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

PLoS One. 2016 Oct 31;11(10):e0165162. doi: 10.1371/journal.pone.0165162. eCollection 2016.

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.

MeSH terms

  • Adrenergic Neurons / metabolism
  • Animals
  • Cell Line, Tumor
  • Cytoplasmic Structures / metabolism*
  • Humans
  • Immunohistochemistry
  • Lamins / metabolism
  • Microtubule-Associated Proteins / metabolism*
  • Microtubules / metabolism*
  • Nuclear Envelope / metabolism*
  • Protein Binding
  • Protein Transport
  • Rats
  • Retina / cytology*
  • Retina / metabolism*
  • Retinoblastoma / metabolism
  • Ribavirin / pharmacology
  • Tubulin / metabolism

Substances

  • Lamins
  • MAP2 protein, human
  • Microtubule-Associated Proteins
  • Tubulin
  • Ribavirin

Grants and funding

Although the authors did not receive specific funding for this project, we did receive salary support for an undergraduate student in our laboratory. We gratefully acknowledge an Undergraduate Student Research Award (USRA) from the National Science and Engineering Research Council of Canada (NSERC) award and the student research award from Trinity Western University that provided summer salary support for Jake W. Noble. A Murdock General Science Grant as a match to the CFI Leaders Opportunity Fund Grant enabled the purchase of the Olympus DSU spinning disk confocal used in this study. The Molecular and Cell Biology laboratory and Cell Culture laboratory in which these studies took place were made possible by a grant from Murdock Trust as a match to the Industry Canada Knowledge Infrastructure Program Award. Dr. Valerie A. White, Department of Pathology and Laboratory Medicine, University of British Columbia is gratefully acknowledged for her provision of the retinoblastoma tissue for these studies. We also thank Dr. Gail M. Seigel, Department of Ophthalmology, University at Buffalo, for her provision of the retinal cell lines used in this study. Finally, we thank Joseph Goody and William K.A. Sikkema for the technical support they provided during the completion of this manuscript.