Indian Association for the Cultivation of Science

Fluorescent probes are indispensable for cellular imaging and for monitoring pH-dependent biological processes. However, the majority of reported probes are effective only in near-neutral or mildly acidic ranges, with very few functioning reliably in strongly acidic environments (pH < 4). Designing water-soluble, biocompatible probes that can operate efficiently under such extreme conditions remains an important analytical challenge. To address this challenge, two pH-responsive fluorescent probes, Xpy and XpyF, based on a xanthone core, were designed and synthesized. Both probes exhibit excellent water solubility, low cytotoxicity, and strong turn-on fluorescence in acidic environments. The probes display distinct emission changes from blue at neutral to moderately acidic pH (3-7) to green at strongly acidic pH (1-2). Absolute quantum yield and TCSPC studies confirmed significant fluorescence enhancement and lifetime prolongation under acidic conditions, consistent with protonation-induced PET inhibition. Cellular imaging studies revealed efficient internalization and strong nuclear localization with high Pearson's correlation coefficients. Importantly, while Xpy showed consistent nuclear staining across pH values, XpyF demonstrated a unique pH-dependent switch from cytoplasmic localization at pH 7.4 to nuclear accumulation at pH 4.3. Overall, Xpy and XpyF represent reliable nuclear-targeting sensors for strongly acidic media, illustrating how subtle structural modifications can yield distinct functional outcomes in bioimaging.

During genotoxic stress, mammalian cells adapt to resume transcription after repair of damaged DNA. However, mechanisms of these adaptations leading to optimal transcriptional restart are poorly known. In this study, we show critical role of EAF1-mediated enhanced phase separation of elongation factor ELL in its interaction with DNA repair factors for efficient repair of damaged DNA and subsequent transcriptional restart. ELL protein has intrinsic ability to phase separate and form liquid condensates both in vitro and in vivo within mammalian cells. Upon association with EAF1, intrinsic phase separation ability of ELL is enhanced resulting in changes in material property of ELL●EAF1 condensates. Physiologically, upon exposure to genotoxic stress, ATM-mediated phosphorylation-dependent increased EAF1 binding leads to enhanced phase separation and changes the material property of ELL. This, in turn, causes its increased interaction with DNA-PKc and associated Ku complex components. This increased interaction is important for their optimal recruitment on chromatin and corresponding repair of damaged DNA and transcriptional restart. An EAF1 knockdown or ELL mutant that fails to show its enhanced interaction with EAF1 during DNA damage, also fails to show efficient DNA damage repair, transcriptional restart and cell survival after exposure to genotoxic stress.