Decoding ultrasensitive self-assembly of the calcium-regulated cytoskeletal protein Tcb2 using optical actuation.

EF-hand calcium binding proteins are key macromolecular components of many unique filament systems and ultrafast contractile structures found in protists. However, our biochemical understanding of these cytoskeletal systems has been hindered by the need for assays that can controllably generate spatiotemporal calcium dynamics to probe their behavior. Here, we define the quantitative requirements for calcium-dependent self-assembly of the cortical cytoskeletal protein Tcb2 using a microscopy-based spatiotemporally controlled optical calcium release assay. Light-driven uncaging of the photolabile calcium chelator DMNP-EDTA stimulates rapid localized self-assembly of Tcb2 into micron-scale protein networks. We quantify how the growth, size, and lifetime of Tcb2 networks is controlled by the duration and intensity of the applied light pulse. Incorporating the fluorescent calcium indicator Rhod-5N allows inference of the spatiotemporal distribution of calcium-bound Tcb2 monomers during the reaction and identifies a sharp, ultrasensitive transition to Tcb2 self-assembly. By applying this assay to mutants in Tcb2's four EF hand domains, we show that D184 is the key calcium binding site that licenses Tcb2 for self-assembly and define quantitative roles for other binding sites in tuning Tcb2's calcium-responsiveness. Our approach reveals a rich space of structures and regulation available to a single-protein system through coupling calcium-binding to ultrasensitive self-assembly, opening new paths forward to understanding other protist filament networks and contractile myonemes.