Spatial distribution of lamin A and B1 in the K562 cell nuclear matrix stabilized with metal ions.
When the nucleus is stripped of most DNA, RNA, and soluble proteins, a structure remains that has been referred to as the nuclear matrix, which acts as a framework to determine the higher order of chromatin organization. However, there is always uncertainty as to whether or not the nuclear matrix, isolated in vitro, could really represent a skeleton of the nucleus in vivo. In fact, the only nuclear framework of which the existence is universally accepted is the nuclear lamina, a continuous thin layer that underlies the inner nuclear membrane and is mainly composed of three related proteins: lamins A, B, and C. Nevertheless, a number of recent investigations performed on different cell types have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nuclear matrix. In most cell types investigated, the nuclear matrix does not spontaneously resist the extraction steps, but must rather be stabilized before the application of extracting agents. In this investigation, by immunochemical and morphological analysis, we studied the effect of stabilization with different divalent cations (Zn(2+), Cu(2+), Cd(2+)) on the distribution of lamin A and B1 in the nuclear matrix obtained from K562 human erythroleukemia cells. In intact cells, antibodies to both lamin A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. The fluorescent lamin A pattern detected in Cu(2+)- and Cd(2+)-stabilized nuclei was markedly modified, whereas Zn(2+)-incubated nuclei showed an unaltered pattern of lamin A distribution. By contrast, the distribution of lamin B1 in isolated nuclei was not modified by the stabilizing cations. When chromatin was removed by nuclease digestion and extraction with solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix. Our results indicate that when metal ions are used as stabilizing agents for the recovery of the nuclear matrix, the distribution of both lamin A and lamin B1 in the final structures, corresponds to the pattern we have very recently reported using different extraction procedures. This observation strengthen the concept that intranuclear lamins may act as structural components of the nuclear matrix.
Neri
L M
LM
Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia Umana, Università di Ferrara, 44100 Ferrara, Italy.
Raymond
Y
Y
Giordano
A
A
Borgatti
P
P
Marchisio
M
M
Capitani
S
S
Martelli
A M
AM
D002104:Cadmium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology*; D002453:Cell Cycle; D003300:Copper / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D020014:K562 Cells; D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D018613:Microscopy, Confocal; D015530:Nuclear Matrix / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D002104:Cadmium; D003300:Copper; D015032:Zinc