Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase IIβ (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37°or 42°C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0°C in a buffer containing spermine/spermidine/KC1 or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0°C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37°or 42°C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KC1, the incubation buffer also contained 5 mM Mg++ and the temperature was 37°C. If the stabilization was performed at 42°C, Mg++ failed to maintain the original distribution of DNA topoisomerase IIβ, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37°C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37°C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0°C incubated nuclei; they showed a certain degree of shrinkage in 0°C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37°or 42°C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37°C, but not at 42°C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.

Redistribution of DNA topoisomerase II β after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy

NERI, Luca Maria
Primo
;
1997

Abstract

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase IIβ (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37°or 42°C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0°C in a buffer containing spermine/spermidine/KC1 or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0°C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37°or 42°C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KC1, the incubation buffer also contained 5 mM Mg++ and the temperature was 37°C. If the stabilization was performed at 42°C, Mg++ failed to maintain the original distribution of DNA topoisomerase IIβ, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37°C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37°C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0°C incubated nuclei; they showed a certain degree of shrinkage in 0°C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37°or 42°C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37°C, but not at 42°C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.
1997
Neri, Luca Maria; A. M., Martelli; N. M., Maraldi
File in questo prodotto:
File Dimensione Formato  
Microscopy Research and Technique 1997-2.pdf

solo gestori archivio

Descrizione: versione editoriale
Tipologia: Full text (versione editoriale)
Licenza: NON PUBBLICO - Accesso privato/ristretto
Dimensione 197.54 kB
Formato Adobe PDF
197.54 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/521561
Citazioni
  • ???jsp.display-item.citation.pmc??? 1
  • Scopus 16
  • ???jsp.display-item.citation.isi??? 15
social impact