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.