Optogenetics

Optogenetics

Natural
1. Rhodopsins
  1. used in rat to control neuronal activity, restore visual function, control cardiac activity
  2. Channelrhodopsin
    1. ChR1 and ChR2 from Chlamydomonas reinhardtii
      1. ChR2 better expressed inmost hosts (10X)
      2. blue light: depolarization
        similar to event that causes neurons to fire
    2. VChR1 from Volvox carteri
      1. green/yellow activated variant
    3. NpHR1 from Natronomonas pharaonis
      1. yellow light 590nm: hyperpolarization
        Simultaneous expression with ChR1 to drive cardiac function in Zebra fish
        their wavelengths do not overlap
        also to turn neuronal activity on and off
        similar to neuronal hyperpolarization, stops neuronal activity
      2. chloride channel; helps maintain osmotic balance during growth
    4. 7-pass transmembrane proteins and long C-terminal region
    5. chromophore: all-trans-retinal covalently linked to the protein through a protonated schiff base to a lysine residue of helix-7
    6. photostimulation causes retinal isomerization from all-trans to 13-cis-retinal and covalent link to 7TM triggering conformational change and gating (opening of the channel pore)
    7. first by Deisseroth and colleagues in 2005
    8. single component unlike chARGe
    9. MChR1 from Mesostigma viride; green light
  3. retinal cofactor readily present in many cell types
  4. Rhodopsin is both an opsin and a G-protein coupled receptor
2. Phytochromes
  1. PAS-GAF-PHY-PAS-PAS-HKRD
    1. N-terminal photosensory light-input domain
    2. C-terminal histidine kinase related domain
  2. bacteriophytochromes
    1. Cph1
      1. binds phycocyanobilin
      2. light regulated HKRD from Synechocystis PCC6803
  3. red light; red(650nm)/far-red light (730nm)
    1. interconversion between Pr and Pfr
  4. Phytochrome A
    1. covalently binds bilin chromophore via a cysteine residue
      1. isomerizationof bilin around C15-C16 double bond between the C and D rings resulting in the flipping of the D ring
  5. Pfr of PhyB reacts with PIF3
    1. Y2H system used to create light controllable post-translational levl switch in Saccharomyces cerevisiae vascular artificially split intein: protein splicing
3. LOV
  1. plants, fungi, bacteria
  2. blue light
  3. FMN chromophore bound non covalently in the dark
  4. Photochemistry: information of covalent bond between C4a of isoalloxazine ring of FMN and conserved cysteine residue in the apoprotein
    1. leads to conformational change with the undocking of the j alpha helix from the beta scaffold
      1. and the autophosphorylation of the ser/thr kinae domain at the C-terminus
  5. Application
    1. Pa-Rac1
      1. Rac1 is a small GTPase protein that regulates actin cytoskeletal dynamics and cell migration in mammalian cells
      2. Lov2-Jalpha fusion with Rac1 sterically inhibits Rac1 active site
        blue light alleviates caging
        allowing it to bind to the P21-activated kinase PAK1 leading to polymerization of the actin filaments and genertion of cell localized protusions and movement
      3. to control motility
        of fibroblasts
        zebrafish embryos
        drosophila ovary cells
    2. LOVTAP
      1. replaced the fixl protein from Bradyrhizobium and changed the PAS domain from voltage regulated to light activable
      2. tryptophan activated protein
    3. Photoactivable caspase 1 to stimulate rapid apoptosis
  6. strengths
    1. small and soluble
    2. ubiquitous cofactor in most cells
    3. tunable/ decay rate variable/ mutagenesis possible
  7. take advantage of dimerization to control gene expression
    1. e.g. Dronpa
    2. yeast hybrid system
      1. FKF1/GI
4. BLUF: Blue light utilizing FAD
  1. Prevalent in prokaryotes/ chromophore: FAD 450nm
  2. photochemistry: rearrangement within the hydrogen bon network between N5 and O4 of the active site of FAD and nearby conseerved tyrosine and glutamine residues
    1. glutamine flips and signalling is propagated through structural changes in the βscaffold interface and protein protein interactions
  3. Poly adenylyl cyclases: PACs
    1. Euglena PAC
      1. photorecceptor for phototaxis
      2. Composed of two PACα and PACβ subunits. Each contains two BLUF domains linked to two AC domains
        PACα has proved a powerful tool in non-invasively controlling cAMP levels in the neurons of Drosophila, Xenopus oocytes, and the nematode Caenorhabditis elegans
        But: large subunit size (> 1000 amino acids), low solubility and high background activity
    2. Beggiatoa PAC
      1. Soluble protein that is much smaller than Euglena PAC (350 amino acids)
      2. Lower dark activity of Beggiatoa PAC compared to Euglena PAC makes it more suitable for controlling intracellular cAMP levels (i.e. less leaky)
      3. Utility in generating cAMP from ATP in cultured neurons
      4. Specificity of Beggiatoa PAC has already been reengineered to modulate cGMP-dependent signalling cascades
Engineered
1. Channelrhodopsin: ChR2
  1. ChETA
    1. changed active site residue E123 to Gln or thr
    2. faster gate closing and 20nm shift in wavelength
    3. rational approach based on rhodopsin knowledge and high conservation of the retinal binding pocket
  2. substitution of C128 by Ser or D156 by Ala or combination led to an extreme extension of open state up to 30 mins and on and off switching with dual wavelength light protocols
2. Rhodopsin
  1. optoXRs
    1. intracellular loops of Gt bovine rhodopsins replaced with those of specific adrenergic receptors; chimera
    2. Gq from human alpha1 adrenergic receptor: IP2, DAG upregulated
    3. Gs from hamster beta2 adrenergic receptor: cGMP upregulated
    4. green light; 500nm
3. Cph1- based
  1. Cph8
    1. chimera of Cph1 photoreceptor domain fused with EnvZ and the two component EnvZ-OmpR HK system; Red light regulated gene expression system
      1. EnvZ-OmpR HK system regulates porin expression in response to osmotic shock
    2. enables autophosphorylation of EnvZ and transfer of phosphate group to OmpR
      1. Phosphorylated OmpR initiates transcription of promoter OmpC expressing LacZ
    3. PCB not synthesized in E. coli, has to be expressed
  2. CcaS
    1. Dual chromatic switch: Green (on-535nm); Red(off-670nm)
    2. phosphorylates its response regulator CcaR in response to green light
    3. also gene expression system
Applications
1. biomedical
  1. Parkinson's
    1. to understand specific regions and pathway responsible for disease
    2. Go pathway restored movemet to preinfection state
    3. stop pathway caused disease in healthy mice
  2. Reinbothe et al., 2014
    1. to control insulin secretion in intact pancreatic islets with Beta-cell specific expression of ChR2
advantages and incentives
1. no added chemicals
2. fast and precise
3. genetically encoded
4. tunable: speed, wavelength, power
5. spectrally diverse tools
6. natural/biological so physiological tolerability
history
1. Crick suggestion in 1979
2. Gero Meisenboeck in 2005
  1. Arabidopsis phototransduction cascade
  2. neural function control of flight in Drosophila in dopaminergic regions
Specificity
1. injection site
2. recombinase or promoter dependent
3. projection targeting
Gain or loss of function
1. loss: role of cholinergic neurons in cocaine conditioning
2. gain: neural codes of awakening
Concerns
1. unprecedented indirect effects
Readouts
1. fMRI: ofMRI
2. phenotypic: e.g parkinsonian approach
3. optrodes
  1. Kevin Ung, 2012
    1. simple protocol for implantable fiber optic
Advances/Advantage/looking forward
1. ion-selective channels: Dunalliela salina: DChR1
2. subcellular targeting
3. molecular engineering to improve use of intrinsic factors such as flavin and biliverdin
4. reverse engineering to understand complex disease states
5. cell type specific readouts
6. genetically encoded readouts e.g.g Ca2+ indicators and voltage sensors to do away with orthogonality and mixed modality of electrical system
7. brain and cardiac research, metabolic diseases, implants of photosensitive cells
Engineering strategies
1. rational and directed
  1. homology modeling e.g. ChETA
2. Random mutagenesis
  1. DNA shuffling/ molecular evolution
3. genome mining
Fluorescence
1. LOV
  1. smaller than GFP 10KDa versus 25KDa
  2. molecular evolution to generate increased fluorescence
  3. ilov
    1. mutagnesis and domain shuffling
    2. improved fluorescence and photostability
    3. used to track TMV spread in tobacco
    4. multiple amino acid changes
    5. foot and mouth disease virus
    6. smaller, so lower genetic payload on the virus, better viral biomarker than GFP
    7. Chapman et al., 2008
    8. time-lapse photography and live cell imaging
  4. tlov
    1. withstand high temp
  5. Drepper et al, LOV as biomarker in oxygen limiting environment: anaerobic
    1. GFP and its derivatives requires oxygen for their amino acid chromophore to fluoresce, also unstable at pH lower than 5
  6. miniSOG: LOV2 engineered to generate singlet oxygen aiding electron microscopy, helped to understand specific synaptic location in mice
2. IFP
  1. engineered from Deinococcus radiodurans bacteriophytochrome
  2. chromophore: biliverdin
  3. when excited by red/far red 684nm, it fluoresces in the infra-red 708nm
  4. suitable for whole body imaging... as IF wavelength penetrates tissue better than visible light

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	Created by Jumai Abioye almost 10 years ago