Intermediate structures in nuclear morphogenesis following metaphase from HeLaS3 cells can be isolated and temporally grouped.
Previously nuclear reformation following metaphase in HeLaS3 cells was conceptualized in terms of a stepwise process which was continuous throughout anaphase and telophase. This concept was based on a three-dimensional visualization by scanning electron microscopy (SEM) of individual, organically prepared chromatid structures (prenuclei) which could be sequentially arranged. Morphologic analysis revealed unique topographies and morphometric properties which suggested that it should be possible to isolate populations of prenuclei aqueously. Such an isolation using detergents and density centrifugation is presented which yields metaphase plates and two populations of prenuclei with distinctive morphology. Essentially, prenuclei are freed from late mitotic cells in suspension cultures of synchronized HeLaS3 cells by treatment with 0.1% Nonidet-P40 followed by treatment with a mixture of Tween 40-desoxycholate (0.5%). Critical for the isolation is the presence of a divalent cation (5 mM Mg(+)+) and an acid pH (approximately 5.8). After density centrifugation, 2N decondensing structures (late intermediates) are recovered from 42% Percoll, and a mixture of 2N predecondensing (early intermediates) and 4N metaphase plates are recovered from 52% Percoll. The latter intermediates can be further separated into highly enriched populations (greater than 94% pure) by fluorescence-activated sorting. Predecondensing structures are of the same overall morphology as prenuclei isolated previously by organic means, can also be ordered sequentially to demonstrate nuclear morphogenesis, and retain centromere/kinetochore loci. These chromosomal loci based on immunostaining of individual structures appear to be positioned centrally during chromatid reassociation and then appear to be dispersed prior to structural rearrangements leading to formation of a disc-like prenucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Hodge
L D
LD
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912.
Martinez
J E
JE
Allsbrook
W C
WC
Pantazis
C G
CG
Welter
D A
DA
D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D002877:Chromosomes, Human / Q000032:analysis; D004247:DNA / Q000032:analysis; D003902:Detergents; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008677:Metaphase; D008858:Microscopy, Phase-Contrast; D009024:Morphogenesis
D003902:Detergents; D004247:DNA