光激活磁共振脑功能成像的研究现状及前景

磁共振脑功能成像经过20年的发展已经成为大脑认知神经科学研究中最主要的技术.光激活磁共振脑功能成像是将它与新发展起来的光遗传学相结合的新兴技术,具有高时空选择性地分析特定神经回路或特定类型的神经元在整个功能网络中的作用的巨大潜力,为理解人类脑功能和揭示神经精神疾病机理可提供新的技术手段.     

Bidirectional Regulation of mRNA Translation in Mammalian Cells by Using PUF Domains

The regulation of gene expression is crucial in diverse areas of biological science, engineering, and medicine. A genetically encoded system based on the RNA binding domain of the Pumilio and FBF (PUF) proteins was developed for the bidirectional regulation (i.e., either upregulation or downregulation) of the translation of a target mRNA. PUF domains serve as designable scaffolds for the recognition of specific RNA elements and the specificity can be easily altered to target any 8‐nucleotide RNA sequence. The expression of a reporter could be varied by over 17‐fold when using PUF‐based activators and repressors. The specificity of the method was established by using wild‐type and mutant PUF domains. Furthermore, this method could be used to activate the translation of target mRNA downstream of PUF binding sites in a light‐dependent manner. Such specific bidirectional control of mRNA translation could be particularly useful in the fields of synthetic biology, developmental biology, and metabolic engineering.     

Optogenetic Engineering: Light‐Directed Cell Motility

Genetically encoded, light‐activatable proteins provide the means to probe biochemical pathways at specific subcellular locations with exquisite temporal control. However, engineering these systems in order to provide a dramatic jump in localized activity, while retaining a low dark‐state background remains a significant challenge. When placed within the framework of a genetically encodable, light‐activatable heterodimerizer system, the actin‐remodelling protein cofilin induces dramatic changes in the F‐actin network and consequent cell motility upon illumination. We demonstrate that the use of a partially impaired mutant of cofilin is critical for maintaining low background activity in the dark. We also show that light‐directed recruitment of the reduced activity cofilin mutants to the cytoskeleton is sufficient to induce F‐actin remodeling, formation of filopodia, and directed cell motility.     

Light-Induced Dimerization Approaches to Control Cellular Processes

Light-inducible approaches provide a means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. New expansions of the toolbox facilitate control of signal transduction, genome editing, “painting” patterns of active molecules onto cellular membranes, and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches have also seen interesting progress. Herein, an overview of optogenetic systems and emerging chemo-optogenetic systems is provided, and recent applications in tackling complex biological problems are discussed.     

Early Formation of the Ion‐Conducting Pore in Channelrhodopsin‐2

Channelrhodopsins (ChRs) are light‐gated ion channels that are widely used in optogenetics. They allow precise control of neuronal activity with light, but a detailed understanding of how the channel is gated and the ions are conducted is still lacking. The recent determination of the X‐ray structural model in the closed state marks an important milestone. Herein the open state structure is presented and the early formation of the ion conducting pore is elucidated in atomic detail using time‐resolved FTIR spectroscopy. Photo‐isomerization of the retinal‐chromophore causes a downward movement of the highly conserved E90, which opens the pore. Molecular dynamic (MD) simulations show that water molecules invade through this opened pore, Helix 2 tilts and the channel fully opens within ms. Since E90 is a highly conserved residue, the proposed E90‐Helix2‐tilt (EHT) model might describe a general activation mechanism and provides a new avenue for further mechanistic studies and engineering.     

A Phytochrome Sensory Domain Permits Receptor Activation by Red Light

Optogenetics and photopharmacology enable the spatio‐temporal control of cell and animal behavior by light. Although red light offers deep‐tissue penetration and minimal phototoxicity, very few red‐light‐sensitive optogenetic methods are currently available. We have now developed a red‐light‐induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases—the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non‐invasively in mammalian tissue and in multicolor cell‐signaling experiments. The light‐controlled dimerizer and red‐light‐activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.     

Optochemical genetics

Transmembrane receptors allow a cell to communicate with its environment in response to a variety of input signals. These can be changes in the concentration of ligands (e.g. hormones or neurotransmitters), temperature, pressure (e.g. acoustic waves or touch), transmembrane potential, or light intensity. Many important receptors have now been characterized in atomic detail and our understanding of their functional properties has markedly increased in recent years. As a consequence, these sophisticated molecular machines can be reprogrammed to respond to unnatural input signals. In this Review, we show how voltage-gated and ligand-gated ion channels can be endowed with synthetic photoswitches, and how the resulting artificial photoreceptors can be used to optically control neurons with exceptional temporal and spatial precision. They work well in animals and might find applications in the restoration of vision and the optical control of other sensations. The combination of synthetic photoswitches and receptor proteins contributes to the field of optogenetics and adds a new functional dimension to chemical genetics. As such, we propose to call it "optochemical genetics".     

Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian Cells

The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA‐synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity‐enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.