Aquaporin Expression in Freeze Tolerance

Abstract

<p>A class of integral membrane proteins known as aquaporins play an essential role in maintaining water balance, by forming selective water pores in the plasma membrane of various cells of animals, plants and microorganisms (Zardoya, 2005; Fu and Lu, 2007; Verkman, 2008). Cope's grey tree frog, Hyla chrysoscelis, is unusual among animals because it can survive freezing. Cold acclimated Hyla chrysoscelis show increased expression of aquaporins compared with Hyla chrysoscelis at warm temperatures. We hypothesized that aquaporins are critical for this animal's cells to survive the freezing process. In this study, aquaporin expression is examined under two conditions: warm frogs (20°e) and cold acclimated frogs (4°e). We will quantify expression of aquaporins by Western blot, and we will use immunofluorescent imaging to examine whether aquaporin expression is localized at the cell membrane, where these proteins provide their function. The correlation between expression of aquaporins and their functionality in liver of H. chrysoscelis that were acclimated to warm and to cold temperatures is also being tested. Osmotic swelling assays are used to quantify total membrane permeability, allowing the correlation of the expression of aquaporins with the presence of functional proteins. Cells will then be frozen at -2.5°C for 24 hours and cell survival measured with trypan blue viability assays. By comparing cells from the warm and cold animals, it will be possible to show if increased aquaporin expression levels is associated with increased survivability of the cells after freezing, showing an important role of aquaporins in the maintenance of cells at low temperatures related to thermal acclimation.</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 16, 2010</p>