Optogenetics Flashcards
Examples of blue light sensor domain, chromophore, organism obtained
Bacteria:
Blue light utilising FAD (BLUF, ~15 kDa) with flavin adenine dinucleotide (FAD) chromophore in prokaryotes.
Photoactivated adenylyl cyclase (PAC) is comprised of a BLUF domain which regulates the activity of the Adenylyl cyclase.
First discovered as an photoactivated biosensor in Euglena algae in 2002 (2 BLUF and 2 AC domains)
PAC from Beggiatoa bacteria is used since smaller (1 BLUF and 1 AC domain) and less dark activity (less leaky/larger dynamic range)
Plant:
LOV (light, oxygen or voltage) sensing domains with flavin mononucleotide (FMN) discovered in plants.
Phototropin (discovered in Arabidopsis thaliana), a light-activated serine/threonine kinase. Has 2 LOV domains and a kinase effector/output domain
Downsides of generating chimeric photosensors
Requires trial and error engineering to generate a product with desirable properties
Examples of plant proteins that can be utilised for their property of photoreversible dimer/oligomerisation
UVR8 (UV)
Phototropin and Crytochrome (blue)
Phytochrome (red)
2x UVR8-POI (induce and reverse dimerisation without/with light)
UVR8 and COP1 (with light the UVR8-POI dimers disassociate and the UVR8-POI can heterodimerise with COP1)
CRY2-POI and CIB-POI dimerises with blue light
CRY2-POI can form homodimer/homo-oligomerise with blue light
PHYB-POI and PIF3-POI are photoswitchable and can form heterodimers with red light and reverse with far-red light
Which general application/s can specific blue light domains be utilised and why
PAC can be used to control neural function. Adenylyl cyclase produces active cAMP which is involved in many cellular events including synaptic transmission and is particularly important for learning and memory.
LOV to control cell signalling by fusing with an enzyme to artificially place under light control. Can also be used to have light regulated degradation.
Example of LOV fusion protein. Describe how it structurally works and it’s function (4)
PA-Rac1: Fused LOV2-Jalpha with Rac1, a GTPase protein. In dark LOV2 obstructs the active site of Rac1 and inhibits. In blue light the J alpha unfolds, releasing the allosteric block on Rac1 which activates multiple downstream targets such as PAK1.
This leads to the polymerisation of actin filaments and generation of localised cell protrusions to allow a control of cell morphology and migration.
Has been implemented in Zebrafish embryos and Drosophila ovary cells to spatially control cell movements
paCaMKII: Fused LOV2 -Jalpha with CaMKIIalpha, a serine/threonine kinase abundant in the cerebrum and hippocampus. In blue light or two photon excitation the J alpha unfolds, releasing the allosteric block on CaMKII which autophosphorylates activates
The activation of paCaMKII induced synaptic plasticity in hippocampal neurons
BLINK1: Blue light induced K+ channel 1 of LOV2-Jalpha fused to potassium ion channel Kcv.
When injected with BLINK as embryos, it inhibits zebrafish escape response in blue light, inducing hyperpolarisation
By combining with a degradation sequence (ornithine decarboxylase-like degradation sequence; cODC1) and fusing to an enzyme of interest, its abundance can be controlled in a temporal manner by blue light. In blue light the allosteric block of LOV2-Jalpha on the degron is removed and the enzyme undergoes proteasomal degradation.
When to use LOV based reporters over GFP
Studies in anaerobic conditions or when oxygen can be limiting as GFP fluorophore needs oxygen to mature and fluoresce (ex. as real time reporter for probing cell biomass in bioreactors, product formation in fermenter culture)
More pH tolerant
Smaller (12 kDa vs 27 kDa) so:
-less genetic payload when inserted into a viral genome so more stably integrated (ex. use as a reporter to confirm viral infection)
-less steric impact when fused to protein
-can be used to report protein secretion of effector targets from E. coli and other gram negative bacteria since won’t get stuck in the Type III secretion system
Difference between LOV and all it’s derivatives (3)
phiLOV: Through mutagenesis of iLOV produced phiLOV exhibiting less photobleaching upon confocal imaging
tLOV: Through genome searching identified tLOV which has improved thermostability and fluorescence
Photocycling: In LOV, I427L has short adduct decay time, I427V has shorter, V416L the fastest (stability of the FMN-cysteinyl adduct relates to adduct decay time relates to the photoproduct lifetime and so can be altered to promote or diminish signalling)
iLOV: Chapman et al. (2008). Through mutagenesis of residues around the chromophore. Smaller with improved fluorescence and photostability
Dark reversion rates for the flavin adduct range from 10s to 1000s of seconds
Red photoprotein examples
IFP: Infra-red fluorescence protein engineered from a phytochrome from bacteria D. radiodurans.
Biliverdin chromophore (breakdown of heme present in some cell types)
Excited by red light and fluoresces infra-red
Govarunova et al. (2020) RubyACR: Bacterial (A. limacinum) channelrhodopsin
Excited by red/IF light to open and allow Cl- in/out, inducing hyperpolarisation.
Both suitable for whole-body imaging since its wavelengths penetrate tissue well and less phototoxicity. Both expresses well in mammalian cells and mice
Definition of Biotechnology
Biotechnology involves the use of biological processes or knowledge of biological systems to produce substances beneficial to agriculture, the environment, industry and medicine such as the techniques that use an organism to make or modify products to improve plants or animals
Benefits of using photoreceptors to control cell activities
Light is non-invasive so simple
Photoreceptors are genetically encoded so can be produced in any cell
Many photoreceptors have a ubiquitous chromophore that can be readily acquired from the cell environment
Photoreceptor activation is extremely rapid compared to chemical induction that requires uptake
Spatial regulation is possible by dosing specific parts of the cell/tissue with light or put under influence of specific promoter
Reversible
Many light sensing domains which can be engineered into an optogenetic tool are:
Small
Soluble
Ubiquitous cofactor
Varys in photochemical properties
What is Optogenetics and early example
Field that exploits the use of natural or engineered photoreceptor proteins (comprised of chromophore; light absorbing pigment and apoprotein; the protein backbone structure that together make holoprotein) for biotechnology and biomedical research.
Combines optical and genetic approaches to control cell activates (ex. use cells as production systems by controlling gene expression/enzyme function, or new treatment in biomedicine controlling complex organs/tissues), or monitor cell activity.
Optogenetics has long been discussed. Francis Crick in 1979 indicated that a major challenge in neuroscience was the need to control one brain cell type while leaving the others unaltered. Later he speculated light may have properties to serve as a control tool but didn’t have a strategy to implement.
Miesenbock (2005) produced the first example of optogenetics. 3 component system called chARGe of photoreceptor components in drosophila eye (rhodopsin, arrestin desensitises receptor to modulate activation and Gq protein alpha subunit which activates PLC) expressed in neural cells. Light activated rhodopsin activates cation channels causing PM depolarisation and generation of an action potential in cultured neurons. Too complex so use ChR2 instead now.
Went on to show expressing the phototrigger P2X2 ion channel in drosophila dopamine neurons causes blue light to increase flight and movement.
How neurons function
Brain is the most complex organ in body made of over 100 billion neurons.
Neuron is made of the cell body, axon (sends signals) and dendrites (receive signals)
At rest the membrane is polarised with positive and negative charged ions on both sides (outside is more positively charged, -70mV).
Sodium ion channels open when the neuron receives a signal (from another neuron or sensory receptor) causing a short-lived localised influx in sodium and so a depolarisation (reduction in charge difference across the membrane). At the threshold potential (-55mV), neighbouring voltage gated sodium channels open allowing more sodium to enter and causing an action potential. At it’s peak (~40mV) the sodium ion channels begin to close and the voltage gated potassium ion channels open allowing potassium ions to flow out the neuron, causing repolarisation. Hyperpolarisation occurs before the potassium ion channels close again and the sodium potassium pump restores the resting membrane potential.
The action potential activates neighbouring sodium ion channels and propagates along the cell in a directional manner from the dendrite to the axon.
Depolarisation at the axon terminus or synapse triggers the release of chemical neurotransmitters that diffuse across the gap from the axon of one neuron to the dendrite of another.
Excitatory neurotransmitters (ex. Glutamate) will open sodium ion channels in the next neuron and initiate depolarisation. Inhibitory neurotransmitters (GABA) will lead to the opening of chloride channels and increase polarisation which prevents depolarisation
Experimental approaches to express and assess performance of an optogenetic tool in neural studies
Model system: For in vitro use mouse cortical neurons or human induced pluripotent stem cell derived neurons. For in vivo use transgenic mice.
Expression:
Clone gene into appropriate viral vector (AAV; adeno-associated virus or Lentivirus) but the maximum genetic payload (DNA length) of viruses is usually small promoter fragments (<4 kb) that are specific and strongly expressed. This is rare so can instead use Cre/loxP
Expression validation: immunocytochemistry and fluorescence microscopy using specific antibodies or fluorescent tags (GFP, mCherry) to confirm localisation
Assess light activation:
Patch clamp experiment to monitor membrane potential under light stimulation.
Use time course experiments to observe the onset, duration and offset of neural activity after light exposure.
Measure phototoxicity by monitoring cell viability with MTT assays
In vivo monitor mice behavioural responses (movement, learning, pain sensation)
Natural photoreceptors
All are 7 TM opsins with all trans retinal chromophore (aldehyde derivative of vitamin A) which absorbs maximally 470 nm (blue). This instigates conformational changes in the transmembrane protein that leads to opening of the channel pore (at least 6A).
Rhodopsin is a green light activated opsin and GPCR
Channelrhodopsin (ChR1 and ChR2; ChR2 expresses 10x better) from Chlamydomonas reinhardtii is a blue light sensitive cation channel. Larger the rhodopsin with long C-terminal extension (unnecessary for function). First optogenetic example with ChR2 demonstrated by Deisseroth et al. (2005).
Implemented by using cell specific promoters to express ChR2 in certain brain cell types. Fibre optics delivers light to brain while electrically recording (optrodes).
Halorhodopsin (NpHR) is a chloride pump activated by yellow light. From N. pharaonis was isolated from soda lakes in Sahara Desert so copes with extreme conditions (specifically high salt conc. and alkaline environment pH11)
Optogenetics in biomedicine examples
ChR2 has revolutionised study of brain function. Has been engineered to modify spectral and kinetic properties:
-Maximise photocurrent so lower levels of light can stimulate neurons
-Improve expression and membrane targeting in host cells
-Increase closure rate after light stimulation (beneficial to repolarising neurons to baseline membrane after action potential and also reduces toxicity/expression levels (E123T in ChETA absorbs longer wavelengths). In some cases slow closure is useful; ex. desensitise addiction pathways)
-Single component (unlike chARGe)
Parkinson’s (affects over 1mil Americans). Induce in mice by injecting neurotoxin like oxidopamine or OHDA (6-hydroxydopamine), since in Parkinson’s the degeneration of neurons in basal ganglia (which helps control motor function) makes them stop producing dopamine.
Scientists long believed the motor circuitry in basal ganglia can be separated into a go pathway (that can be affected by Parkinson’s) and stop pathway (dominant in Parkinson’s). Optogenetics allowed this to be tested and more precision stimulating brain.
ChR2 delivered to neurons of the go pathway (D1) or the stop pathway (D2) in the basal ganglia with cell specific promoters. Stimulating D1 neurons restored mice mobility to pre-toxin levels whereas stimulating D2 caused Parkinson’s in untreated mice. Stimulation was done with a laser coupled probe implanted in the brain.
Optogenetics can be used to study other movement and behavioural disorders (depression)
ChETA: engineered by homology modelling of ChR2 on the structure of microbial opsins and targeted residues close to Schiff base (E123 that stabilises Schiff Base mutated to Q quickens channel closing but D is slower than WT)
NpHR: Yellow absorbing halorhosopsin (chloride pump) causing hyperpolarisation. Absorption spectra does not overlap with ChR2 (blue cation channel) so both can be expressed in the same cell to activate and inhibit/silence neural activation. Since NpHR only one chloride ion is transported per photocycle, it requires constant illumination of high intensities which can produce side effects. Instead, ChR2 has been engineered to functions as a chloride channel (Brendt et al., 2014) and some naturally do (RubyACR)
Optogenetics in cAMP signalling
Photoactivated adenylyl cyclase (PAC) is comprised of a BLUF domain which regulates the activity of the Adenylyl cyclase.
Blue light utilising FAD (BLUF, ~15 kDa) with flavin adenine dinucleotide (FAD) chromophore in prokaryotes.
First discovered as an photoactivated biosensor in Euglena algae in 2002 (2 BLUF and 2 AC domains)
PAC from Beggiatoa bacteria is used since smaller (1 BLUF and 1 AC domain) and less dark activity (less leaky/larger dynamic range)
OptoXRs: Mammalian rhodopsins (opsin (photoreceptor) and GPCR) is coupled to heterotrimeric G protein transducin. Since the structure and function of GPCRs are well known, can convert chemically activated GPCRs into light activated GPCRs to regulate well defined pathways (manipulate levels of cAMP, cGMP, IP3, etc).
The intracellular loops of rhodopsin (Gt that decreases GMP) are replaced with those from the beta2 adrenergic receptor from hamsters (Gs to increase cAMP), or the human alpha1 adrenergic receptor (Gq to increase IP3 and DAG)
Cre/loxP
Cyclisation recombination is a small recombinase protein from bacteriophage P1 which mediates excise, inversional, and integrative site specific recombination
Between two loxP sites (locus of X over P1) which comprise DNA sequences with two 13bp inverted repeats separated by an 8bp asymmetric spacer region.
Two transgenic mice strains are generated and mate to produce cell specific expression
