Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca²⁺ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca²⁺-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca²⁺-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca²⁺ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca²⁺-responsive proteins, such as parasite Ca²⁺-dependent protein kinases. Our approach also identified numerous Ca²⁺-responsive proteins that are not predicted to bind Ca²⁺, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca²⁺-responsive enzyme that relocalized to the parasite apex upon Ca²⁺ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca²⁺ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca²⁺-regulated serine/threonine phosphatase activity in apicomplexan parasites.