Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca²⁺) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca²⁺ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca²⁺ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca²⁺ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca²⁺ imaging demonstrated lower Ca²⁺ basal levels, reduced magnitude, and slower Ca²⁺ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca²⁺-ATPases involved in intracellular Ca²⁺ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca²⁺ rapidly restored their intracellular Ca²⁺ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca²⁺ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (V_m) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FAST^M). Different from present multiplexing approaches, FAST^M uses phase-sensitive signal detection, which renders various combinations of common probes for V_m and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FAST^M allows simultaneous recording of rapid changes in Ca²⁺, pH, Na⁺, and V_m with high sensitivity and minimal crosstalk. FAST^M is also suited for multiplexing using single-cell microscopy and genetically-encoded FRET biosensors. Moreover, FAST^M is compatible with opto-chemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FAST^M also allows resolving rapid chemical reactions. Altogether, FAST^M opens new opportunities for interrogating cellular signaling.