skip to primary navigationskip to content
 

Single molecule translation imaging

last modified Nov 15, 2017 12:45 PM
Single molecule translation imaging, SMTI, is a novel technique development by the Laser Analytics group to measure the rate and spatial distribution of protein synthesis [1,2]. Together with scientists form the Department of Physiology and Development of the University of Cambridge, we study the processes underlying neurodevelopment and the formation of neuronal networks in vivo.

 Image caption: The SMTI procedure in xenopus embyos. (a) A fusion construct between a protein of interest and the YFP Venus is (b) introduced into retinal ganglion cell. (c) The cells are dissected and cultured in a dish to study the effect of various chemicals, so-called guidance cues, on local translation in axon tips. (d) This is quantified via the SMTI procedure: segmentation, bleaching, and localisation. (e) Changes can be measured upon cue application even during SMTI acquisitions. Click on the picture for an enlarged version. 

The technique SMTI requires a single molecule sensitive microscope in combination with a fusion protein construct between a target protein of interest and the read-out fluorescent protein Venus. Venus is a yellow fluorescent protein (YFP) that is known for its extremely fast folding time, brightness, yet low photo-stability. The combination of these parameters makes it an excellent probe for SMTI. The fusion protein is introduced into a model system, xenopus leavis in our case, and renders the sample fluorescent. Using a short but intense light pulse the existing fluorescence is bleached, effectively providing a zero background system. At lower illumination powers a readout of newly translated protein can be generated resembling photoactivated localisation microscopy data sets.

The simplicity of the technique allows rapid progress on scientific questions and quantification of protein translation with unprecedented precision. The use of only a single fluorophore per event is a great advantage to alternative techniques, most prominently the SunTag approach that relies on multi-epitope tagging. There, a single newly translated protein must recruit up to 50 fluorescent proteins to provide sufficient signal-to-background ratios. This overloading also has the potential to invalidate the biological significance of produced results. Proper controls are hence paramount, but can be provided by SMTI as an orthogonal read-out.

We are constantly developing the SMTI technique itself further and are also starting to employ it in a much wider range of applications – from developmental biology to molecular changes during neurodegeneration.

 

References

[1] Ströhl F, Lin JQ, Laine RF, Wong HH, Urbančič V, Cagnetta R, Holt CE, Kaminski CF, "Single Molecule Translation Imaging Visualizes the Dynamics of Local β-Actin Synthesis in Retinal Axons"Sci. Rep. (2017), 7, 709.

[2] Wong HH-W, Lin J Q, Ströhl F, Roque CG, Cioni J-M, Cagnetta R, Turner-Bridger B, Laine R F, Harris WH, Kaminski CF, Holt CE (2017). "RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo" Neuron 95 (4) 852-868.