Optogenetics II, sponsored by JST: Report for the session 13

In the session of “Optogenetics II, sponsored by JST”, 4 speakers gave a talk about their recent studies.

Dr. Peter Hegemann (Humboldt-Universitaet zu Berlin, Germany) talked on “The Wondrous Flexibility of Microbial Rhodopsins” Since the discovery of Bacteriorhodopsin in 1971 the friendly competition of engineers and genome miners unearthed an enormous variety of microbial rhodopsins, which might act as outward or inward directed mostly procaryotic pumps, highly specific for H⁺, Na⁺ or Cl^–. Second, channelrhodopsins with intrinsic light-activated conductance with overlapping selectivity for H⁺, Na⁺. K⁺ or anions have been engineered by nature or biochemists in such a way that they for optogenetic applications mainly conduct a single sort of ions with the recent appearance of Ca²⁺ and K⁺-selected ChRs (CapChR, KCRs and WitChR). In parallel, nature developed enzyme-rhodopsins which may be bimodular as the fungal Rh-cyclases (RGCs), flagellate Rh-phosphodiesterases (R-PDEs), or multimodular in case of algal histidine-kinase rhodopsins (HKRs). Other rhodopsins only transiently interact with signaling molecules either within the membrane (archeal sensory rhodopsins) or via hydrophilic interactions as realized in cyanobacterial sensory rhodopsins (Anabeana rhodopsin). The rhodopsin complexity recently culminated with the “maxi-rhodopsins”, named Bestrhodopsins, which form pentameric ion channel complexes with 5 or 10 covalently linked single or tandem rhodopsin modules. The rhodopsin absorption can be tuned - from what we know so far - from 380 nm in case of HKRs from Chlamydomonas or Ochromonas all the way to 800 nm in case of A2-retinal linked NeoRs. The speaker discussed some principles that are responsible or at least intimately linked to the special characteristics.

Dr. Haruhiko Bito (The University of Tokyo, Japan) talked on “Multiplexed recording and manipulation of neural activity and synaptic signaling”. The aim of neuroscience is to understand how information is processed and encoded at different resolutions in the brain, such as at the circuit, neuronal ensemble, single neuron, or subcellular level. Calcium imaging allows for the observation of neuronal activity at the cell soma level and within subcellular compartments. The speaker introduced a new suite of multicolor genetically encoded calcium indicators, XCaMPs, which offer improved accuracy and resolution. Using XCaMPs, the speaker demonstrated fast single AP detection, non-invasive subcortical imaging, fiber photometric recording of multiple neuronal ensembles, and paired recording of pre- and postsynaptic firing. The XCaMPs, combined with photochemical and optogenetic tools, provide a powerful toolkit for investigating the molecular and circuit mechanisms behind information processing and cognitive behavior in the brain.

Dr. Elena G. Govorunova (The University of Texas Health Science Center at Houston McGovern Medical School, USA) talked on “Wavelength regulation and ion selectivity in channelrhodopsins” Channelrhodopsins (ChRs) are light-gated ion channels that guide phototaxis in protists. Recent advances in polynucleotide sequencing have led to identification of hundreds of ChR homologs in many phylogenetic lineages, and there are indications that ion channel function has evolved within the superfamily of microbial rhodopsins by several convergent routes. The diversity of ChRs provides an exceptional platform for the study of structure-function relationships in membrane proteins. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective channelrhodopsins (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by de- and hyper-polarization of the membrane, respectively. Red-absorbing ChRs are in demand for optogenetics, because red light more deeply penetrates biological tissue. Red-shifted CCRs have been known for some time, and red-shifted ACRs have been discovered only recently. These proteins, named RubyACRs, have a unique retinal-binding pocket responsible for their red light absorption. Potassium-selective ChRs (“kalium channelrhodopsins” or KCRs), which they recently discovered in non-photosynthetic stramenopile and alveolate microbes, mediate photoinduced potassium efflux and thus present a new opportunity for optogenetic inhibition. The speaker presented their current advances on RubyACRs and structural foundations of potassium selectivity, including identification of structural motifs that determine the K⁺ selectivity of KCRs.

Dr. Moritoshi Sato (The University of Tokyo, Japan) talked on “Optical control of the genome” The genome consists of more than 20,000 genes and is essential for most of biological phenomena. To understand these biological phenomena, including diseases, and to utilize or modify them, approaches that enable optical control of the genome are required. Recently, they developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). They divided the Cas9 nuclease from the CRISPR–Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequence of the genome by light stimulation. Additionally, they developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR–Cas9 system. They demonstrated that this optogenetic tool allowed rapid and reversible targeted gene activation by light. Using this tool, they exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR–Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR–Cas9-based optogenetic tools, we also have developed a photoactivatable Cre–loxP system. This new tool, named PA-Cre, allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. Genome engineering technology and optogenetics technology have emerged as different technologies from each other so far. Their studies described above merge these emerging research fields together.

Dr. Takeharu Nagai (Osaka University, Japan) talked on “Development of the shortest wavelength fluorescent protein for multifunctional imaging” Fluorescent proteins, such as Aequorea victoria green fluorescent protein (avGFP), are indispensable tools for life science research as markers for reporter assays as well as materials for making functional probes to measure the functional molecules in living cells. To understand the causal relationships among diverse physiological phenomena occurring in living cells, it is necessary to simultaneously observe the multiple events. For this purpose, diversification of wavelength properties of fluorescent proteins and recombinant functional indicators is underway. However, the shortest emission wavelength of fluorescent proteins has not been updated for more than 10 years since the ultramarine color fluorescent protein, Sirius with an emission peak at 424 nm was developed in 2009. They reported the development of Sumire, a fluorescent protein emitting 414 nm violet fluorescence from a hydrated chromophore., and a FRET-type indicator for multifunctional analysis using Sumire, thus, successfully extending the observation channel on the short wavelength side.

In general, these presentations raised a lot of discussions.