Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization

Abstract

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A.In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts.Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.

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

“…However, in contrast to previous studies reporting lamin aggregates described as intranuclear [18,[25][26][27], we found that lamin A aggregates mainly localized at the nuclear periphery of the nucleus, suggesting that although unable to properly organize in the nuclear rim and form the lamina, lamin A mutants keep the capability of associating with the nuclear envelope. Alternatively, other mutants (L85R, R482W) behaved in a way that was indistinguishable from the wild type.…”

“…1A and B), which is consistent with results already described in C2C12 or COS7 cells [11,26,27]. Several immunofluorescence microscopy studies performed on cells lines transfected with lamin A/C have reported the aggregation of lamin A as intra-nuclear foci or nuclear aggregates inside the nucleoplasm [18,[25][26][27]. To confirm this statement in our analyses, we used confocal immunofluorescence microscopy and 3D reconstruction of the cell (Fig.…”

“…Mice expressing M371K lamin A exhibit cardiomyocytes with convoluted nuclear envelopes, intranuclear inclusions and chromatin clumps in nuclei [24]. Several LMNA mutations are known to result in the aggregation of lamin A in vitro [18,[25][26][27]. In a previous report, we found DCM-causingLMNA mutations leading to the aggregation of nuclear lamin C as well [11].…”

“…Interestingly, only the mutations responsible for skeletal and cardiac diseases were found to result in this atypical aggregation whereas mutation R482W involved in human lipodystrophy, did not result in noticeable abnormalities in the location or distribution of either lamin A or lamin C. This finding suggests that the pathophysiological mechanism of laminopathies with cardiac involvement is distinct from lipodystrophy and engages lamin C but not lamin A as made evident by the absence of abnormal phenotype in cells expressing lamin A with the dilated cardiomyopathy mutation L85R. Other mutations responsible for cardiomyopathy or muscular dystrophy have also been reported to result in aberrant lamin A or C aggregation such as mutations E358K, M358K [27], or R453W [25]. However, all mutations should be tested to corroborate this conclusion and determine if this lamin C aggregation inside the nucleoplasm could be regarded as a potential hallmark of laminopathies with cardiac or muscular involvement.…”

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

“…Plasmid pHM990, coding for a fusion protein of IE2p86 and GFP, was kindly provided by Dr. N. Tavalai (Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg) (28). The plasmids pEGFP-N1-lamin A and pEGFP-N1-lamin C code for lamin A and C, respectively, which are fused to EGFP (kindly provided by Dr. J. Broers, Cardiovascular Research Institute, University of Maastricht, Maastricht, The Netherlands) (29).…”