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© The Rockefeller University Press, 0021-9525/1997//275 $5.00
The Journal of Cell Biology, Volume 137, Number 2, , 1997 275-289

Article

Structural Protein 4.1 in the Nucleus of Human Cells: Dynamic Rearrangements during Cell Division



Sharon Wald Krauss*, Carolyn A. Larabell*, Stephen Lockett*, Philippe Gascard*, Sheldon Penman{ddagger}, Narla Mohandas*, and Joel Anne Chasis*

* Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, Berkeley, California 94720; and {ddagger} Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

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.

1. Abbreviations used in this paper: CMV, cytomegalovirus; DAPI, 4'6- diamidino-2-phenylindole; GST, glutathione-S-transferase; NuMA, nuclear mitotic apparatus protein; PCNA, proliferating cell nuclear antigen.

We are very grateful to Ms. Gabriela Krockmalnic (Massachusetts Institute of Technology, Cambridge, MA) for her expertise in providing the resinless section electron micrographs of nuclear matrix, to Ms. Gloria Lee for red cell experiments, and to Ms. Marilyn Parra for engineering the 4.1 epitope-tagged construct. We particularly appreciate the important suggestions of Dr. John Conboy. We thank Drs. Judith Campisi and Jeffrey A. Nickerson for many valuable discussions during the course of this work. Ms. Alicia Sheppard and Mr. Derek Clark were most helpful in the preparation of this manuscript and its figures.

Address all correspondence to Sharon Wald Krauss, University of California, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 74-157, Berkeley, CA 94720. Tel.: (510) 486-6439. Fax: (510) 486-6746.


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