The Detection and Localization of Frost Protein in Drosophila

In response to cold stress, Drosophila Melanogaster increase their expression of Frost, a candidate gene involved in cold response. The direct role of Frost in cold tolerance is yet to be determined, and its importance for survival in cold environments has been questioned. In this study, I attempt to better understand the molecular machinery of Frost by selecting for its protein in fly lysate knowing only its RNA has been found. Detection of Frost expression and subsequently studying its protein will hopefully lead to enhanced comprehension of cold responses in insects, which is the longterm goal of this research. I predict that Frost will be expressed in flies that undergo cold stress at 0C for 2 hours before recovering at 22C for 3 hours before lysing. Two Western blots were executed using fly lysate that underwent the above treatment, whose antibodies were specific to Frost. No bands were seen in any of the lanes containing treated samples in either of the Immunoblots, indicating that Frost was either not expressed or not detected in fly lysate. Introduction The distribution and abundance of insects are largely affected by temperature. As temperatures plummet, insects undergo a reversible process called chill coma, where they temporarily lose the ability to move (Denlinger and Lee, 2010). As such, Chill susceptibility is a term used for species that succumb to cold exposure without the formation of ice crystals (Denlinger & Lee, 2010). However, it was found that for many insect species, exposure to a nonlethal short lived cold stress was beneficial, as it raised tolerance levels for more extreme cold stresses (Denlinger & Lee, 2010). This was termed rapid cold hardening and is essential to the survival of insect species in colder environments (Denlinger and Lee, 2010). Due to its varying cold tolerances and phenotypic plasticity, Drosophila Melanogaster is used as the genetic model species for studies involving the effects of cold exposures (Udaka et al. 2013). Genes that show increased up-regulation after cold exposure may contribute in the enhancement of cold tolerance. Previous studies have found a gene whose expression was considerably increased after exposure to cold in Drosophila melanogaster, which they called Frost (Goto, 2001). Consisting of 278 amino acids, Frost appears to be a stress-related disordered protein whose modular structure indicates that it is directed into the endoplasmic reticulum and secreted into the extracellular space (Goto, 2001). Other notable characteristics include a proline rich region and an unusually high number of internal repeats in both amino acid and nucleotide sequences (Goto, 2001). The exact mechanism by which Frost enhances cold response remains unknown and its importance for cold tolerance is being questioned. Conflicting reports have emerged concerning whether Drosophila needs to express frost to survive cold environments. In one study, RNAi was used to knock down Frost expression in fruit flies which were subsequently exposed to cold but yielded a similar number of survivors regardless of Frost expression levels (Udaka et al. 2013). Another study, testing the same hypotheses using a similar methodology found that the absence of Frost was lethal in Drosophila when exposed to colder conditions (Colinet et al. 2010). The discrepancy in the results led researchers to question whether different life stages in flies had varying cold tolerances. The answer was a resounding yes, as studies have shown that significant up-regulation of Frost occurs in eggs, third instar larvae, 2 and 5 day old flies (Bing et al. 2012). Also, much like in heat tolerance, cold tolerance sees a rapid decline as flies age indicating that studies should be conducted on flies in the same life stage (Colinet et al. 2013). While stress genes Hsp22 and Hsp70Aa saw a similar decline in function with increasing age, previous studies have concluded that no correlation is evident between stress genes and cold tolerance (Colinet et al. 2013; Udaka et al. 2010). Clearly, consensus on the importance and function of Frost is difficult to come by, as the genetic machinery by which it works remains, for the most part, a mystery. Additionally, only its RNA has been identified, making the task of piecing together this protein’s function more challenging. This study will attempt to select for Frost protein in fly lysate. We predict that, under optimized conditions that includes both cold shock and recovery, Frost protein will be expressed in fly lysate if it is essential for cold tolerance and will therefore be detected by an immunoblot.