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

Abstract

“Rods and rings” (RR) and loukoumasomes are similarly shaped, subcellular macromolecular structures with as yet unknown function. RR, so named because of their shape, are formed in response to inhibition in the GTP or CTP synthetic pathways and are highly enriched in the two key enzymes of the nucleotide synthetic pathway. Loukoumasomes also occur as linear and toroidal bodies and were initially inferred to be the same as RR, largely due to their shared shape and size and the fact that it was unclear if they shared the same subcomponents. In human retinoblastoma tissue and cells we have observed toroidal, perinuclear, macromolecular structures of similar size and antigenicity to those previously reported in neurons (neuronal-loukoumasomes). To further characterize the subcomponents of the retinal-loukoumasomes, confocal analysis following immunocytochemical staining for alpha-tubulin, beta-III tubulin and detyrosinated tubulin was performed. These studies indicate that retinal-loukoumasomes are enriched for beta-III tubulin and other tubulins associated with microtubules. Immunofluorescence together with the in situ proximity ligation assay (PLA), confirmed that beta-III tubulin colocalized with detyrosinated tubulin within loukoumasomes. Our results indicate that these tissues contain only loukoumasomes because these macromolecular structures are immunoreactive with an anti-tubulin antibody but are not recognized by the prototype anti-RR/inosine monophosphate dehydrogenase (IMPDH) antibody (It2006). To further compare the RR and retinal-loukoumasomes, retinoblastoma cells were exposed to the IMPDH-inhibitor ribavirin, a drug known to induce the formation of RR. In contrast to RR, the production of retinal-loukoumasomes was unaffected. Coimmunostaining of Y79 cells for beta-III tubulin and IMPDH indicate that these cells, when treated with ribavirin, can contain both retinal-loukoumasomes and RR and that these structures are antigenically distinct. Subcellular fractionation studies indicate that ribavirin increased the RR subcomponent, IMPDH, in the nuclear fraction of Y79 cells from 21.3 ± 5.8% (0 mM ribavirin) to 122.8 ± 7.9% (1 mM ribavirin) while the subcellular localization of the retinal-loukoumasome subcomponent tubulin went unaltered. Further characterization of retinal-loukoumasomes in retinoblastoma cells reveals that they are intimately associated with lamin folds within the nuclear envelope. Using immunofluorescence and the in situ PLA in this cell type, we have observed colocalization of beta-III tubulin with MAP2. As MAP2 is a microtubule-associated protein implicated in microtubule crosslinking, this supports a role for microtubule crosslinkers in the formation of retinal-loukoumasomes. Together, these results suggest that loukoumasomes and RR are distinct subcellular macromolecular structures, formed by different cellular processes and that there are other loukoumasome-like structures within retinal tissues and cells.

The value of anti‐rods and rings antibodies in Western China population: A retrospective study

“…Observing by fluorescence microscope, the anti‐RR has a variety of structures, but there are two basic forms—rods 3‐10μm length and rings 2‐5μm diameter. And the formations between rods and rings are also been reported, looking like ‘sewing pin’ and ‘figure eight’ . Although the exact components of RR are still not very clear, inosine monophosphate dehydrogenase type 2 (IMPDH2) and cytidine triphosphate synthase 1 (CTPS1) are definitely the components of RR .…”

“…And the formations between rods and rings are also been reported, looking like 'sewing pin' and 'figure eight'. 4,5 Although the exact components of RR are still not very clear, inosine monophosphate dehydrogenase type 2 (IMPDH2) and cytidine triphosphate synthase 1 (CTPS1) are definitely the components of RR. 6 It is reported that IMPDH2 can catalyse the conversion of inosine monophosphate into xanthosine monophosphate in the GTP biosynthesis pathway and CTPS1 can catalyse the conversion of uridine triphosphate into cytidine triphosphate.…”

Compositional complexity of rods and rings

“…Cytoophidia are also smaller than RRs and lack ring-shaped structures ( Figure 6 ). Loukomasomes, another large cellular structure similar in appearance to RRs, have been reported ( Noble et al. , 2016 ).…”

Microtubular and Nuclear Functions of γ-Tubulin: Are They LINCed?

“…Our STED analyses suggested that the ability of γ-tubulin to oligomerize and aggregate might be behind the rod formation. Linear inclusions positive for γ-tubulin and actin observed in nuclei and the perinuclear area in mature neurons were suggested to serve as a supply of the centrosomal material in non-dividing cells [56].…”

“…It is most likely the fibrillar nature of γ-tubulin that is behind its interaction with other cytoskeletal systems: with microtubules [37], actin cytoskeleton [39] and with intermediate filaments [40,41]. Rod-like intranuclear inclusions containing γ-tubulin are present in neuronal and retinoblastoma cells [56,76], as well as in plant cells [2]. γ-Tubulin positive nuclear inclusions are closely associated with lamins in cancer cells [56].…”