Cell cycle-dependent translocations of a major nucleolar phosphoprotein, B23, and some characteristics of its variants.
A major nucleolar phosphoprotein, B23, is thought to play several apparently unrelated roles and appears to be associated with other cell compartments besides the nucleolus. However, characteristic properties of B23 variants still remain to be established. Here, we raised a new monoclonal antibody against B23 (20B2) and used it to address the issue particularly focusing on the events during mitosis. Also, we made an attempt to generalize the data on the cell cycle-dependent translocations of B23 by the use of three mammalian cell lines (HeLa, PK, RAMT) which were found to be immunoreactive for 20B2. In all the cell strains studied, B23 was chiefly located within the nucleolus at interphase, and was associated with a few cellular domains during mitosis. They were: the nucleoplasm (at prophase before the nuclear envelope breakdown), the cytoplasm (from prometaphase until mid telophase), the perichromosomal layer (from prometaphase till early telophase), cytoplasmic B23-containing bodies (at anaphase and telophase) and prenucleolar bodies, PNBs (at telophase). On Western blots, electrophoretic mobility of B23 was found to be the same at G1, S and G2 periods of interphase, but became slower at mitosis. In situ and cell extraction experiments showed that like the nucleolar B23, B23 of the perichromosomal layer and that of PNBs was highly resistant to extraction with Triton X-100, but could be released with Triton X-100/RNase A. These findings indicated that these portions of B23 were most likely to be associated with RNA. The cytoplasmic B23 was the major intracellular variant of B23 during mitosis. It had a slightly lower electrophoretic mobility than the perichromosomal B23 and could readily be extracted with Triton X-100 without addition of RNase A, a fact indicating that the cytoplasmic B23 was mainly in an RNA-free state. Mitosis-like translocations of B23 from the nucleolus to the nucleoplasm induced by actinomycin D increased its extractability but did not affect the electrophoretic mobility. The phosphorylation status of different B23 variants at interphase and mitosis both in controls and following the drug, is discussed.
Zatsepina
O V
OV
A. N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Russia.
Todorov
I T
IT
Philipova
R N
RN
Krachmarov
C P
CP
Trendelenburg
M F
MF
Jordan
E G
EG
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects / Q000276:immunology; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003609:Dactinomycin / Q000494:pharmacology; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000187:drug effects; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D009687:Nuclear Proteins / Q000737:chemistry / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D000090243:Nucleophosmin; D017830:Octoxynol / Q000494:pharmacology; D051381:Rats; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D013552:Swine
D000911:Antibodies, Monoclonal; C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D003609:Dactinomycin; D004004:Dichlororibofuranosylbenzimidazole; D017830:Octoxynol; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic