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Mechanisms that govern anhydrobiosis involve the accumulation of highly hydrophilic macromolecules, such as late embryogenesis abundant (LEA) proteins. Group 1 LEA proteins comprised of 181 (AfLEA1.1) and 197 (AfLEA1.3) amino acids were cloned from embryos of Artemia franciscana and expressed in Drosophila melanogaster cells (Kc167). Confocal microscopy revealed accumulations of green fluorescence protein (GFP) and AfLEA1.3 constructs in the mitochondria (AfLEA1.3-GFP) and AfLEA1.1-GFP constructs in the cytoplasm. In the presence of mixed substrates, oxygen consumption was similar for permeabilized Kc167 control and Kc167-AfLEA1.3 cells. Acute titrations of NaCl (up to 500 mM) led to successive drops in oxygen flux of permeabilized cells and were ameliorated by 18% in Kc167-AfLEA1.3 cells compared to Kc167 controls. Mitochondria were isolated from both cell types and resuspended in a sucrose-based buffer solution. The purified mitochondria from Kc167 control cells exhibited larger reductions in respiratory capacities after one freeze-thaw cycle (-80°C) compared to mitochondria isolated from Kc167-AfLEA1.3 cells. My data demonstrates that AfLEA1.3 exerts a protective influence on mitochondrial function during freezing and osmotically challenging events.
Marunde, Matthew R., "Improved Tolerance to Freeze and Salt Stress in Mitochondria of Drosophilia melanogaster Cells Conferred by LEA Protein" (2013). Undergraduate Honors Theses. 61.