No abstract

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging.

From gene expression to nanotechnology, understanding and controlling DNA requires a detailed knowledge of its higher order structure and dynamics. Here we take advantage of the environment-sensitive photoisomerization of cyanine dyes to probe local and global changes in DNA structure. We report that a covalently attached Cy3 dye undergoes strong enhancement of fluorescence intensity and lifetime when stacked in a nick, gap or overhang region in duplex DNA. This is used to probe hybridization dynamics of a DNA hairpin down to the single-molecule level. We also show that varying the position of a single abasic site up to 20 base pairs away modulates the dye–DNA interaction, indicative of through-backbone allosteric interactions. The phenomenon of stacking-induced fluorescence increase (SIFI) should find widespread use in the study of the structure, dynamics and reactivity of nucleic acids.

The cytoplasm represents a crowded environment whose properties may change according to physiological or developmental states. Although the effects of crowding and viscosity on in vitro reactions have been well studied, if and how the biophysical properties of the cytoplasm impact cellular functions in vivo remain poorly understood. Here, we probed the effects of cytoplasmic concentration on microtubule (MT) dynamics by studying the effects of osmotic shifts in the fission yeast Schizosaccharomyces pombe. Increasing cytoplasmic concentration by hyperosmotic shock led to proportionate reductions in the rates of interphase MT growth and shrinkage. Conversely, dilution of the cytoplasm in hypoosmotic shifts led to proportionately faster rates. Numerous lines of evidence indicate that these effects were due to biophysical properties of the cytoplasm. These effects were recapitulated in in vitro reconstituted MT assays by modulating viscosity, not by crowding. Our findings suggest that even at normal conditions, the viscous properties of cytoplasm modulate the dynamic reactions of MT polymerization and depolymerization.