phaidra.vetmeduni.ac.at o:4943 DOI 10.1038/s41586-025-09141-5 Probing condensate microenvironments with a micropeptide killswitch Biomolecular condensates are thought to create subcellular microenvironments that have different physicochemical properties compared with their surrounding nucleoplasm or cytoplasm. However, probing the microenvironments of condensates and their relationship to biological function is a major challenge because tools to selectively manipulate specific condensates in living cells are limited. Here, we develop a non-natural micropeptide (that is, the killswitch) and a nanobody-based recruitment system as a universal approach to probe endogenous condensates, and demonstrate direct links between condensate microenvironments and function in cells. The killswitch is a hydrophobic, aromatic-rich sequence with the ability to self-associate, and has no homology to human proteins. When recruited to endogenous and disease-specific condensates in human cells, the killswitch immobilized condensate-forming proteins, leading to both predicted and unexpected effects. Targeting the killswitch to the nucleolar protein NPM1 altered nucleolar composition and reduced the mobility of a ribosomal protein in nucleoli. Targeting the killswitch to fusion oncoprotein condensates altered condensate compositions and inhibited the proliferation of condensate-driven leukaemia cells. In adenoviral nuclear condensates, the killswitch inhibited partitioning of capsid proteins into condensates and suppressed viral particle assembly. The results suggest that the microenvironment within cellular condensates has an essential contribution to non-stoichiometric enrichment and mobility of effector proteins. The killswitch is a widely applicable tool to alter the material properties of endogenous condensates and, as a consequence, to probe functions of condensates linked to diverse physiological and pathological processes. eng Cell Biology Imaging Mechanisms of Disease Nuclear Organization 2026-02-24T09:35:50.893Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Zhang;Yaotian; FN:Yaotian Zhang X-ORCID:https://orcid.org/0000-0003-3802-0843 END:VCARD BEGIN:VCARD VERSION:3.0 N:Stöppelkamp;Ida; FN:Ida Stöppelkamp END:VCARD BEGIN:VCARD VERSION:3.0 N:Fernandez-Pernas;Pablo; FN:Pablo Fernandez-Pernas X-ORCID:https://orcid.org/0000-0002-1305-1339 END:VCARD BEGIN:VCARD VERSION:3.0 N:Allram;Melanie; FN:Melanie Allram X-ORCID:https://orcid.org/0009-0009-9241-4107 END:VCARD BEGIN:VCARD VERSION:3.0 N:Charman;Matthew; FN:Matthew Charman END:VCARD BEGIN:VCARD VERSION:3.0 N:Magalhaes;Alexandre P.; FN:Alexandre P. Magalhaes X-ORCID:https://orcid.org/0000-0002-4097-338X END:VCARD BEGIN:VCARD VERSION:3.0 N:Piedavent-Salomon;Melanie; FN:Melanie Piedavent-Salomon END:VCARD BEGIN:VCARD VERSION:3.0 N:Sommer;Gregor; FN:Gregor Sommer X-ORCID:https://orcid.org/0009-0008-3685-010X END:VCARD BEGIN:VCARD VERSION:3.0 N:Sung;Yu-Chieh; FN:Yu-Chieh Sung X-ORCID:https://orcid.org/0009-0001-4576-152X END:VCARD BEGIN:VCARD VERSION:3.0 N:Meyer;Katrina; FN:Katrina Meyer X-ORCID:https://orcid.org/0000-0002-8244-6887 END:VCARD BEGIN:VCARD VERSION:3.0 N:Grams;Nicholas; FN:Nicholas Grams END:VCARD BEGIN:VCARD VERSION:3.0 N:Halko;Edwin; FN:Edwin Halko END:VCARD BEGIN:VCARD VERSION:3.0 N:Dongre;Shivali; FN:Shivali Dongre END:VCARD BEGIN:VCARD VERSION:3.0 N:Meierhofer;David; FN:David Meierhofer X-ORCID:https://orcid.org/0000-0002-0170-868X END:VCARD BEGIN:VCARD VERSION:3.0 N:Malszycki;Michal; FN:Michal Malszycki X-ORCID:https://orcid.org/0000-0003-1780-5596 END:VCARD BEGIN:VCARD VERSION:3.0 N:Ilik;Ibrahim A.; FN:Ibrahim A. Ilik END:VCARD BEGIN:VCARD VERSION:3.0 N:Aktas;Tugce; FN:Tugce Aktas X-ORCID:https://orcid.org/0000-0003-1599-9454 END:VCARD BEGIN:VCARD VERSION:3.0 N:Kraushar;Matthew L.; FN:Matthew L. Kraushar X-ORCID:https://orcid.org/0000-0002-7359-0318 END:VCARD BEGIN:VCARD VERSION:3.0 N:Vastenhouw;Nadine; FN:Nadine Vastenhouw X-ORCID:https://orcid.org/0000-0001-8782-9775 END:VCARD BEGIN:VCARD VERSION:3.0 N:Weitzman;Matthew D.; FN:Matthew D. Weitzman END:VCARD BEGIN:VCARD VERSION:3.0 N:Grebien;Florian; FN:Florian Grebien X-ORCID:https://orcid.org/0000-0003-4289-2281 END:VCARD BEGIN:VCARD VERSION:3.0 N:Niskanen;Henri; FN:Henri Niskanen X-ORCID:https://orcid.org/0000-0002-8361-5531 END:VCARD BEGIN:VCARD VERSION:3.0 N:Hnisz;Denes; FN:Denes Hnisz X-ORCID:https://orcid.org/0000-0002-6256-1693 END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by/4.0 application/pdf 20925536 https://phaidra.vetmeduni.ac.at/o:4943 https://phaidra.vetmeduni.ac.at/api/object/o:4943/thumbnail LOMv1.0 discipline