241 EGFP-emerin (25 z-sections taken in total) 35 hours after being released from nocodazole treatment. All z-sections were collected and represented as x-z slices through the cells. The projections of all the z-sections taken show emerin in green, lamin A/C in red and colocalisation in yellow. The white lines show where the z-sections were collected: the centre of the nucleus 0, 0+5 µm and 0-5 µm. The line thickness was ±0.3 µm for wild-type cells at 28 hours, ±0.3 µm for Del236-241 at 28 hours and ±0.6 µm for Del236-241 at 35 hours. Each x-z slice is shown separately in black and white for clarity. Cells expressing Del236-241 EGFP-emerin 28 and 35 hours after synchronisation were microscopically similar. The correct localisation of emerin and lamin A/C to the nuclear membrane is shown to be important, as the mutant form of emerin (Del236-241) redistributes lamin A/C with resultant defects in the nuclear architecture. Radiance Confocal Laser Scanning System; bar, 5 µm.