Abstract
Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range.
Highlights
Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei
To label RNA polymerase II (RNAPII) with electron dense probes suitable for electron microscopy (EM) localization studies we employed a monoclonal antibody directed against the C-terminal domain (CTD) of its largest subunit
It was here coupled to 6 nm colloidal gold particles and introduced into living HeLa cells using a comwww.nature.com/scientificreports mercially available lipid-based antibody delivery system
Summary
Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. We have developed a method to internalize immunogold labels into living cells and allow their nuclear import. For more general use we employed lipid-based protein delivery agents compatible with cell viability to internalize the probes[7]. Antibodies do not diffuse freely into the nucleus and fluoresceinlabelled anti tubulin antibodies delivered by lipid vehicles into HeLa cells remain cytoplasmic as expected for molecules devoid of Nuclear Localization Signals[12] (Fig. S1). Antibodies directed against nuclear targets such as RNA polymerase II (RNAPII) can be imported into the nucleus presumably because they recognize their target in the cytoplasm and are transported into the nucleus by a piggyback mechanism
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