Biology subjects

Eapen, V. V.

Publications and source records attributed to Eapen, V. V..

4 recordsLinked to original sources

bioRxiv2023

Prolonged DNA Damage Checkpoint Arrest Requires Constant Renewal and the Spindle Assembly Checkpoint

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hours these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2s binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

genetics↗

bioRxiv2022

Nuclear autophagy interactome unveils WSTF as a constitutive nuclear inhibitor of inflammation

Macroautophagy (hereafter referred to as autophagy) degrades a variety of cellular components. A poorly understood area is autophagic degradation of nuclear substrates, or "nuclear autophagy". It remains unclear what can be degraded by autophagy from the mammalian nuclei. We began our study by investigating the nuclear binding partners of ATG8 family proteins that play important roles in recognizing autophagy substrates. We systematically evaluated the ATG8 nuclear interactome in primary human cells and in mouse brain, identifying hundreds of novel interactions. We continued our study by evaluating the nuclear proteomes of cellular senescence, a stable form of cell cycle arrest program associated with inflammation, in which nuclear autophagy is involved. Combined with the ATG8 nuclear interactome data, we identified WSTF, a component of the ISWI chromatin remodeling complex, as a novel substrate of nuclear autophagy. The degradation of WSTF, mediated by a direct interaction with the GABARAP isoform of ATG8, promotes chromatin accessibility of inflammatory genes and induces senescence-associated inflammation. Furthermore, WSTF directly binds the p65 subunit of NF-{kappa}B and inhibits its acetylation, thus blocking inflammatory gene expression in the setting of senescence, cancer, and pathogen infection. In addition, we show that loss of WSTF is required for the immuno-surveillance of oncogenic Ras in mouse liver; forced expression of WSTF inhibited tumor-suppressive inflammation and led to the development of liver tumors. Taken together, our study provides a global view of mammalian nuclear autophagy and reveals a novel nuclear inhibitor of inflammation implicated in diverse pathological contexts. Targeting WSTF may be broadly valuable as therapeutic intervention of inflammatory diseases.

cell biology↗

bioRxiv2018

PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex

Autophagy is orchestrated by the Atg1-Atg13 complex in budding yeast. Under nutrient-rich conditions, Atg13 is maintained in a hyperphosphorylated state by TORC1 kinase. After nutrient starvation, Atg13 is dephosphorylated, triggering Atg1 kinase activity and autophagy induction. The phosphatases that dephosphorylate Atg13 remain uncharacterized. We show that two redundant PP2C phosphatases, Ptc2 and Ptc3, regulate autophagy via dephosphorylating both Atg13 and Atg1. In the absence of these phosphatases, starvation-induced macroautophagy is inhibited, as is the cytoplasm-to-vacuole targeting (Cvt) pathway, and the recruitment of the essential autophagy machinery to phagophore assembly sites (PAS) is impaired. Despite prolongation of the DNA damage-induced checkpoint in ptc2{Delta} ptc3{Delta} cells, genotoxin-induced autophagy is also blocked. Creating a genomic atg13-8SA allele under its endogenous promoter, lacking key TORC1 phosphorylation sites, bypasses the autophagy defect in ptc2{Delta} ptc3{Delta} strains. Taken together, these results imply that PP2C type phosphatases promote autophagy by regulating Atg1 complex.

cell biology↗

bioRxiv2018

Live cell monitoring of double strand breaks in S. cerevisiae

We have used two different live-cell fluorescent protein markers to monitor the formation and localization of double-strand breaks (DSBs) in budding yeast. Using GFP derivatives of the Rad51 recombination protein or the Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on different chromosomes, usually display 2 or 3 foci that coalesce and dissociate. Rad51-GFP, by itself, is unable to repair DSBs by homologous recombination in mitotic cells, but is able to form foci and allow repair when heterozygous with a wild type Rad51 protein. The kinetics of disappearance of Rad51-GFP foci parallels the completion of DSB repair. However, in meiosis, Rad51-GFP is proficient when homozygous. Using Ddc2-GFP, we conclude that co-localization of foci following 3 DSBs does not represent formation of a homologous recombination \"repair center,\" as the same distribution of Ddc2-GFP foci was found in the presence or absence of the Rad52 protein. The maintenance of separate DSB foci and much of their dynamics depend on functional microtubules, as addition of nocodazole resulted in a greater population of cells displaying a single focus.\n\nAuthor SummaryDouble strand breaks (DSBs) pose the greatest threat to the fidelity of an organisms genome. While much work has been done on the mechanisms of DSB repair, the arrangement and interaction of multiple DSBs within a single cell remain unclear. Using two live-cell fluorescent DSB markers, we show that cells with 3 site-specific DSBs usually form 2 or 3 foci what can coalesce into fewer foci but also dissociate. The aggregation of DSBs into a single focus does not depend on the Rad52 recombination protein, suggesting that there is no \"repair center\" for homologous recombination. DSB foci are highly dynamic and their dynamic nature is dependent on microtubules.

molecular biology↗