Evaluation of mammalian cell-free systems of nuclear assembly and disassembly

Abstract

Mammalian cell-free systems are very useful for the biochemical and structural study of nuclear disassembly and assembly. Through manipulation, the role of specific proteins in these processes can be investigated. I first intended to examine the involvement of integral and peripheral inner nuclear membrane proteins in nuclear disassembly and assembly. However, I was unable to achieve disassembly when isolated interphase HeLa nuclei were exposed to mitotic soluble extracts containing cyclin B1. Homogenates of synchronized mitotic HeLa cells left to reassemble nuclei resulted in incomplete nuclear envelope assembly 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 the experimental evaluation of mammalian cell-free systems. Synchronization itself induced incomplete nuclear assembly. This may be caused by the prior aberrant nuclear disassembly, or by the abnormal number of mitotic spindles.