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.
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.
Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue twophoton microscopy. Surprisingly little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (σ 2 ), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, σ 2 = 315 GM, and brightness, σ 2 φ = 130 GM, where φ is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, σ 2 = 216 GM and σ 2 φ = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.Fluorescent proteins (FPs) have revolutionized the way that we image biological systems 1 . Because FPs can be genetically encoded, they can be targeted to specific organelles, cells, and tissues with the precision in ways that dyes cannot, and since they do not damage the cells, they can be imaged repeatedly in living systems. To take full advantage of the opportunities these probes offer, it is often necessary to use two-photon laser scanning microscopy (TPLSM) 2,3 , whose longer wavelengths are better suited for deep imaging. In addition, TPLSM offers less photodamage, less photobleaching, and less autofluorescence background compared to one-photon confocal microscopy. Surprisingly, little is known about which fluorescent proteins are best suited for two-photon induced fluorescence.Choosing the best FP for TPLSM is a critical issue that requires, among other things, knowledge of the optimum excitation wavelength, λ opt , as well as the two-photon brightness, which is defined as the product of the peak two-photon absorption (2PA) cross section σ 2 and the fluorescence quantum yield φ: σ 2 ' = σ 2 φ. It is important to characterize the 2PA properties of the fluorescent proteins because σ 2 cannot be readily predicted from the one-photon absorption (1PA) strength alone. Furthermore, the peak wavelength of 2PA does not always coincide with twice the wavelength of the 1PA peak. In particular, the short-wavelength shift of 2PA transition maximum with respect to 1PA transition maximum is observed in centrosymmetric and slightly asymmetric molecules with pronounced vibronic structure 4 .The few available reports on the 2PA properties of FPs 2,3,5-11 explore a narrow range of excitation wavelengths with often inconsistent results 6,10,11 . Because the orange and red fluorescent proteins are particularly useful for imaging in thick tissues, we decided to *Corresponding author email: drobizhev@physics.montana.edu. Figure 1 shows the 2PA spectra (symbols) of a series of orange and red FPs together with their corresponding fluorescence emission (blue solid line) ...
BackgroundGenetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores.ResultsHere we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.ConclusionK-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-018-0480-0) contains supplementary material, which is available to authorized users.
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