Abstract
Mitotic catastrophe is an oncosuppressive mechanism that drives cells toward senescence or death when an error occurs during mitosis. Eukaryotic cells have developed adaptive signaling pathways to cope with stress. The phosphorylation on serine 51 of the eukaryotic translation initiation factor (eIF2α) is a highly conserved event in stress responses, including the one that is activated upon treatment with mitotic catastrophe inducing agents, such as microtubular poisons or actin blockers. The protocol described herein details a method to quantify the phosphorylation of eIF2α by high-throughput immunofluorescence microscopy. This method is useful to capture the ‘integrated stress response’, which is characterized by eIF2α phosphorylation in the context of mitotic catastrophe.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Donnelly N, Gorman AM, Gupta S, Samali A (2013) The eIF2alpha kinases: their structures and functions. Cell Mol Life Sci 70(19):3493–3511. https://doi.org/10.1007/s00018-012-1252-6
Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC, Durchschlag M, Joza N, Pierron G, van Endert P, Yuan J, Zitvogel L, Madeo F, Williams DB, Kroemer G (2009) Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J 28(5):578–590. https://doi.org/10.1038/emboj.2009.1
Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059):1081–1086. https://doi.org/10.1126/science.1209038
Vitale I, Galluzzi L, Castedo M, Kroemer G (2011) Mitotic catastrophe: a mechanism for avoiding genomic instability. Nat Rev Mol Cell Biol 12(6):385–392. https://doi.org/10.1038/nrm3115
Denisenko TV, Sorokina IV, Gogvadze V, Zhivotovsky B (2016) Mitotic catastrophe and cancer drug resistance: a link that must to be broken. Drug Resist Updat 24:1–12. https://doi.org/10.1016/j.drup.2015.11.002
Portugal J, Mansilla S, Bataller M (2010) Mechanisms of drug-induced mitotic catastrophe in cancer cells. Curr Pharm Des 16(1):69–78
Ganguly A, Yang H, Sharma R, Patel KD, Cabral F (2012) The role of microtubules and their dynamics in cell migration. J Biol Chem 287(52):43359–43369. https://doi.org/10.1074/jbc.M112.423905
Bezu L, Sauvat A, Humeau J, Gomes-da-Silva LC, Iribarren K, Forveille S, Garcia P, Zhao L, Liu P, Senovilla L, Kepp O, Kroemer G (2018) eIF2α phosphorylation is pathognomonic for immunogenic cell death. Cell Death Differ 25(8):1375–1393
Coldwell MJ, Cowan JL, Vlasak M, Mead A, Willett M, Perry LS, Morley SJ (2013) Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis. Cell Cycle 12(23):3615–3628. https://doi.org/10.4161/cc.26588
Senovilla L, Vitale I, Martins I, Tailler M, Pailleret C, Michaud M, Galluzzi L, Adjemian S, Kepp O, Niso-Santano M, Shen S, Marino G, Criollo A, Boileve A, Job B, Ladoire S, Ghiringhelli F, Sistigu A, Yamazaki T, Rello-Varona S, Locher C, Poirier-Colame V, Talbot M, Valent A, Berardinelli F, Antoccia A, Ciccosanti F, Fimia GM, Piacentini M, Fueyo A, Messina NL, Li M, Chan CJ, Sigl V, Pourcher G, Ruckenstuhl C, Carmona-Gutierrez D, Lazar V, Penninger JM, Madeo F, Lopez-Otin C, Smyth MJ, Zitvogel L, Castedo M, Kroemer G (2012) An immunosurveillance mechanism controls cancer cell ploidy. Science 337(6102):1678–1684. https://doi.org/10.1126/science.1224922
Senovilla L, Demont Y, Humeau J, Bloy N, Kroemer G (2017) Image cytofluorometry for the quantification of ploidy and endoplasmic reticulum stress in cancer cells. Methods Mol Biol 1524:53–64. https://doi.org/10.1007/978-1-4939-6603-5_3
Szaflarski W, Fay MM, Kedersha N, Zabel M, Anderson P, Ivanov P (2016) Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation. Oncotarget 7(21):30307–30322. https://doi.org/10.18632/oncotarget.8728
Tanimukai H, Kanayama D, Omi T, Takeda M, Kudo T (2013) Paclitaxel induces neurotoxicity through endoplasmic reticulum stress. Biochem Biophys Res Commun 437(1):151–155. https://doi.org/10.1016/j.bbrc.2013.06.057
Perlman ZE, Slack MD, Feng Y, Mitchison TJ, Wu LF, Altschuler SJ (2004) Multidimensional drug profiling by automated microscopy. Science 306(5699):1194–1198
Acknowledgments
GK is supported by the Ligue contre le Cancer Comité de Charente-Maritime (équipe labelisée); Agence National de la Recherche (ANR)—Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); a donation by Elior; the European Commission (ArtForce); the European Research Council (ERC); Fondation Carrefour; Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière; the Searave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI). JH is supported by the Fondation Philanthropia. LB is supported by Bristol Myers Squibb Foundation for Research in Immuno-Oncology (BMS). PL is supported by the China Scholarship Council.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s)
About this protocol
Cite this protocol
Humeau, J., Bezu, L., Kepp, O., Senovilla, L., Liu, P., Kroemer, G. (2021). Quantification of eIF2α Phosphorylation Associated with Mitotic Catastrophe by Immunofluorescence Microscopy. In: Manfredi, J.J. (eds) Cell Cycle Checkpoints. Methods in Molecular Biology, vol 2267. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1217-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1217-0_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1216-3
Online ISBN: 978-1-0716-1217-0
eBook Packages: Springer Protocols