{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:46:25Z","timestamp":1785415585085,"version":"3.56.0"},"reference-count":38,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T00:00:00Z","timestamp":1779235200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["R01AG061086"],"award-info":[{"award-number":["R01AG061086"]}]},{"award":["R01AG061086"],"award-info":[{"award-number":["R01AG061086"]}],"id":[{"id":"https:\/\/ror.org\/044zkrt47","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>SUMOylation is a rapid and dynamic process that orchestrates the switch between complex assembly and disassembly and between protein stabilization and turnover, making it particularly suitable for regulating stress responses. While proteomic methodologies exist for analyzing SUMOylated proteins under stress conditions, methods\/tools for visualizing polySUMOylation dynamics have not been established. Here, we develop a polySUMOylation tracking tool by fluorescently labeling the polySIM domains derived from RNF4, which can reliably track polySUMO location and relate polySUMOylation levels to puncta number and intensity under various stress conditions, such as serum starvation, oxidative stress, and genotoxic stress. Furthermore, we extend its utility for tracking polySUMOylation across multiple cellular contexts in both control and stressed states. Collectively, this tracking tool enables deeper investigation of polySUMOylation dynamics and advances our understanding of how polySUMOylation regulates cellular processes in stress responses and disease pathogenesis.<\/jats:p>","DOI":"10.3390\/biom16050748","type":"journal-article","created":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T10:07:47Z","timestamp":1779444467000},"page":"748","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An RNF4-Based Tool for Tracking Subcellular Localization of PolySUMOylation During Cellular Stress"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-0776-1637","authenticated-orcid":false,"given":"Joseph S.","family":"Floramo","sequence":"first","affiliation":[{"name":"Department of Cell Biology, Van Andel Institute, Grand Rapids, MI 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-5385-6770","authenticated-orcid":false,"given":"Yaguang","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Van Andel Institute, Grand Rapids, MI 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorna","family":"Cohen","sequence":"additional","affiliation":[{"name":"Optical Imaging Core, Van Andel Institute, Grand Rapids, MI 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3587-1028","authenticated-orcid":false,"given":"Kristin","family":"Gallik","sequence":"additional","affiliation":[{"name":"Optical Imaging Core, Van Andel Institute, Grand Rapids, MI 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Brass","sequence":"additional","affiliation":[{"name":"Research Development, Van Andel Institute, Grand Rapids, MI 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9856-9929","authenticated-orcid":false,"given":"Tao","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Van Andel Institute, Grand Rapids, MI 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1006\/jmbi.1998.1839","article-title":"Structure determination of the small ubiquitin-related modifier SUMO-1","volume":"280","author":"Bayer","year":"1998","journal-title":"J. Mol. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1042\/BST20190357","article-title":"Molecular mechanisms in SUMO conjugation","volume":"48","author":"Varejao","year":"2020","journal-title":"Biochem. Soc. Trans."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1016\/j.molcel.2018.07.027","article-title":"SUMO-Mediated Regulation of Nuclear Functions and Signaling Processes","volume":"71","author":"Zhao","year":"2018","journal-title":"Mol. Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1038\/nsmb.2890","article-title":"Uncovering global SUMOylation signaling networks in a site-specific manner","volume":"21","author":"Hendriks","year":"2014","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/j.molcel.2020.05.017","article-title":"The Nuclear SUMO-Targeted Ubiquitin Quality Control Network Regulates the Dynamics of Cytoplasmic Stress Granules","volume":"79","author":"Wagner","year":"2020","journal-title":"Mol. Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1042\/BJ20100158","article-title":"Mechanisms, regulation and consequences of protein SUMOylation","volume":"428","author":"Wilkinson","year":"2010","journal-title":"Biochem. J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"jcs211904","DOI":"10.1242\/jcs.211904","article-title":"SUMO-specific proteases and isopeptidases of the SENP family at a glance","volume":"131","author":"Kunz","year":"2018","journal-title":"J. Cell Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1042\/BST0360874","article-title":"SUMO under stress","volume":"36","author":"Tempe","year":"2008","journal-title":"Biochem. Soc. Trans."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"668","DOI":"10.1016\/j.tcb.2016.05.004","article-title":"Principles and Properties of Stress Granules","volume":"26","author":"Protter","year":"2016","journal-title":"Trends Cell Biol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Floramo, J.S., Molchanov, V., Liu, H., Liu, Y., Craig, S.E.L., and Yang, T. (2023). An Integrated View of Stressors as Causative Agents in OA Pathogenesis. Biomolecules, 13.","DOI":"10.3390\/biom13050721"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Liu, H., Craig, S.E.L., Molchanov, V., Floramo, J.S., Zhao, Y., and Yang, T. (2022). SUMOylation in Skeletal Development, Homeostasis, and Disease. Cells, 11.","DOI":"10.3390\/cells11172710"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Karhausen, J., Ulloa, L., and Yang, W. (2021). SUMOylation Connects Cell Stress Responses and Inflammatory Control: Lessons From the Gut as a Model Organ. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.646633"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"ra24","DOI":"10.1126\/scisignal.2000282","article-title":"System-wide changes to SUMO modifications in response to heat shock","volume":"2","author":"Golebiowski","year":"2009","journal-title":"Sci. Signal."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s13008-015-0010-1","article-title":"Sumo and the cellular stress response","volume":"10","author":"Enserink","year":"2015","journal-title":"Cell Div."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1038\/s41467-017-02413-3","article-title":"Desumoylase SENP6 maintains osteochondroprogenitor homeostasis by suppressing the p53 pathway","volume":"9","author":"Li","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1958","DOI":"10.1016\/j.bbamcr.2012.07.010","article-title":"Heat shock induces a massive but differential inactivation of SUMO-specific proteases","volume":"1823","author":"Pinto","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1016\/j.bbadis.2010.12.008","article-title":"Transcriptional control of metabolic and inflammatory pathways by nuclear receptor SUMOylation","volume":"1812","author":"Treuter","year":"2011","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"21343","DOI":"10.1038\/s41598-022-25665-6","article-title":"SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect types of SUMO conjugate","volume":"12","author":"Garvin","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"886","DOI":"10.1002\/mc.23353","article-title":"Detection of rapidly accumulating stress-induced SUMO in prostate cancer cells by a fluorescent SUMO biosensor","volume":"60","author":"Yin","year":"2021","journal-title":"Mol. Carcinog."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Stirling, D.R., Swain-Bowden, M.J., Lucas, A.M., Carpenter, A.E., Cimini, B.A., and Goodman, A. (2021). CellProfiler 4: Improvements in speed, utility and usability. BMC Bioinform., 22.","DOI":"10.1186\/s12859-021-04344-9"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"538","DOI":"10.1038\/ncb1716","article-title":"RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation","volume":"10","author":"Tatham","year":"2008","journal-title":"Nat. Cell Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1038\/embor.2010.206","article-title":"Purification and identification of endogenous polySUMO conjugates","volume":"12","author":"Bruderer","year":"2011","journal-title":"EMBO Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"rs2","DOI":"10.1126\/scisignal.2005146","article-title":"Proteome-wide identification of SUMO2 modification sites","volume":"7","author":"Tammsalu","year":"2014","journal-title":"Sci. Signal."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1074\/mcp.M700173-MCP200","article-title":"In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy","volume":"7","author":"Matic","year":"2008","journal-title":"Mol. Cell Proteom."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3678","DOI":"10.1038\/s41467-019-11549-3","article-title":"Arkadia\/RNF111 is a SUMO-targeted ubiquitin ligase with preference for substrates marked with SUMO1-capped SUMO2\/3 chain","volume":"10","author":"Sriramachandran","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3174","DOI":"10.1016\/j.febslet.2008.08.008","article-title":"Arsenic trioxide stimulates SUMO-2\/3 modification leading to RNF4-dependent proteolytic targeting of PML","volume":"582","author":"Weisshaar","year":"2008","journal-title":"FEBS Lett."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.ijbiomac.2008.05.002","article-title":"Oxyradical-induced GFP damage and loss of fluorescence","volume":"43","author":"Alnuami","year":"2008","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"100199","DOI":"10.1016\/j.cellin.2024.100199","article-title":"The role of SUMOylation in biomolecular condensate dynamics and protein localization","volume":"3","author":"Wang","year":"2024","journal-title":"Cell Insight"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Wu, Z., Huang, H., Han, Q., Hu, Z., Teng, X.L., Ding, R., Ye, Y., Yu, X., Zhao, R., and Wang, Z. (2022). SENP7 senses oxidative stress to sustain metabolic fitness and antitumor functions of CD8+ T cells. J. Clin. Investig., 132.","DOI":"10.1172\/JCI155224"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1080\/21541264.2017.1311829","article-title":"Regulation of transcription factors by sumoylation","volume":"8","author":"Rosonina","year":"2017","journal-title":"Transcription"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/nrm.2016.81","article-title":"A comprehensive compilation of SUMO proteomics","volume":"17","author":"Hendriks","year":"2016","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Jang, M., Pete, E.S., and Bruheim, P. (2022). The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles. Front. Bioeng. Biotechnol., 10.","DOI":"10.3389\/fbioe.2022.964397"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1038\/nature08657","article-title":"Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks","volume":"462","author":"Galanty","year":"2009","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"5497","DOI":"10.1093\/nar\/gks222","article-title":"CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage","volume":"40","author":"Ismail","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/j.molcel.2005.12.019","article-title":"Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes","volume":"21","author":"Bossis","year":"2006","journal-title":"Mol. Cell"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1312","DOI":"10.15252\/embj.201593404","article-title":"Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress","volume":"35","author":"Drzewicka","year":"2016","journal-title":"EMBO J."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1016\/j.cels.2022.11.005","article-title":"The sound of silence: Transgene silencing in mammalian cell engineering","volume":"13","author":"Cabrera","year":"2022","journal-title":"Cell Syst."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/5\/748\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T04:19:39Z","timestamp":1779509979000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/5\/748"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,20]]},"references-count":38,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["biom16050748"],"URL":"https:\/\/doi.org\/10.3390\/biom16050748","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,20]]}}}