Pitfalls of Using ANS Dye Under Molecular Crowding Conditions.

The 1-anilino-8-naphthalenesulfonate (ANS) fluorescent dye is widely used in protein folding studies due to the significant increase in its fluorescence quantum yield upon binding to protein hydrophobic regions that become accessible during protein unfolding. However, when modeling cellular macromolecular crowding conditions in protein folding experiments in vitro using crowding agents with guanidine hydrochloride (GdnHCl) as the denaturant, the observed changes in ANS spectral characteristics require careful consideration. This study demonstrates that crowding agents can form clusters that interact differently with ANS.

Time-resolved fluorescence of ANS dye as a sensor of proteins LLPS.

The explosive growth in the number of works addressing the phase separation of intrinsically disordered proteins has driven both the development of new approaches and the optimization of existing methods for biomolecular condensate visualization. In this work, we studied the potential use of the fluorescent dye ANS as a sensor for liquid-liquid phase separation (LLPS), focusing on visualizing condensates formed by the stress-granules scaffold protein G3BP1. Using fluorescence lifetime imaging microscopy (FLIM), we demonstrated that ANS can accumulate in RNA-induced G3BP1 condensates in aqueous solutions, but not in G3BP1 condensates formed under macromolecular crowding conditions in highly concentrated PEG solutions.

Ageing drop by drop: Disturbance of the membrane-less organelle biogenesis as an aging hallmark.

Despite extensive research, the features associated with the aging phenotype are not all-inclusive and need to be updated on a regular basis to incorporate new findings. We propose to include the dysfunction of membrane-less organelle (MLO) as a new aging hallmark. Special scaffold proteins with a high degree of intrinsic disorder drive the formation of MLOs via the liquid-liquid phase separation (LLPS) process.

Safari with an Electron Gun: Visualization of Protein and Membrane Interactions in Mitochondria in Natural Environment.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari.