A light-switching pyrene probe to detect phase-separated biomolecules

Abstract

Summary 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.

Enhancing scanning electrochemical microscopy's potential to probe dynamic co-culture systems via hyperspectral assisted-imaging

“…Thus, our system allows for richer insight into dynamic systems of interest compared to conventional microscope platforms. For example, the system outlined here could be used to detect spectral shifts that occur when chromophores encounter a phase within a cell that is very different from continuous water (e.g., liquid droplets 34,35 ). Use of our hyperspectral imaging system in coordination with bio- logical scanning electrochemical microscopy allows for differentiation of cells based on cell location, cell type, and cellular reactivity given use of an appropriate fluorescence label (or substance) and a redox mediator.…”

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells

“…HeLa cells were obtained from ATCC [ 18 ], and maintained in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin/streptomycin (P/S). All cells were cultured at 37C and 5% CO2 in a humidified incubator.…”

Discovery of a Novel Aminocyclopropenone Compound That Inhibits BRD4-Driven Nucleoporin NUP210 Expression and Attenuates Colorectal Cancer Growth

“…Recombinant mEGFP protein was obtained from ORIGENE (Sku#TP790050). Recombinant mEGFP fusion protein (BRD4aa674-1351; BRD4-IDR-mEGFP) was generated as described previously [35] and concentrated to an appropriate level using Amicon Ultra centrifugal filters (50K MWCO, Millipore, Burlington, MA, USA). Recombinant protein was diluted to a final concentration of 5 µM in droplet buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, and 10% PEG) containing 10 µM of each aminocyclopropenone, and then immediately loaded on a glass-bottomed dish (MATSUNAMI) and covered with a coverslip.…”

“…Recombinant protein was diluted to a final concentration of 5 µM in droplet buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, and 10% PEG) containing 10 µM of each aminocyclopropenone, and then immediately loaded on a glass-bottomed dish (MATSUNAMI) and covered with a coverslip. Samples were imaged using a Zeiss LSM5 EXCITER microscope with a mercury lamp and a Plan-Apochromat 100×/1.4 oil objective, as previously described [35]. Axio Vision software (version 4.8) was used for image acquisition.…”

“…We next elucidated the functions of ACP-1n against cancer growth [13,35,39,40] and its functional significance in the maintenance of CRC in vitro by targeting BRD4 and BRD4driven gene expression in colorectal cancer HCT116 cells. Consistent with the previous results from an in vitro droplet formation assay (Figure 1C), immunofluorescent confocal imaging of BRD4 proteins demonstrated that the numbers of BRD4 puncta (representing BRD4 assembly) significantly decreased following ACP-1n treatment (Figure 2A,B).…”