Biophysical mechanism and synthetic engineering of optically-controlled Ca2+-powered supramolecular engines

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PROJECT ABSTRACT Myonemes are calcium-powered supramolecular protein `springs’ that form the force-generating cytoskeletal structure in some protozoan ciliates such as Spirostomum ambiguum. In Spirostomum, myonemes extraordinarily high-power outputs (equivalent to a 2-stroke diesel engine) that enable Spirostomum to contract to 1/4th of its body length in less than 5 milliseconds (one of the fastest motions at the single cell level). In terms of power per unit mass, myonemes generate six orders of magnitude more force than conventional ATP-powered molecular motors such as myosin or kinesin. Myonemes do not contain conventional cytoskeletal elements such as actin, microtubules or myosin. Rather, myonemes comprise of self-assemblies of two-components: centrin proteins that are calcium-responsive and Sfi1, an elastic backbone protein. Thus, myonemes offer attractive features such as non-ATP dependent actuation, ultrafast and high-power delivery and a simple two-component system, that could enable potentially transformational synthetic biology applications, such as design of artificial cytoskeletons for synthetic or biohybrid cells to enable them to divide, move or transport cargo similar to their living counterparts. However, there exists key gaps in our knowledge on the governing biophysical mechanism of force generation in these springs, how calcium ions act as chemical latches to control and synchronize force deliver over millimeter length scales, and how these supramolecular assemblies can be synthetically engineered and self-assembled in-vitro for harnessing them for desired functionalities. To address these gaps in understanding, the proposed research over the next 5 years will take a two-pronged approach: i) combine biophysical experiments, live microscopy and soft matter physics-based models to uncover the biophysical mechanism of force-generation in myonemes in-vivo in living cells, and ii) engineer, self- assemble and incorporate light-control in synthetic myonemes (synMyo) in-vitro in microfluidic devices and lipid vesicles. Finally, this work will also utilize mathematical theory and numerical simulations to support our findings. Long-term, this research will open up a fundamentally new class of nanoscale, Ca2+-based, and light-actuatable synthetic force generating cytoskeletal assemblies, with applications in intracellular actuation and sensing, therapeutic drug-delivery devices and artificial cytoskeletons in synthetic cells. For synthetic cells, these supramolecular springs can enable new mechanical functionalities, such as faster contraction than any microtubule or actin based system could offer; localized force generation free from polymer tracks; controllability that is orthogonalized from cell-specific biochemistry; and a novel, non-ATP- or GTP-based energy source to power movement inside cells.