Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive. In this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required. Together, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.

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. 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. RR 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. This 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.

Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate‐limiting steps in the de novo synthesis of purine and pyrimidine nucleotides, respectively. It has been shown that IMPDH and CTPS can comprise a filamentous macrostructure termed the cytoophidium, which may play a role in regulation of their catalytic activity. Although these two proteins may colocalise in the same cytoophidium, how they associate with one another is still elusive. As reported herein, we established a model HeLa cell line coexpressing OFP‐tagged IMPDH2 and GFP‐tagged CTPS1 and recorded the assembly, disassembly and movement of the cytoophidium in live cells. Moreover, by using super‐resolution confocal imaging, we demonstrate how IMPDH‐ and CTPS‐based filaments are aligned or intertwined in the mixed cytoophidium. Collectively, our findings provide a panorama of cytoophidium dynamics and suggest that IMPDH and CTPS cytoophidia may coordinate by interfilament interaction.

A novel pattern in the indirect immunofluorescence antinuclear antibody assay on HEp-2 cells (IIF-HEp-2) characterized by cytoplasmic rods and rings (RR) was reported in HCV patients, but stringent disease specificity studies and longitudinal analysis are lacking. We investigated the clinical significance of anti-RR in an HCV cohort with up to a 12-month treatment follow up. 597 patients (342 HCV, 55 HCV/HIV, 200 non-HCV) were screened and titered for anti-RR. Serial samples were available from 78 of 176 treated and 27 of 166 untreated patients. Anti-RR was detected in 14.1% of 342 HCV patients, 9.1% of 55 HCV/HIV, 3.4% of 29 Hepatitis B, and none of 171 non-HCV (p<0.0001; HCV versus non-HCV). Anti-RR was present in 38% of 108 patients receiving interferon-α/ribavirin, but none in 26 receiving either interferon-α or ribavirin, or 166 untreated patients (p47% tested positive for anti-RR. The anti-RR titer generally increased with sustained treatment and remained high in 53% of patients. After treatment, anti-RR titer was negative in 41%. Non-responders to HCV therapy were 77% in anti-RR-positive versus 64% in anti-RR-negative patients. Response to treatment was not associated with anti-RR titer or the dynamics of anti-RR reactivity during and after treatment. The exquisite association of anti-RR reactivity with combined interferon-α/ribavirin therapy in HCV patients represents a unique model for drug-induced autoantibody generation in humans as demonstrated by the fact that a significant fraction of patients who have anti-RR during therapy becomes anti-RR-negative after completion of therapy.