Evaluation of mammalian cell-free systems of nuclear disassembly and assembly.
Mammalian cell-free systems are very useful for the biochemical and structural study of nuclear disassembly and assembly. Through experimental manipulations, the role of specific proteins in these processes can be studied. Recently, we intended to examine the involvement of integral and peripheral inner nuclear membrane proteins in nuclear disassembly and assembly. However, we could not achieve proper disassembly when isolated interphase HeLa nuclei were exposed to mitotic soluble extracts obtained from the same cell line and containing cyclin B1. Homogenates of synchronized mitotic HeLa cells left to reassemble their nuclei generated incomplete nuclear envelopes on chromatin masses. Digitonin-permeabilized mitotic cells also assembled incomplete nuclei, generating a lot of cytoplasmic inclusions of inner nuclear membrane proteins as an intermediate. These results were therefore used as a basis for a critical evaluation of mammalian cell-free systems. We present here evidence that cell synchronization itself can interfere with the progress of nuclear assembly, possibly by causing aberrant nuclear disassembly and/or by inducing the formation of an abnormal number of mitotic spindles.
Vaillant
Dominique C
DC
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5.
Paulin-Levasseur
Micheline
M
D000818:Animals; D016466:CHO Cells; D002457:Cell Extracts; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure; D002474:Cell-Free System; D006224:Cricetinae; D003412:Cricetulus; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D009685:Nuclear Envelope / Q000502:physiology / Q000648:ultrastructure; D010539:Permeability
D002457:Cell Extracts; D004072:Digitonin; D015739:Nocodazole