Abstract
BackgroundOur laboratory previously reported interesting rods 3–10 μm long and rings 2–5 μm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. In this study, we aimed to investigate the dynamic behavior of these RR in live cells.ResultsRR were detected in >90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. In the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. The specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2.ConclusionsThis is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. The disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-5-1) contains supplementary material, which is available to authorized users.
Highlights
Unique intracellular rods 3–10 μm long and rings 2–5 μm diameter (RR) have been characterized in the cytoplasm of many cell types [1,2,3,4]
Experimental evidence indicates that inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) is a major component of the RR structures, but cytidine triphosphate synthetase (CTPS), another enzyme involved in nucleotide metabolism, has been shown to be part of RR structures [2,4,5]
Human prototype anti-RR serum and rabbit polyclonal anti-IMPDH2 antibody were shown to recognize the characteristic set of RR structures in ribavirin-treated COS-7 cells (Figure 1A-C)
Summary
Unique intracellular rods 3–10 μm long and rings 2–5 μm diameter (RR) have been characterized in the cytoplasm of many cell types [1,2,3,4]. Experimental evidence indicates that inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) is a major component of the RR structures, but cytidine triphosphate synthetase (CTPS), another enzyme involved in nucleotide metabolism, has been shown to be part of RR structures [2,4,5]. Our laboratory previously reported interesting rods 3–10 μm long and rings 2–5 μm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. We aimed to investigate the dynamic behavior of these RR in live cells
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