Specific contribution of lamin A and lamin C in the development of laminopathies

Abstract

Mutations in the lamin A/C gene are involved in multiple human disorders for which the pathophysiological mechanisms are partially understood. Conflicting results prevail regarding the organization of lamin A and C mutants within the nuclear envelope (NE) and on the interactions of each lamin to its counterpart. We over-expressed various lamin A and C mutants both independently and together in COS7 cells. When expressed alone, lamin A with cardiac/muscular disorder mutations forms abnormal aggregates inside the NE and not inside the nucleoplasm. Conversely, the equivalent lamin C organizes as intranucleoplasmic aggregates that never connect to the NE as opposed to wild type lamin C. Interestingly, the lamin C molecules present within these aggregates exhibit an abnormal increased mobility. When co-expressed, the complex formed by lamin A/C aggregates in the NE. Lamin A and C mutants for lipodystrophy behave similarly to the wild type. These findings reveal that lamins A and C may be differentially affected depending on the mutation. This results in multiple possible physiological consequences which likely contribute in the phenotypic variability of laminopathies. The inability of lamin C mutants to join the nuclear rim in the absence of lamin A is a potential pathophysiological mechanism for laminopathies.

Fig. 1

A. Nuclei expressing wild type or the various lamin A mutant constructs transiently expressed as ECFP fusion protein in COS7 cells. Cells were visualized by wide-field fluorescence microscopy with an excitation wavelength of 433 nm. Wild type lamin A, as well as L85R and R482W lamin A mutants homogeneously organize throughout the nucleus. In contrast, D192G, N195K and R386K lamin A mutants accumulate in abnormal aggregates. The nuclear membrane appears granular and discontinued. B. The abnormal lamin A aggregation previously found in COS7 cells expressing D192G, N195K and R386K lamin A, was confirmed in nuclei of rat cardiomyoblast H9C2 cells as opposed to cells expressing wild type or L85R lamin A (not shown).

Fig. 2

Confocal microscopy picture of H9C2 cell nuclei transiently expressing D192G lamin A as ECFP fusion protein. Mutated lamin A organizes as aberrant aggregates. However, the spherical organization of these aggregates reveal that they are likely embedded in the nuclear envelope.

Fig. 3

Nuclei expressing wild type or the various mutated lamin C constructs transiently expressed as ECFP fusion protein in COS7 cells. Prior to visualization by wide-field fluorescence microscopy, Hoechst 33258 dye was used to locate the nuclei (in blue). Excitation wavelengths were 433 nm for lamin C-ECFP and 365 nm for Hoechst 33258. Note that the aberrant aggregation of the lamin C (in red) within the nucleus visualized by Hoechst 33258 dye is common to several LMNA mutations. Only R482W lamin C mutants responsible for lipodystrophy gave rise to a phenotype similar to the wild type.

Fig. 4

A. Transient transfections of COS7 cells with varying quantity of either wild type or mutated lamin C-FP constructs. Cells were visualized by wide-field fluorescence microscopy with an excitation wavelength of 433 nm. The phenotype observed with mutated lamin C-FP is not due to a too elevated quantity of vectors used to transfect the cells. B. Representative Western blot analysis of COS7 cells transfected with the different lamin C-FP variants and using anti lamin A/C antibody. Results show equal over-expression for all construct.

Fig. 5

A. Electron micrographs of COS7 cells transiently transfected with lamin C-FP constructs. In 60% of cells, wild type lamin C nuclear aggregates were localized in close contact with the nuclear envelope (a–c). Notably, wild type aggregates were able to establish close contact with the nuclear envelope (circle) (c). Similarly, L85R lamin C aggregates organized in contact with the nuclear envelope (d–e). Conversely, in 88% and 87.5% of cells respectively, D192G and R386K lamin C aggregates were found within the nucleoplasm without any contact with the nuclear envelope (f–i). In 30% of cells expressing R386K lamin C, nuclei presented with lamin C aggregates localized outside the nuclear envelope (j–k). B. Percentage of transfected cells displaying lamin C nuclear aggregates in direct contact with the nuclear envelope. χ² test showed that the number of cells displaying lamin C aggregates in close contact with the nuclear envelope is significantly different in transfected cells expressing D192G or R386K lamin C mutants compared to the wild type (p<0.01).

Fig. 6

A. COS7 cell nuclei transiently co-expressing wild type or mutated lamin A and lamin C constructs. Lamins A and C were inserted into pECFP-C1 and pDsRed2-C1 fluorescent expression vectors respectively. Cells were visualized by wide-field fluorescence microscopy with excitation wavelengths of 433 nm for lamin A-FP and 558 nm for DsRed2-lamin C. Compared to the wild type, the complex lamin A/C forms aggregates and the membrane appears granular and discontinued. B. Laser-scanning confocal microscopy of COS7 cells nuclei transiently co-transfected with either wild type or mutated lamin A and lamin C. Lamin A (in red) and lamin C (in green) were inserted into pECFP-C1 and pEYFP-C1 fluorescent expression vectors; respectively. Excitation wavelengths were 433 nm for lamin A-FP and 558 nm for DsRed2-lamin C.

Fig. 7

Confocal immunofluorescence microscopy pictures of COS7 cells nuclei transiently co-expressing wild type or mutated lamin A-FP and lamin C-FP and immunostained with the anti RanGaP1 polyclonal antibody (N-19). RanGap appears normally distributed in the nuclear envelope. Arrows indicate nuclei with R386K lamin C aggregates localized outside the nuclear envelope.

Fig. 8

A. FRAP experiments on wild type and L85R, D192G and/or R386K lamin C nuclear aggregates of COS7 cells. The boxed regions were bleached and the fluorescence recovery was monitored over a 210 s period. B. Quantitative experiments showing normalized fluorescence recovery after photobleaching of a targeted region of the lamin C nuclear aggregates in COS7 cells. (a) The fluorescence intensity in the bleached area is expressed as a relative recovery. Error bars indicates SEM, n=7. 1 is the level of fluorescence before bleaching. (b) We assessed the time after photobleaching required for fluorescence to recover the median value between the prebleach and just after the bleach (t_1/2) as a mean to reflect the dynamics of molecule.

Fig. 8

A. FRAP experiments on wild type and L85R, D192G and/or R386K lamin C nuclear aggregates of COS7 cells. The boxed regions were bleached and the fluorescence recovery was monitored over a 210 s period. B. Quantitative experiments showing normalized fluorescence recovery after photobleaching of a targeted region of the lamin C nuclear aggregates in COS7 cells. (a) The fluorescence intensity in the bleached area is expressed as a relative recovery. Error bars indicates SEM, n=7. 1 is the level of fluorescence before bleaching. (b) We assessed the time after photobleaching required for fluorescence to recover the median value between the prebleach and just after the bleach (t_1/2) as a mean to reflect the dynamics of molecule.