Chandrasekharan Nithesh P, Lei Xiangting, Honts Jerry, Bhamla Saad, Coyle Scott M
Department of Biochemistry, University of Wisconsin-Madison.
Integrated Program in Biochemistry Graduate Program.
bioRxiv. 2025 May 29:2025.05.26.656216. doi: 10.1101/2025.05.26.656216.
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.
EF手型钙结合蛋白是原生生物中许多独特的丝状系统和超快收缩结构的关键大分子成分。然而，由于需要能够可控地产生时空钙动力学以探究其行为的检测方法，我们对这些细胞骨架系统的生化理解受到了阻碍。在这里，我们使用基于显微镜的时空可控光学钙释放检测方法，确定了皮质细胞骨架蛋白Tcb2钙依赖性自组装的定量要求。光驱动的光不稳定钙螯合剂DMNP-EDTA的解笼作用刺激了Tcb2快速局部自组装成微米级蛋白质网络。我们量化了Tcb2网络的生长、大小和寿命是如何由施加的光脉冲的持续时间和强度控制的。加入荧光钙指示剂Rhod-5N可以推断反应过程中钙结合的Tcb2单体的时空分布，并确定向Tcb2自组装的急剧、超敏感转变。通过将这种检测方法应用于Tcb2的四个EF手型结构域中的突变体，我们表明D184是许可Tcb2进行自组装的关键钙结合位点，并确定了其他结合位点在调节Tcb2钙反应性方面的定量作用。我们的方法揭示了通过将钙结合与超敏感自组装耦合，单个蛋白质系统可获得的丰富结构和调节空间，为理解其他原生生物丝状网络和收缩肌丝开辟了新的道路。
