Abstract
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.
Highlights
In 2002, we reported the identification and cloning of a novel 182 kDa protein named GW182, which we isolated using a patient serum to immunoscreen a HeLa cDNA library (Eystathioy et al, 2002a)
GWBs are distinct cytoplasmic entities with their number and size varying during the cell cycle Cytoplasmic GWBs were first observed by indirect immunofluorescence (IIF) on HEp-2 and HeLa cells by using the index human autoimmune serum
In some cells there were more strongly stained structures than in other cells. To address whether this variation is associated with the cell cycle, we first performed costaining with conventional markers of the cell cycle that were readily available to us, including CENP-F (Fig. 1B,C) and PCNA
Summary
In 2002, we reported the identification and cloning of a novel 182 kDa protein named GW182, which we isolated using a patient serum to immunoscreen a HeLa cDNA library (Eystathioy et al, 2002a). GW182 appears to react with a subset of mRNAs and localizes to discrete cytoplasmic domains that do not colocalize to any of the known conventional organelles in the cytoplasm (Eystathioy et al, 2002a). We tentatively identified the cytoplasmic domain marked by antibodies directed against GW182 as GW bodies or GWBs. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within GWBs. We proposed that the GW182 ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. Epitopes bound by the human autoantibodies were mapped to the GW-rich mid-part of the protein, the non-
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