A normally masked nuclear matrix antigen that appears at mitosis on cytoskeleton filaments adjoining chromosomes, centrioles, and midbodies.
mAbs were generated against HeLa nuclear matrix proteins and one, HIB2, which selectively stained mitotic cells, was selected for further study. Western blot analysis showed H1B2 antibody detected a protein of 240 kD in the nuclear matrix fractions. The H1B2 antigen was completely masked in immunofluorescently stained interphase cells. However, removing chromatin with DNase I digestion and 0.25 M ammonium sulfate extraction exposed the protein epitope. The resulting fluorescence pattern was bright, highly punctate, and entirely nuclear. Further extraction of the nuclear matrix with 2 M NaCl uncovers an underlying, anastomosing network of 9-13 nm core filaments. Most of the H1B2 antigen was retained in the fibrogranular masses enmeshed in the core filament network and not in the filaments themselves. The H1B2 antigen showed remarkable behavior at mitosis. As cells approached prophase the antigen became unmasked to immunofluorescent staining without the removal of chromatin. First appearing as a bright spot, the antibody staining spread through the nucleus finally concentrating in the region around the condensed chromosomes. The antibody also brightly stained the spindle poles and, more weakly, in a punctate pattern in the cytoskeleton around the spindle. As the chromosomes separated at anaphase, H1B2 remained with the separating daughter sets of chromosomes. The H1B2 antigen returned to the reforming nucleus at telophase, but left a bright staining region in the midbody. Immunoelectron microscopy of resinless sections showed that, in the mitotic cell, the H1B2 antibody did not stain chromosomes and centrioles themselves, but decorated a fibrogranular network surrounding and connected to the chromosomes and a fibrogranular structure surrounding the centriole.
Nickerson
J A
JA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Krockmalnic
G
G
Wan
K M
KM
Turner
C D
CD
Penman
S
S
D034961:Antigens, Nuclear; D002467:Cell Nucleus / Q000737:chemistry*; D002502:Centrioles / Q000737:chemistry; D002875:Chromosomes / Q000737:chemistry*; D003599:Cytoskeleton / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D016253:Microscopy, Immunoelectron; D008938:Mitosis; D015530:Nuclear Matrix / Q000737:chemistry*; D009687:Nuclear Proteins / Q000032:analysis*; D011418:Prophase; D008941:Spindle Apparatus / Q000737:chemistry; D013692:Telophase
D034961:Antigens, Nuclear; D009687:Nuclear Proteins