Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase.
The nuclear envelope of eukaryotic cells provides a barrier separating nucleus from cytoplasm, thereby regulating the exchange of macromolecules between both compartments. However, in cells exposed to severe forms of stress this barrier may break down, resulting in the mixing of nuclear and cytoplasmic contents. We show here that the fusion protein GFP-beta-galactosidase can be used to evaluate the intactness of nuclear envelopes in HeLa cells that have been exposed to heat and oxidative stress. GFP-beta-galactosidase is restricted to the cytoplasm of interphase cells, but enters the nucleus when nuclear membranes are disrupted. For comparison, we have analyzed the barrier function of nuclear membranes with antibodies against lamin B. Treatment of fixed cells with digitonin permeabilizes the plasma membrane, but leaves nuclear envelopes intact. Consequently, after digitonin incubation antibodies to lamin B can bind their antigen only if nuclear membranes are damaged. For various heat and oxidative stress conditions, we have compared the distribution of GFP-beta-galactosidase with the accessibility of lamin B to antibodies. Our results demonstrate that nuclear envelopes are permeable to antibodies whenever GFP-beta-galactosidase enters the nucleus. GFP-beta-galactosidase is therefore a useful tool for evaluating the disintegration of the nuclear envelope and identifying cells in which a mixing of nuclear and cytoplasmic material takes place.
Sánchez
Leticia
L
Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, H3G 1Y6, Canada.
Kodiha
Mohamed
M
Stochaj
Ursula
U
D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D009685:Nuclear Envelope / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase