As already anticipated by many, the next big step of optogenetics will consist in multiplexing optogenetic tools in the same experiment (see the page Combining Optogenetic Tools). Tools used in parallel will need to have well-separated spectral characteristics, a requirement that FRET sensors will have a hard time meeting. The problems of FRET sensors might run deeper than their bandwidth of operation, with issues including differential donor/acceptor photobleaching, worse 2P performance & more scattering of the excitation wavelength (usually blue-shifted), the difficulty to fuse them with other proteins and their signal-to-noise ratio being lower than for 1-FP sensors. The race for diversifying 1-FP sensors has already begun, with new hue-variants of the GCaMP scaffold (the GECOs series and RCaMP), of kinase activity sensors (Cyan Sinphos) and of voltage-sensitive fluorescent proteins (VSFP3s).
The lab of Richard Tsien has just added a new member to this emerging family of hue-shifted single-FP sensors: a novel red pH-sensitive red fluorescent protein called pHTomato. In a nicely-done 2-author paper published in Nature Neuroscience, Li and Tsien demonstrate the usefulness and efficiency of pHTomato by fusing it to the vesicular membrane protein synaptophysin. The resulting protein, SypHTomato, can report vesicle fusion and recycling as well as its green counterpart. In addition, the authors show that SypHTomato can be used in conjunction with GCaMP3 (the genetically-encoded calcium indicator) or ChR2 (channelrhodopsin-2).
To generate pHTomato, Li and Tsien built upon two monomeric red fluorescent proteins: mRFP and mStrawberry. Mutants were generated by error-prone PCR (epPCR) with equal amounts of mRFP and mStrawberry as templates. After three rounds of “evolution”, the authors identified a bright pH-sensitive mutant that they named pHTomato, which contains six amino acid differences from mStrawberry (F41T, F83L, S182K, I194K, V195T and G196D). pHTomato shows all the characteristic required for a good pH sensor (no photoswitching behavior, no change in wavelength of excitation and emission peaks with pH and a clear pH dependence of its fluorescence intensity).
To generate sensors of vesicle fusion and recycling, the authors fused pHTomato to VAMP2 (VAMP2-pHTomato) and synaptophysin (sypHTomato). Although they show no direct comparison of the two probes, sypHTomato is likely to perform better than VAMP2-pHTomato given that synaptophysin displays less background expression on the plasma membrane than VAMP2. SypHTomato was also not directly compared to the existing and already popular VAMP2-pHluorin (aka synaptopHluorin) engineered in the late 90s by Miesenböck (original paper here). Instead, sypHTomato was shown to perform as well as sypH (pHluorin + synaptophysin) in reporting presynaptic activity of cultured hippocampal neurons.
Most importantly, Li and Tsien provide the proof-of-principle of a dual-color optical imaging approach where presynaptic calcium signalling and presynaptic release are compared directly at the same synapses or in distinct pre- and postsynaptic elements. Both processes were imaged concurrently by coexpressing sypHTomato and GCaMP3 in the same or in distinct hippocampal neurons in culture.
In a final set of experiments, the authors propose an all-optical control and readout system by coexpressing channelrhodopsin variants and sensors of presynaptic activity. This strategy was implemented in two ways. In the first one, ChR2 was coexpressed with sypHTomato in the same neurons. SypHTomato was interrogated using green light (546-566 nm) while ChR2 was (almost) independently activated using blue light (457-482 nm). ChR2-driven vesicular turnover was clearly visible in the SypHTomato channel. The small caveat in this experiment is the direct excitation (20-25% of peak excitation) of SypHTomato by these blue wavelengths. In the second one, the same wavelength is used to excite channelrhodopsin variants and presynaptic activity sensors. An increase in presynaptic activity is simply induced by raising the frequency of episodes of illumination (which rather inconveniently also increases the sampling frequency of the probe signal). The originality of this second method is that two pairs of actuator/indicator were used in a multiplex fashion: ChR2/vGluTpH on the one hand (vGluTpH is a pHluorin-tagged vesicular glutamate transporter that specifically labels glutamate-containing vesicles) and VChR1/sypHTomato on the other. Despite again some overlap of the different action spectra, the activity of two separate sets of presynaptic terminals could be stimulated and monitored independently.
Overall, sypHTomato looks very promising. Let’s wait to see how it performs in vivo compared to synaptopHluorin. Stay tuned.