All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and v-ATPase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag GTPases and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Km and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

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We here present a new way to engineer complex proteins toward multidimensional specifications, through a simple yet scalable directed evolution strategy. By robotically picking mammalian cells that are identified, under a microscope, to express proteins that simultaneously exhibit several specific properties, we can screen through hundreds of thousands of proteins in a library in a matter of a few hours, evaluating each along multiple performance axes. We demonstrate the power of this approach by identifying a novel genetically encoded fluorescent voltage indicator, simultaneously optimizing brightness and membrane localization of the protein using our microscopy-guided cell picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1, and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices as well as in larval zebrafish in vivo. We also demonstrate measurement of postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.