Nuclear lamina assembly, synthesis and disaggregation during the cell cycle in synchronized HeLa cells.

Abstract

ABSTRACT The pattern of assembly, synthesis and disaggregation of the nuclear envelope-associated lamina in synchronized HeLa cells has been examined by means of immunofluorescence microscopy of cell preparations treated with antibody to lamina polypeptide LP67 or lamin B, using the nomenclature of Gerace and Blobel. During the cell cycle the distribution of lamina polypeptide varies dramatically. Three distinct stages can be detected with respect to its location and synthesis: Lamina polypeptide is cytoplasmically distributed during cell division particularly at the time of complete nuclear envelope breakdown. At telophase it is reassembled and becomes associated with regions of the chromosome surface. This process continues into early G1, lamina polypeptide associating progressively with the outer surface of the chromosomes; simultaneously its cytoplasmic concentration is reduced. The traffic of antigen from cytoplasm to the chromosomes is highest between telophase and G1 but continues at a reduced rate during G1. Inhibition of protein synthesis during division and G1 does not inhibit G1 lamina reformation, suggesting that the G1 lamina is constructed from depolymerized cytoplasmically-stored polypeptide derived from the previous cell cycle.Lamin B is synthesized in S-phase. The protein is rapidly accumulated at the nuclear periphery during the doubling of the nuclear surface. There is very little if any cytoplasmic location of lamina polypeptide in S-phase cells. The S-phase pattern of lamina distribution persists through early and mid G2Towards the end of G2, and particularly with the onset of prophase, the nuclear membrane-bound lamina is released into the cytoplasm over approximately 30 min. (Fusion between metaphase and either G1 or S-phase cells also results in the depolymerization of lamin B within 30 min.) Lamina polypeptides appear in the cytoplasm before the nuclear envelope has disappeared, and protein synthesis in G2, is required for complete lamina disaggregation. Lamina reformation in late mitosis and the polarity of its appearance in the telophase cell can be perturbed by treatment with colcemid or low temperature so that lamina is induced to associate with metaphase and anaphase chromosomes. In the absence of spindle microtubules lamina reformation may proceed around the surface of whole chromosomes, leading to the formation of micronuclei in G1 cells. We propose that in the normal cell nuclear envelope formation at telophase is related to the terminal distribution of cytoskeleton structures such that lamina is laid down only on the outer surface of the telophase genome beginning at the telomeric chromosome ends adjacent to the remaining mitotic fibres of the cleavage furrow.