Disruption of a ∼23–24 nucleotide small RNA pathway elevates DNA damage responses in <i>Tetrahymena thermophila</i>

Lee, Suzanne R.; Pollard, Daniel A.; Galati, Domenico F.; Kelly, Megan L.; Miller, Brian; Mong, Christina; Morris, Megan N; Roberts-Nygren, Kerry; Kapler, Geoffrey M.; Zinkgraf, Matthew S.; Dang, Hung Quang; Branham, Erica; Sasser, Jason; Tessier, Erin; Yoshiyama, Courtney; Matsumoto, Mitsuhiko; Turman, Gaea

doi:10.1091/mbc.e20-10-0631

Cited by 4 publications

(1 citation statement)

References 65 publications

(163 reference statements)

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“…To identify S-phase cells, EdU (Jena Bioscience, Jena, Thuringia, Germany) was added into culture medium at 25 μM and incubated for 30 min. Incorporated EdU was visualized by a fluorescent dye through a Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction, by which terminal alkyne group of EdU is covalently attached with azide group of a fluorescent dye, as described previously with minor modifications [ 39 , 40 ]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.5% Triton X-100 for 3 min.…”

Section: Methodsmentioning

confidence: 99%

Impact of cell cycle on repair of ruptured nuclear envelope and sensitivity to nuclear envelope stress in glioblastoma

Kamikawa

Nakazawa

et al. 2023

Cell Death Discov.

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The nuclear envelope (NE) is often challenged by various stresses (known as “NE stress”), leading to its dysfunction. Accumulating evidence has proven the pathological relevance of NE stress in numerous diseases ranging from cancer to neurodegenerative diseases. Although several proteins involved in the reassembly of the NE after mitosis have been identified as the NE repair factors, the regulatory mechanisms modulating the efficiency of NE repair remain unclear. Here, we showed that response to NE stress varied among different types of cancer cell lines. U251MG derived from glioblastoma exhibited severe nuclear deformation and massive DNA damage at the deformed nuclear region upon mechanical NE stress. In contrast, another cell line derived from glioblastoma, U87MG, only presented mild nuclear deformation without DNA damage. Time-lapse imaging demonstrated that repairing of ruptured NE often failed in U251MG, but not in U87MG. These differences were unlikely to have been due to weakened NE in U251MG because the expression levels of lamin A/C, determinants of the physical property of the NE, were comparable and loss of compartmentalization across the NE was observed just after laser ablation of the NE in both cell lines. U251MG proliferated more rapidly than U87MG concomitant with reduced expression of p21, a major inhibitor of cyclin-dependent kinases, suggesting a correlation between NE stress response and cell cycle progression. Indeed, visualization of cell cycle stages using fluorescent ubiquitination-based cell cycle indicator reporters revealed greater resistance of U251MG to NE stress at G1 phase than at S and G2 phases. Furthermore, attenuation of cell cycle progression by inducing p21 in U251MG counteracted the nuclear deformation and DNA damage upon NE stress. These findings imply that dysregulation of cell cycle progression in cancer cells causes loss of the NE integrity and its consequences such as DNA damage and cell death upon mechanical NE stress.

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Section: Methodsmentioning

confidence: 99%

Impact of cell cycle on repair of ruptured nuclear envelope and sensitivity to nuclear envelope stress in glioblastoma

Kamikawa

Nakazawa

et al. 2023

Cell Death Discov.

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Tetrahymena

Cole¹

2022

Encyclopedia of Life Sciences

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The Tetrahymena genus is composed of mostly free‐living ciliated protozoa that have helped researchers gain insight into fundamental problems of cell biology. As all ciliates, Tetrahymena contain separate germline and somatic nuclei known as the micronucleus and macronucleus, respectively. The macronucleus is derived from a copy of the micronucleus through a process that involves extensive whole‐genome rearrangement and that is under intensive study. Tetrahymena thermophila can be readily manipulated using the tools of genetics, molecular biology, cell biology and biochemistry. Recently, next‐generation, whole‐genome sequencing has made possible the identification of previously unidentified genes responsible for a rich gallery of mutant phenotypes. Notable discoveries made using Tetrahymena include the structure of telomeres and telomerase, self‐splicing RNA, the first microtubular motor, and the link between histone acetylation and gene regulation. The approximately 104 Megabase macronuclear genome of T. thermophila has been sequenced and annotated; the micronuclear genome has been sequenced as well. Key Concepts Tetrahymena are large, complex eukaryotic cells well‐suited both for university research and for the undergraduate teaching‐laboratory. Tetrahymena, have been studied for nearly a century and have been the source of numerous ground‐breaking discoveries. Applying modern tools of genomics, proteomics, and next‐generation, whole‐genome sequencing has ushered in a new era of research to such topics as the assembly of basal bodies and cilia, pattern formation, novel roles for small ribonucleic acids ( RNAs ), membrane trafficking, secretion, phagocytosis, pinocytosis, cytoskeletal motility and chromosome dynamics including studies on the origins of deoxyribonucleic acid ( DNA ) replication, telomere synthesis, meiosis, recombination, apoptosis and the regulation of gene expression through epigenetic chromatin marking. Tetrahymena offer special insights into cell biological processes due to their many original and extraordinary capabilities including the use of a unique genetic code, novel twists on metabolism and protein trafficking, self‐splicing RNA, programmed genome rearrangement involved in genome surveillance, cortical patterning and cellular regeneration. Tetrahymena exhibit both conserved and evolutionarily divergent solutions to fundamental cell biological problems.

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Recent Advances in Ciliate Biology

Howard-Till

Kar

Fabritius

et al. 2022

Annu. Rev. Cell Dev. Biol.

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Ciliates are a diverse group of unicellular eukaryotes that vary widely in size, shape, body plan, and ecological niche. Here, we review recent research advances achieved with ciliate models. Studies on patterning and regeneration have been revived in the giant ciliate Stentor, facilitated by modern omics methods. Cryo-electron microscopy and tomography have revolutionized the structural study of complex macromolecules such as telomerase, ribozymes, and axonemes. DNA elimination, gene scrambling, and mating type determination have been deciphered, revealing interesting adaptations of processes that have parallels in other kingdoms of life. Studies of common eukaryotic processes, such as intracellular trafficking, meiosis, and histone modification, reveal conservation as well as unique adaptations in these organisms that are evolutionarily distant from other models. Continual improvement of genetic and molecular tools makes ciliates accessible models for all levels of education and research. Such advances open new avenues of research and highlight the importance of ciliate research. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 38 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Disruption of a ∼23–24 nucleotide small RNA pathway elevates DNA damage responses in Tetrahymena thermophila

Cited by 4 publications

References 65 publications

Impact of cell cycle on repair of ruptured nuclear envelope and sensitivity to nuclear envelope stress in glioblastoma

Impact of cell cycle on repair of ruptured nuclear envelope and sensitivity to nuclear envelope stress in glioblastoma

Tetrahymena

Recent Advances in Ciliate Biology

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