Results and discussion Expression of mutant lamin A in human cells To investigate the biological affect of different mutations on lamin A nuclear localisation and dynamics we expressed epitope tagged forms of the protein, carrying disease-associated missense mutations, in human HT1080 cells. The mutations chosen were; L85R (DCM), N195K (DCM), R482W (FPLD), and L530P (AD-EDMD). Although L85R and N195K are both located within the coiled-coil domain of lamin A, and associated with DCM (Figure 1A), they have been shown to have different behaviours when transiently expressed [ 27, 28]. R482W and L530P are associated with different disease phenotypes (lipodystrophy and myopathy, respectively), and although they are both within the globular domain, R482W is a surface residue, whilst L530P is internal (Figure 1B). Since these mutations are responsible for autosomal dominant forms of disease they should still exert their molecular phenotype in the cell in the presence of wild-type (wt) protein. Both FLAG-tagged and GFP-tagged prelamin As were transiently transfected into human fibrosarcoma cells. Each protein was processed into mature lamin A [ 29] and incorporated into the nuclear lamina, as evident by the bright nuclear ring of staining visualised either by immunofluorescence with anti-FLAG antibody or from the GFP signal (Figure 2). The mutant forms of lamin A generally had a more uneven distribution at the nuclear periphery, compared to wt, as has been reported previously [ 30]. We saw high levels of N195K lamin A in the nucleoplasm in addition to the nuclear periphery, but we did not see much evidence for its aggregation into intra-nuclear foci, as has been reported in mouse myoblasts and embryonic fibroblasts [ 27, 28],. This might reflect differences in cell-type or relative expression levels of the mutant protein. Apparently internal sites of epitope-tagged lamin As are seen, but analysis of 3D image stacks (Figure 2B) shows that these are invaginations of the nuclear periphery and not intra-nuclear foci. Such invaginations has previously been reported in many types of cultured cells [ 31- 33]. | Figure 2Sub-cellular localisation of epitope-tagged lamin As.A) Detection of FLAG-tagged wt and mutant lamin As transfected into human HT1080 fibrosarcoma cells. The FLAG tag was detected by immunofluorescence with M2 anti-FLAG (red in merge), in DAPI stained (more ...) |
Analysis of lamin A dynamics by FRAP The lamin A mutations that we have studied are within different domains of the protein (Figure 1A), or within different parts of the same structural domain (Figure 1B). Therefore they likely have different interactions, either with other molecules of lamin A, or with other proteins of the nuclear periphery or nucleoplasm. Such interactions affect the kinetic properties of a protein, and photobleaching and time-lapse imaging can probe this [ 34]. We therefore analysed the mobility of GFP tagged wt and mutant lamin As by FRAP in transiently transfected human cells. In each case a region at the nuclear lamina was bleached. The fluorescence within a 1.8 × 1.8 μm region of interest (ROI) of this bleach region was then followed every 5 minutes over a period of up to 65 minutes. To calculate the loss of fluorescence attributed to the imaging process alone, the sum of pixel intensities was also calculated for a control (unbleached) cell in each case. This was used to normalise the fluorescence intensity for each ROI [ 35]. The mean relative fluorescence intensity for each time point was then calculated for 9 cells of each of the GFP-lamin A proteins (WT, L85R, N195K, R482W and L530P). For wild-type lamin A, fluorescence at the nuclear lamina is visibly bleached (t = 0 in Figure 3A), and only about 20% of the signal recovers over the time course of the experiment (Figure 3C). This indicates that a large proportion (~80%) of lamin A at the nuclear periphery is immobile, at least within the time-frame of these experiments. This is similar to the reported immobility of 60% of lamin B receptor (LBR) in the INM [ 36]. The recovery curve shows that wt lamin A moves back into the bleach area only very slowly (Figure 3C). The extrapolated t1/2 is ~140 minutes, similar to that reported for lamin B1 (>180 mins) [ 37]. GFP-tagged lamin C expressed in CHO cells has also been reported to show very little recovery after 1 hour [ 33]. Most nuclear proteins e.g. transcription factors, and even chromatin-associated proteins such as HP1, are very dynamic with t1/2 values in the range of a few seconds [ 38]. Even the INM proteins emerin, Lap2β, and Man1 have recovery halftimes of about 1 minute [ 39]. The slow recovery of lamin A is compatible with its incorporation into a stable polymer that only exchanges subunits very slowly. | Figure 3FRAP analysis of wild type and mutant lamin As. A and B) Single z-plane confocal images of GFP-tagged (A) wt and (B) N195K lamin A expressing cells. Images were captured before (t = -5) and immediately after (t = 0) photobleaching of an area of the nuclear (more ...) |
The recovery kinetics for the R482W lamin A mutant are indistinguishable from wt and the extrapolated t1/2 = 145 mins (Figure 3C). However, the other lamin A mutants analysed show significant differences. The L85R and L530P mutant proteins appear to be more mobile than wild-type lamin A. They recover more rapidly: t1/2 L85R = 75 mins, L530P = 80 mins. Compared to wt, a higher proportion of the L85R fluorescence (35%) also recovers, suggesting that less of this mutant lamin A is in an immobile fraction. The most dramatic difference in dynamics was seen for the N195K mutant. Compared to the other lamin As it does not bleach to the same extent, and this is attributable to rapid diffusion of the high levels of nucleoplasmic protein, since at t = 0 recovery of fluorescence can be seen in the nucleoplasmic part of the bleach region, but not in the nuclear periphery itself (Figure 3B). It is known that in early G1 cells the nucleoplasmic pools of lamin A recovery their fluorescence immediately following bleaching [ 37]. However, even within the nuclear periphery fluorescence recovers within the observation period (Figure 3B, t = 15) and the t1/2 = 30 mins (Figure 3C). Therefore the N195K lamin A mutant is considerably more mobile within the nuclear lamina than wt lamin A, or indeed the other lamin A mutants studied here. Analysis of lamin A dynamics by FLIP To further analyse the movement of lamin A within the nuclear lamina, and between the lamina and the nucleoplasm, FLIP experiments were performed on wt, and N195K and L530P mutant GFP-lamin A expressing cells. After successive rounds of photobleaching at a region of the nuclear periphery, the fluorescence at a region of the nuclear periphery distant from the bleach, and at a region within the nucleoplasm were measured (Figure 4) in 10 cells each. As in FRAP, the data was normalised for the loss of fluorescence caused by the successive rounds of imaging. | Figure 4FLIP analysis of wild type and mutant lamin As. A) Single z-plane confocal images of a GFP-tagged wt lamin A expressing cell captured before (left) and immediately after (right) a round of photobleaching of an area of the nuclear periphery (red box). (more ...) |
For both wt and L530P lamin A there is little loss of fluorescence from either a distant region of the nuclear periphery, or the nucleoplasm after repeated rounds of photobleaching (Figure 4B). This reflects the slow FRAP recovery kinetics of these forms of lamin A (Figure 3). In contrast, the nucleoplasmic fraction of the N195K mutant lamin A shows a substantial decrease (24%) in fluorescence after successive rounds of bleaching at the nuclear periphery. This may reflect diffusion into the small region of nucleoplasm contained within the bleach region, but could also be due to exchange of protein between the nucleoplasm and the lamina. A 10% decrease in fluorescence is also seen at a non-bleached part of the nuclear periphery. This suggests that there is enhanced lateral movement of mutant lamin A within the nuclear lamina compared to wild-type protein. |
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