Heat-induced stabilization of the nuclear matrix: a morphological and biochemical analysis in murine erythroleukemia cells.
Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.
Martelli
A M
AM
Istituto di Anatomia Umana Normale, Bologna, Italy.
Falcieri
E
E
Gobbi
P
P
Manzoli
L
L
Gilmour
R S
RS
Cocco
L
L
D000818:Animals; D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure; D003850:Deoxyribonuclease I; D006358:Hot Temperature; D034882:Lamins; D004915:Leukemia, Erythroblastic, Acute; D007942:Leukemia, Experimental; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D015530:Nuclear Matrix / Q000648:ultrastructure*; D009687:Nuclear Proteins / Q000032:analysis / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D034882:Lamins; D009687:Nuclear Proteins; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic