Abstract
BackgroundCytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.Methodology/Principal FindingsDistinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.Conclusions/SignificanceRR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.
Highlights
Over the past few decades many human autoantibodies have emerged as significant disease-specific markers for systemic rheumatic diseases [1]
The current study reports the identification of cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) as components associated with these mammalian rods and rings (RR)
RR are expressed in some mitotic cells; co-staining studies with anti-centromere protein F (CENP-F), showed that the expression of either rods or rings were independent of the cell cycle, as they appear in all stages (Figure 3)
Summary
Over the past few decades many human autoantibodies have emerged as significant disease-specific markers for systemic rheumatic diseases [1]. These autoantibodies are mainly directed against intracellular macromolecular complexes or particles, such as nucleosomes and small nuclear/cytoplasmic ribonucleoproteins [1]. Human autoantibodies have served as useful probes for exploring subcellular structures and functions because of their unexpected specificity to novel self-antigens. Examples of significant uses of human autoantibodies in further characterization of novel subcellular structures included the identification of p80coilin in Cajal bodies (formerly known as coiled bodies) [2] and GW182 in GW bodies [3]. Within the past few years, our laboratories identified novel human autoantibodies that recognized unique cytoplasmic structures described provisionally as rods and rings. The formation of these conserved structures in mammalian cells and functions linked to these structures were examined
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