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First published online 19 October 2004
doi: 10.1242/jcs.01477
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Research Article |
1 Department of Oral Biology, University of Florida, PO Box 100424, Gainesville, FL 32610-0424, USA
2 The Core Microscopy Facility, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
3 Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive, Calgary, Alberta, T2N 4N1, Canada
4 Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, 915 Camino de Salud NE, Albuquerque, NM 87131, USA
Author for correspondence (e-mail: echan{at}ufl.edu)
Accepted 2 August 2004
A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.
Key words: mRNA degradation complex, GW repeats, GW bodies
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