Ultrastructure of Cytoplasmic and Nuclear Inosine-5’-Monophosphate Dehydrogenase 2 “Rods and Rings” Inclusions

Abstract

Inosine-5′-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.

Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction

“…2). The recently reported intranuclear RR structures Gou et al, 2014;Juda et al, 2014) seem to show similar composition as in the cytoplasm (double-arrows, Fig. 2F).…”

Ensemble cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation

“…Under these conditions IMPDH2 is bound to both adenine and guanine nucleotides, but not IMP, forming free octamers rather than filaments. In vivo, filaments are typically not observed in quiescent cells; rather, as in our model, their assembly is associated with increased intracellular IMP and decreased intracellular GTP (Schiavon et al 2018; Keppeke et al 2018; Calise et al 2014; Juda et al 2014). Depletion of guanine allows Bateman domain extension, and transient assembly into enzymatically active filaments.…”

“…IMPDH reversibly assembles into filaments in vertebrate cells and tissues, providing an additional layer of regulation. Guanine deprivation induces assembly of IMPDH into micron-scale ultrastructures composed of bundled filaments, which disassemble once homeostasis is restored (Labesse et al 2013; Calise et al 2014; Thomas et al 2012; Juda et al 2014). Additional stimuli that alter IMPDH catalytic flux result in assembly, including increased intracellular IMP, and treatment with IMPDH inhibitors or other anti-proliferative drugs (Keppeke et al 2018; Chang et al 2015; Ji et al 2006; Keppeke et al 2015).…”

Loukoumasomes Are Distinct Subcellular Structures from Rods and Rings and Are Structurally Associated with MAP2 and the Nuclear Envelope in Retinal Cells

“…Structures, thought to be intermediate between rods and rings, with a “sewing pin” and “figure eight” shape, have also been reported [2]. These structures are found predominantly in the cytoplasm, although smaller versions have been found in the nucleus [5] (for review see [3]). The enzyme inosine monophosphate dehydrogenase (IMPDH) is a major component of RR [1, 4, 6–8] and functions primarily in the GTP biosynthesis pathway by catalyzing the rate-limiting conversion of inosine monophosphate into xanthosine monophosphate.…”

“…Interestingly, mutations in the IMPDH1 gene, but not IMPDH2, result in the occurrence of a number of closely related retinal diseases [16–19]. Studies on RR have used cell cultures in which IMPDH2 is the higher expressed isotype [3–5, 8, 20]. However, IMPDH1 has also been shown to form RR structures [21, 22].…”