Gilat, T. | Search bioRxiv

bioRxiv2020

Overcoming efflux of fluorescent probes for actin imaging in living cells

Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

biochemistry↗

bioRxiv2019

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

Bioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.\n\nSignificance statementBioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only two bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present, for the first time, autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the six enzymes from the bacterial bioluminescence system.

biochemistry↗