Journal: Nature Communications
Article Title: Live-cell protein labelling with nanometre precision by cell squeezing
Figure Lengend Snippet: ( a ) Combination of tris NTA labelling with SNAP f -tag labelling in living cells. HeLa Kyoto cells co-expressing His10 LaminA and H2B-SNAP f were squeezed in the presence of 100 nM tris NTA Alexa647 (magenta) and subsequently incubated with cell-permeable TMR-Star (red) for SNAP f -tag labelling. Confocal images were taken 1 h after squeezing and demonstrate simultaneous specific labelling of the His-tagged LaminA and the SNAP f -tagged H2B. ( b ) tris NTA Alexa64 7 concentration scan for tunable labelling of TAP1 mVenus-His10 in HeLa Kyoto cells. High labelling density was obtained even at 1 nM tris NTA Alexa647 during squeezing. ( c ) Light-activated labelling of His10-mEGFP Lamin A with PA- tris NTA ATTO565 in living HeLa Kyoto cells. Before photoactivation, no decoration of Lamin A was observed 24 h after squeezing (top), whereas on illumination fluorescence increase and specific labelling were monitored (bottom). ( d ) Reconstructed d STORM image of His10-mEGFP Lamin A labelled with tris NTA ATTO655 (100 nM) in a living HeLa Kyoto cell. Increased spatial resolution (≤40 nm) was obtained in live-cell super-resolution imaging of tris NTA ATTO655 by d STORM (left and magnification right) compared with the wide-field image (left corner, bottom). Images were taken by CLSM ( a – c ) or d STORM ( d ) 1 h ( a , b , d ) or 24 h ( c ) after squeezing. Dashed lines indicate the cell border. Scale bars, 5 μm ( a , b ), 10 μm ( c ) and 2 μm ( d ).
Article Snippet: The pSNAP f -H2B and pSNAP f -Cox8A plasmids (New England Biolabs) were used for SNAP f -tag labelling.
Techniques: Expressing, Incubation, Concentration Assay, Fluorescence, Imaging