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I leverage molecular to organismal biodiversity to advance novel genetic tools using computational and experimental methods for research and medicine
Biodiversity and Evolution
Biodiversity is rich in innovation from molecules to organisms but is often overlooked in fields using powerful genetic models. If there is a question, biodiversity can offer novel places to look for answers. Critically, genome sequencing and new genetic tools like genome editing have democratized functional mechanistic approaches and enable growing use of novel organisms and exploration of diverse biologies.
Evolution is a simple but relentless engineer, endlessly building, testing, and redesigning by mutation and natural selection to optimize and fulfill diverse functions. Though impressive, the brain is but one of its innumerable products, ranging from simple to sophisticated. Critically, evolution builds off of whatever is at hand. Components, systems, organisms it produces are chimeric mixes of juxtaposed elements tracing back to various origins and shared to varying degrees across diversity. This richness and relatedness in design offers a unique opportunity to gain footholds in reverse engineering, for instance, the brain, as components embedded in brain complexity can be examined in other cell-types, systems, and species to elucidate basic principles and core functions using a comparative mechanistic integrative framework. Similarly, evolution's engineering of proteins and systems offers diverse optimized products that can potentially be leveraged to create, advance, or guide technologies in research and medicine.
Sonogenetics
Sonogenetics uses non-invasive ultrasound to activate force-gated or mechanosensitive ion channels and control downstream physiology and behavior.
The technology is analogous to optogenetics but without limitations of transparency for light.
Sonogenetics was established by the Chalasani Lab at Salk in 2015. Expression of the TRP-4 mechanosensitive ion channel was targeted to the AWC neuron that controls crawling reversal in the worm Caenorhabditis elegans. Excitingly, worms crawling forward switched to reverse when exposed to ultrasound and then resumed crawling forward when ultrasound stopped (see gif). This work by the lab demonstrated the ability to modulate ion channels with ultrasound and control downstream neural circuits and behavior. A 2022 Chalasani Lab publication, and one where I contributed comparative sequence analysis of vertebrate TRPA1 channels, established ultrasound activation of human TRPA1 expressed in mammalian cells and mouse brain regions. Work is now underway in lab to push the technology deeper into mouse and gain control of circuits and behavior in freely-moving animals, similar to worm.
Sonogenetic technology is young but holds great promise to become a genetic therapeutic in medicine, particularly when targeted modulation of ion channels is required, including the ability to reset circuits in neurological disorders, like epilepsy or PTSD, or drive bioelectric systems, like an arrhythmic heart pacemaker. Critically, sonogenetic channels should activate at frequencies and thresholds distinct from - and should be absent or highly sequence-divergent relative to - native channels in humans to avoid unwanted channel activation and biological interference in treatment. To achieve this goal, discovery and engineering of diverse ultrasound-sensitive ion channels is needed!
Research Models
Identification of tens to thousands of new ion channel homologs belonging to gene families of interest in distant species is now straight-forward using genome-scale sequencing and phylogenomic pipelines. This would seem to solve the general challenge of identifying new proteins that are likely to provide desired functionality in human yet are novel or distinct at the sequence level from native human proteins and, and given their distinctness, would potentially not interfere with greater biological processes in human. However, screening new proteins in mammalian cells is a critical next step after discovery or computational engineering and represents a major bottleneck in developing new or novel genetic tools in research and medicine, particularly for sonogenetics, where time and cost do not currently scale well. Thus, it would be useful if numerous newly identified proteins could be functionally prescreened in the source species upstream of testing a selected few in mammalian cells - and in a way that is time and cost effective in the source species.
To address this challenge, I have leveraged my deep and often first-hand knowledge of molecular to organismal biodiversity and both my own experience and expertise of my friends, collaborators, and contacts that are similarly developing genomic and functional genetic tools in novel new/emerging model organisms. This has lead me to focus on bioluminescent dinoflagellates (see images and gifs), marine invertebrate larvae, and the cephalopod dynamic skin color system, with dinoflagellates my main focus to date (see below - but see also the No Brains and Big Brains write-ups on my research).
Dinoflagellates are famous for their symbiotic relationship with corals (and in coral bleaching) and for their bioluminescence in oceans. Importantly, dino bioluminescence is mechanosensitive - activated by environmental forces generated by, for instance, crashing waves, swimming dolphins, or surfing surfers. Components of the upstream sensory system include homologs to human mechanosensitive ion channels, such as TRPA, TRPV, and TRPP. I found that bioluminescence can be triggered using ultrasound and that the bioluminescence response can be imaged using standard sonogenetic / calcium imaging rigs, in addition to a PMT-based ultrasound assay system I built. Thus, dinoflagellates offer an ultrasound-sensitive model system that includes native fluorescent readouts for free and can potentially be assayed relatively cheaply and easily upstream of sonogenetic testing of select channels in mammalian cells.
OF NOTE: Force activation of upstream mechanosensitive ion channels in dinoflagellates leads to downstream generation of action potentials in subcellular scintillons and luciferase-based release of light. Thus, classic human brain components (ion channels) and processes (action potentials) are fundamental to bioluminescence in single-cell algae, highlighting deep origins of the brain and nervous system and the largely untapped potential of molecular to organismal biodiversity for comparative insights and discovery of specific components potentially relevant to human biology, research, and medicine.
My work in dinoflagellates is focused on two species I identified as ideal for distinct aspects of prescreening - and for which I have initiated genome sequencing projects. The genomes are likely large - potentially much larger than human - and may have sequence-identical arrays of gene paralogs numbering in the hundreds per array. Using CRISPR for functional studies is likely to be challenging in such species and dinos in general. Fortunately, siRNA was recently established in armored dinos (all bioluminescent species are armored) - with the clever insight that cold or brief light centrifugation will cause dinos to drop their extracellular armor (see black and white image of dropping armor and timelapse of rebuilding armor), providing a window of access to the cell membrane for transfection. I am now working to establish dino siRNA in lab and for mechanosensitive ion channels. More generally, dinoflagellates and marine species in general are highly amenable to pharmacological testing, as chemicals are often readily dissolved and taken up in seawater (see video of TRP ion channel agonist). From existing genomes and transcriptomes in species outside our two lab models, I have also identified homologs to human channels (see pink branches on tree image) that we are now moving into HEK cells for sonogenetic testing (see vector map image). Additional methods in the works include a more classical mutagenesis x sequencing approach that could discover novel channels that would go unnoticed in a phylogenomic analysis based on current functional knowledge of gene families.
Ocean dinoflagellate gif is from Mind Over Matter Media video https://youtu.be/QTjg12km2Nk.
