Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins.
The behavior of chimeric proteins consisting of A-type lamins and green fluorescent protein (GFP) was studied to investigate the localization and dynamics of nuclear lamins in living cells. Cell line CHO-K1 was transfected with cDNA constructs encoding fusion proteins of lamin A-GFP, lamin Adelta10-GFP, or lamin C-GFP. In the interphase nucleus lamin-GFP fluorescence showed a perinuclear localization and incorporation into the lamina for all three constructs. Our findings show for the first time that the newly discovered lamin A 10 protein is localized to the nuclear membrane. The GFP-tagged lamins were processed and behaved similarly to the endogenous lamin molecules, at least in cells that expressed physiological levels of the GFP-lamins. In addition to the typical perinuclear localization, in the majority of transfected cells each individual A-type lamin-GFP revealed an extensive collection of branching intra- and trans-nuclear tubular structures, which showed a clear preference for a vertical orientation. Time-lapse studies of 3-D reconstructed interphase cells showed a remarkable stability in both number and location of these structures over time, while the lamina showed considerable dynamic movements, consisting of folding and indentation of large parts of the lamina. Fluorescence recovery after bleaching studies revealed a low protein turnover of both tubular and lamina-associated lamins. Repetitive bleaching of intranuclear areas revealed the presence of an insoluble intranuclear fraction of A-type lamins. Time-lapse studies of mitotic cells showed that reformation of the lamina and the tubular structures consisting of A-type lamins did not occur until after cytokinesis was completed.
Broers
J L
JL
Department of Molecular Cell Biology and Genetics, University of Maastricht, PO Box 616, The Netherlands. Jos.Broers@molcelb.unimaas.nl
Machiels
B M
BM
van Eys
G J
GJ
Kuijpers
H J
HJ
Manders
E M
EM
van Driel
R
R
Ramaekers
F C
FC
D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007399:Interphase; D034904:Lamin Type A; D034882:Lamins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D015530:Nuclear Matrix / Q000378:metabolism / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D056489:Nucleoplasmins; D010750:Phosphoproteins / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection
D034904:Lamin Type A; D034882:Lamins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C440308:lamin C; D049452:Green Fluorescent Proteins