Quantitative analysis of localization and nuclear aggregate formation induced by GFP-lamin A mutant proteins in living HeLa cells.
Although A-type lamins are ubiquitously expressed, their role in the tissue-specificity of human laminopathies remains enigmatic. In this study, we generate a series of transfection constructs encoding missense lamin A mutant proteins fused to green fluorescent protein and investigate their subnuclear localization using quantitative live cell imaging. The mutant constructs used included the laminopathy-inducing lamin A rod domain mutants N195K, E358K, M371K, R386K, the tail domain mutants G465D, R482L, and R527P, and the Hutchinson-Gilford progeria syndrome-causing deletion mutant, progerin (LaA delta50). All mutant derivatives induced nuclear aggregates, except for progerin, which caused a more lobulated phenotype of the nucleus. Quantitative analysis revealed that the frequency of nuclear aggregate formation was significantly higher (two to four times) for the mutants compared to the wild type, although the level of lamin fusion proteins within nuclear aggregates was not. The distribution of endogenous A-type lamins was altered by overexpression of the lamin A mutants, coexpression experiments revealing that aberrant localization of the N195K and R386K mutants had no effect on the subnuclear distribution of histones H2A or H2B, or on nuclear accumulation of H2A overexpressed as a DsRed2 fusion protein. The GFP-lamin fusion protein-expressing constructs will have important applications in the future, enabling live cell imaging of nuclear processes involving lamins and how this may relate to the pathogenesis of laminopathies.
Hübner
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S
Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, PO Box 13D, Monash University, Clayton, Victoria 3800, Australia. stefan.huebner@mail.uni-wuerzburg.de
Eam
J E
JE
Wagstaff
K M
KM
Jans
D A
DA
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D034904:Lamin Type A / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D020125:Mutation, Missense; D034881:Nuclear Lamina / Q000235:genetics / Q000378:metabolism*; D011371:Progeria / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D006657:Histones; D034904:Lamin Type A; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins