ABSTRACT
Biomolecules may undergo liquid-liquid phase separation (LLPS) to spatiotemporally compartmentalize and regulate diverse biological processes. Because the number of tools to directly probe LLPS is limited (ie. FRAP, FRET, fluorescence microscopy, fluorescence anisotropy, circular dichroism, etc.), the physicochemical traits of phase-separated condensates remain largely elusive. Here, we introduce a light-switching dipyrene probe (Pyr-A) that forms monomers in either hydrophobic or viscous environments, and intramolecular excimers in aqueous solutions. By exploiting their distinct fluorescence emission spectra, we used fluorescent microscopic imaging to study phase-separated condensates formed by in vitro protein droplets and membraneless intracellular organelles (centrosomes). Ratiometric measurement of excimer and monomer fluorescence intensities showed that protein droplets became hydrophobic and viscous as their size increased. Moreover, centrosomes became hydrophobic and viscous during maturation. Our results show that Pyr-A is a valuable tool to characterize LLPS and enhance our understanding of phase separation underlying biological functions.
ABSTRACT
Nuclear import, mediated in part by karyopherin-α (KPNA)/importin-α subtypes, regulates transcription factor access to the genome and determines cell fate. However, the cancer-specific changes of KPNA subtypes and the relevancy in cancer biology remain largely unknown. Here, we report that KPNA4, encoding karyopherin-α4 (KPNA4), is exclusively amplified and overexpressed in head and neck of squamous cell carcinoma (HNSCC). Depletion of KPNA4 attenuated nuclear localization signal-dependent transport activity and suppressed malignant phenotypes and induced epidermal differentiation. Mechanistically, KPNA4-mediated nuclear transport of Ras-responsive element-binding protein (RREB1), which sustains Ras/ERK pathway signaling through repressing miR-143/145 expression. Notably, MAPK signaling enhanced trafficking activity of KPNA4 via phosphorylation of KPNA4 at Ser60. These data reveal that KPNA4 establishes a feed-forward cascade that potentiates Ras/ERK signaling in HNSCC.