The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells

Abstract

LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

Keywords: Emery-Dreifuss muscular dystrophy; Human embryonic kidney 293; Hutchinson-Gilford progeria syndrome; Lamin A/C; Laminopathy; Nuclear envelope.

Fig. 1

A scheme of lamins A and C indicating the location of the analyzed mutations within their domains and position of epitopes recognized by anti lamin A/C antibodies used in this study (Machowska et al. 2015)

Fig. 2

A characterization of the HEK 293, NHDF, and HeLa cell lines. a Lamins A and C were located within the NE and nucleoplasm of all of cell types, but their level was lowest in HEK 293 cells. Emerin localization differed between cell lines: in NHDF, it is located mostly in the nucleus; in HEK 293, it is mostly outside the nucleus; and in HeLa, it is mostly within the nuclear rim. The scale bar is 10 μm long. b A comparison of the mRNA levels for HEK 293, NHDF, and HeLa cells, measured separately for spliced forms of lamin A and C, performed using absolute quantitative RT-PCR. HEK 293 cells had the lowest levels of lamins A and C, and they were found to be similar. c Western blot analysis of protein extracts from HEK 293 and NHDF cells. The primary antibody recognizing the epitope common for lamins A and C was used. For NHDFs, the lamin A and C levels correspond to the mRNA level. For HEK 293 cells, the level of lamin C corresponded to the mRNA level, but the level of lamin A is much lower than expected from mRNA measurement. No signal was detected for HEK 293 lamin A, although there was ten times more cells per lane than for NHDFs. In addition, lamin C migrated faster for HEK 293 cells than for NHDFs. HeLa lamin C migration was the same as for NHDF lamin C (data not shown)

Fig. 3

HEK 293 transfected with plasmids encoding EGFP, fusion protein EGFP-prelamin A, and its mutants, 24 h (left panel) and 72 h (right panel) posttransfection. Cells with different levels of exogenous lamin A proteins were typically found. There was a broad spectrum of phenotypes, depending on both mutation type and protein level. Arrows indicates cells with different levels of exogenous wild-type lamin A showing that the higher the lamin A level, the lower the lamin C level

Fig. 4

HEK 293 transfected with plasmids encoding EGFP, fusion protein EGFP-prelamin A, and its mutants, 72 h posttransfection. The scale bar is 10 μm long. The images show representative nuclei for each mutant and for comparison, at the same field of view, there is a control nucleus (not transfected cell). The far right column shows signal intensity as a function of distance [nm] along drown line for EGFP (green) and lamin C (red) channels with transfected cell first and control cell afterward

Fig. 5

Western blot analysis of HEK 293 (a) and HeLa (b) cells, collected 72 h posttransfection with plasmids encoding EGFP, EGFP-prelamin A, and its mutants. Transfection efficiency was around 90 % for HEK 293 cells and around 50 % for HeLa cells. Exogenous lamin A underwent degradation in HEK 293 cells, but not in HeLa cells. Progerin detection was impaired due to the epitope not being recognized by the anti-lamin A antibody

Fig. 6

The location of endogenous emerin is not changed in HEK 293 sublines expressing EGFP, lamin A, progerin or lamin A D446 in comparison to control (not transfected cells). Scale bar is 10 μm long

Fig. 7

Analysis of HEK 293 sublines stably expressing EGFP alone and lamin A and its mutants D446V and Δ50 (progerin). a Confocal microscopy shows that all lamin A variants localize within the NE. Protein aggregates are much smaller than during transient transfection studies due to the lower level of exogenous proteins; however, some foci are still clearly visible. Progerin causes NE lobulations and the strongest deformation of the nuclear shape. b A proliferation comparison of the sublines was performed with the SRB assay 5 days after seeding. EGFP and lamin A expression significantly inhibits proliferation relative to the control, suggesting that the general effect of introducing exogenous protein may have a major role in this process. D446V and Δ50 abolished this effect, so they were able to increase the proliferation rate in embryonic cells. n/s not significant. c A comparison of protein mobility was performed using FRAP analysis. EGFP alone is very mobile: it immediately refilled the bleached area. By contrast, wild-type lamin A, progerin, and D446V proteins did not replace bleached lamin A. It shows that mutations did not significantly change protein mobility within the NE. Measurements were performed for up to 120 seconds. The insert nucleus picture shows the typical bleached area with a red circle (the merge of the EGFP and transmission light channels)