Abstract
Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.
Highlights
Fluorescence microscopy of the living cells is often achieved through specific labeling of proteins by antibody, nanobody, or bio-specific ligand conjugated to a fluorophore
To demonstrate specific labeling of intracellular structures, we delivered three different membrane impermeant fluorescent probes (DAPI, < 1 kDa; Phalloidin-Alexa647, 1.95 kDa; Anti-PMP70ATTO488, ~150 kDa) simultaneously into live CHO-K1 cells that have been permeabilized with streptolysin O (SLO) (Figure 1b)
Cells that are permeabilized by SLO and labeled by the fluorescent probes should show nucleus stained by DAPI, actin filament stained by phalloidin-Alexa647, and peroxisomes stained by anti-PMP70-ATTO488
Summary
Fluorescence microscopy of the living cells is often achieved through specific labeling of proteins by antibody, nanobody, or bio-specific ligand conjugated to a fluorophore. There are a few exceptions to this barrier, namely, by using few selected cell-permeant fluorophores that have been attached to membrane-permeant entities (Lukinavicius et al, 2014; Grimm et al, 2015; Wombacher et al, 2010) These have obvious trade-offs of limited choice in emission wavelengths and available class of ligands that they can be covalently attached to and maintain their selectivity for the particular intracellular protein. There has been mixed success in delivering fluorescent probes by appending a membrane-permeant small peptide such as the TAT-TAR HIV peptide (Silhol et al, 2002; Richard et al, 2002) Another method to overcome the permeability issue is by transfecting the cells with plasmid DNA encoding the intracellular protein of interest appended to a fluorescent protein (FP). The detection of FPs is less than optimal because of their limited photostability
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