Cell function control represents one of the main areas of synthetic biology. The versatility of calcium ions in cellular processes offers opportunities for precise regulation of many cellular properties and functions. An important characteristic of calcium signaling is also the fact that calcium entry can be induced by non-invasive methods such as ultrasound and heating. Regulating the concentration of calcium ions is crucial for cell function. When we interfere with this balance through chemical or physical stimulation, the cell responds and prevents easy stimulation.
In the course of our work, we developed a system for regulating the influx of calcium ions into cells based on proteolysis and a system for enhancing ultrasound signals using protein gas vesicles, which enhances the uptake of Ca2+ into mammalian cells.
To regulate Ca2+ influx, we relied on a protein pair that controls the tightly regulated intake of calcium: STIM1 and Orai. Based on the active domain of the STIM1 protein and coiled coils, we prepared a protein called PACE, which is activated by proteolytic cleavage. Activation of the protein leads to the entry of Ca2+ ions into the cytosol via Orai channels. The achieved concentration of Ca2+ ions, through calmodulin and calcineurin, results in the nuclear entry of synthetic calcium-dependent transcription factors and the expression of target genes. Based on the original PACE protein, we also prepared a version that can be activated by two different proteases. By expressing the PACE protein and proteases that are activated by small molecules in T cells, we demonstrated that we can control the entry of Ca2+ and the expression of cytokines IL2 and TNFα.
A different approach to enhancing Ca2+ uptake into cells was taken using protein gas vesicles. Gas vesicles from the organism Bacillus megaterium, which can be expressed in E. coli bacteria, behave differently under acoustic stimulation due to their hollow structure compared to mammalian cells. Because of this property, they can amplify the mechanical force induced by ultrasound, leading to the opening of calcium channels on the membrane. To better understand the composition of protein gas vesicles, we found through gene knockout experiments that the proteins GvpR, GvpT, and GvpU are not essential for building functional vesicles. Using flow cytometry and cryo-electron microscopy, we determined that the protein GvpJ tightly binds to gas vesicles, and the N-terminal fusion of the GvpN protein with a fluorescent protein causes the gas vesicles to retain a spindle-like shape. By using the GvpC protein from the gene cluster of the organism Anabaena flos aquae, we successfully attached gas vesicles to the membrane of mammalian cells. Cells with gas vesicles on their surface expressed more reporter protein when stimulated with ultrasound compared to cells without gas vesicles.
Both genetically encoded modified gas vesicles and synthetic intracellular amplifiers are newly developed tools for increasing sensitivity to mild mechanical stimuli. Their activity extends the duration and increases the amplitude of cytosolic Ca2+ and, consequently, enhances the activation of synthetic gene transcription.
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