We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths...

Cellular activity is defined by the precise spatiotemporal regulation of various components, such as ions, small molecules or proteins. Studying cell physiology consequently requires the optical recording of these processes, notably by using fluorescent biosensors. The recent developments of various fluorogenic systems greatly expanded the palette of reporters to be included in these sensors design. Fluorogenic reporters consist in a protein or RNA tag that can complex either an endogenous or a synthetic fluorogenic dye (socalled fluorogen). The intrinsic nature of these tags, along with the high tunability of their cognate chromophore provide interesting features such as far-red to near-infrared emission, oxygen independence or unprecedented color versatility. These engineered photoreceptors, self-labelling proteins, or non-covalent aptamers and protein-tags were rapidly identified as promising reporters to observe biological events. This review focuses on the new perspectives they offer to design unique and innovative biosensors, thus pushing the boundaries of cellular imaging.

A variety of protein tags are available for genetically encoded protein labeling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and, finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example, within cell−cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.

We report on new fluorogenic HaloTag probes based on a molecular rotor design. Thanks to their viscosity-sensitive emission, the probes light-up upon reaction with the protein self-labeling tag HaloTag. The palette of probes cover an emission range from green to red and exhibit remarkably low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast in live Hela cells.