Yaotian Zhang Yaotian Zhang Ida Stöppelkamp Ida Stöppelkamp Pablo Fernandez-Pernas Pablo Fernandez-Pernas Melanie Allram Melanie Allram Matthew Charman Matthew Charman Alexandre P. Magalhaes Alexandre P. Magalhaes Melanie Piedavent-Salomon Melanie Piedavent-Salomon Gregor Sommer Gregor Sommer Yu-Chieh Sung Yu-Chieh Sung Katrina Meyer Katrina Meyer Nicholas Grams Nicholas Grams Edwin Halko Edwin Halko Shivali Dongre Shivali Dongre David Meierhofer David Meierhofer Michal Malszycki Michal Malszycki Ibrahim A. Ilik Ibrahim A. Ilik Tugce Aktas Tugce Aktas Matthew L. Kraushar Matthew L. Kraushar Nadine Vastenhouw Nadine Vastenhouw Matthew D. Weitzman Matthew D. Weitzman Florian Grebien Florian Grebien Henri Niskanen Henri Niskanen Denes Hnisz Denes Hnisz Springer 2025 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. PDFDocument eng 2026-02-24T09:35:50.600031Z 2025 Cell Biology Imaging Mechanisms of Disease Nuclear Organization 20925536 b application/pdf http://creativecommons.org/licenses/by/4.0/