title: Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.
abstract: Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.
authors.affiliation: Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, 94720, USA.
authors.forename: S W
authors.initials: SW
authors.lastname: Krauss
authors.forename: C A
authors.initials: CA
authors.lastname: Larabell
authors.forename: S
authors.initials: S
authors.lastname: Lockett
authors.forename: P
authors.initials: P
authors.lastname: Gascard
authors.forename: S
authors.initials: S
authors.lastname: Penman
authors.forename: N
authors.initials: N
authors.lastname: Mohandas
authors.forename: J A
authors.initials: JA
authors.lastname: Chasis
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002294:Carcinoma, Squamous Cell; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D003598:Cytoskeletal Proteins; D004261:DNA Replication; D004171:Diploidy; D000939:Epitopes / Q000032:analysis; D004910:Erythrocyte Membrane / Q000737:chemistry; D005260:Female; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008969:Molecular Sequence Data; D009479:Neuropeptides; D015530:Nuclear Matrix; D009687:Nuclear Proteins / Q000032:analysis; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D012326:RNA Splicing; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D008941:Spindle Apparatus / Q000737:chemistry; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms
chemical_list: D003598:Cytoskeletal Proteins; D000939:Epitopes; D008565:Membrane Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; D012261:Ribonucleoproteins; C499372:SRSF2 protein, mouse; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors