KSEF RDE: Calcium-Dependent Signaling Mechanisms Governing Rapid Cold Hardening in Insects

The ability to quickly respond to changes in environmental temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold-hardening (RCH), brief chilling (minutes to hours) significantly enhances insects’ ability to survive cold shock. RCH is one of the fastest known physiological responses to temperature, but its underlying mechanisms are poorly understood. Isolated cells and tissues retain the capacity for RCH ex vivo, suggesting that hardening occurs at the cellular level without nervous or hormonal input. Insect cells sense low temperature via an influx of calcium into the cytoplasm, and blocking this calcium movement prevents cold hardening. However, the downstream signaling mechanisms that are activated by calcium to protect cells against cold injury are unknown. Reversible protein phosphorylation is the most common mechanism for regulating protein function and relaying cell signals. Thus, my objective for this project is to identify calcium-dependent protein phosphorylation events that contribute to RCH. Cultured Drosophila S-2 cells will be exposed to two temperature treatments (room temperature or brief chilling) in the presence or absence of BAPTA-AM, a chelating agent that blocks intracellular calcium signaling. Changes in global protein phosphorylation will be measured using quantitative phosphoproteomics, a technique that simultaneously identifies and quantifies proteome-wide phosphorylation events. The proteins identified via phosphoproteomics will be functionally tested in vivo by knocking down gene expression in flies with transgenic RNAi and testing for changes in RCH capacity at the organismal and tissue level. These results will significantly expand our knowledge of how insects and other cold-blooded animals sense and cope with cold stress at the cellular and molecular level. Understanding these mechanisms gives us new targets to manipulate the overwintering survival of pest insects, and insights on how insect cells survive extreme cold may inform human organ cryopreservation efforts.