Killswitch: A universal tool to link condensate microenvironments and functions

Biomolecular condensates precisely regulate a variety of cellular processes, ranging from gene transcription and RNA splicing to the coordination of stress responses, which have attracted widespread attention over the past two decades.1 These membraneless compartments with unique microenvironments exhibit diverse material properties and biological functions. However, tools for selectively manipulating endogenous condensates are less explored. Traditional methods, such as genetic perturbation and small-molecule inhibitors, either dissolve all condensates in a non-specific manner or readily trigger adaptive cellular changes.2,3 Directly probing condensates in living cells and exploring their relationships to cell functions remain major challenges. Recently, Zhang et al. reported a groundbreaking tool: an unnatural micropeptide termed “killswitch” with a nanobody-based recruitment system.4 It is the first universal tool capable of selectively altering the properties of endogenous condensates in living cells, which could be used to reveal the direct association between condensate microenvironment and cellular functions. Killswitch is demonstrated to be applicable to diverse studies of physiologically and pathologically relevant condensates.

Killswitch: A phenylalanine-driven self-associating micropeptide

Essentially, killswitch is a non-natural micropeptide (17 amino acids) enriched in hydrophobic aromatic residues, with three phenylalanines as the functional core. Notably, it shares no homology with human proteins. Killswitch is originally identified as a pathogenic frameshift variant of the nuclear protein HMGB1, which causes mislocalization of mutant HMGB1 to the nucleolus (the largest biomolecular condensate in human cells). This event leads to nucleolar arrest and eventual cell death.5 Building on the observation, Zhang et al. hypothesized that the micropeptide could be engineered into a universal tool for selectively altering the properties of specific condensates, enabling the interrogation of the condensate microenvironment and functions in living cells.4

Mutational analysis reveals that substituting the three phenylalanines with glycines, alanines, or negatively charged residues (F to G/A/E&D) rescues nucleolar dynamics to the same extent as deleting the entire killswitch sequence. In contrast, mutations of individual phenylalanines or other residues (e.g., C16 to A and M to E&D) cannot abrogate the activity of the killswitch, indicating that the three phenylalanines are essential for nucleolar arrest and that activity requires the context of the remaining 14 amino acids. In vitro droplet formation assays show that purified GFP-tagged killswitch forms droplets in a concentration-dependent manner in the presence of physiological salt and crowding agents, whereas the F-to-G mutant fails to form the droplets (Figure 1A). Size-exclusion chromatography (SEC) analysis further reveals that about 18% of killswitch elutes as multimers, compared to less than 1% of the F-to-G mutant. AlphaFold 3 modeling also suggests that killswitch undergoes self-association in a phenylalanine-dependent manner.