Honors Theses

Author(s)

Maria P. LaBello

Advisor

Carissa M. Krane, Ph.D.

Department

Biology

Publication Date

4-1-2019

Document Type

Honors Thesis

Abstract

Dryophytes chrysoscelis, commonly known as Cope’s gray treefrog, is a freeze tolerant anuran that freezes up to 65% of extracellular fluid during winter to survive. Glycerol is presumably used as a cryoprotectant during a period of cold-acclimation to protect cells from permanent damage due to hypoosmotic stress upon freezing and thawing. The passage of glycerol and water during cold-acclimation is mediated through aquaglyceroporin HC-3 in the nucleated erythrocytes (RBCs) of D. chrysoscelis. This thesis analyzes the mechanisms in which D. chrysoscelis prepares for cold-acclimation and glycerol synthesis. Cortisol is a stress hormone known to respond to osmolarity and metabolic challenges and regulate aquaporins; however, the role of cortisol in regulating anuran HC-3 protein expression and subcellular localization, and implications for mediating anticipatory glycerol synthesis and freeze tolerance remain to be determined. We hypothesize that cortisol exposure regulates HC-3 protein expression and subcellular localization. Freshly isolated RBCs were cultured in complete cell culture media (CCCM) and cortisol for 2, 4, and 6 hours at two separate concentrations, 1.0 and 0.1 μg/ml. Another group of RBCs was incubated with CCCM for 24 hours in culture before the 4 and 8-hour incubation with cortisol concentrations of 0.01, 0.1, and 1.0 μg/ml. Densitometric analyses of immunoblots specific for HC-3 for RBCs that had undergone the 24-hour incubation before cortisol exposure showed a 3.7-fold increase in native HC3 from RBCs cultured in 0.01 μg/ml cortisol for 4 hours, 40-fold increase in native HC-3 from RBCs cultured in 0.1 μg/ml cortisol for 4 hours, and 21-fold increase in native HC3 from RBCs cultured in 1.0 μg/ml cortisol for 4 hours compared to RBCs cultured in 0 μg/ml for 4 hours. HC-3 protein abundance increased in RBCs exposed to 0.01 μg/ml cortisol for 8 hours by 2.4-fold from the 4-hour time point. The abundance of Page | ii glycosylated HC-3 (60-150kDa) increased by 3.1-fold, 5.8-fold, and 5.3-fold for RBCs exposed to 0.01, 0.1, and 1.0 μg/ml cortisol for 4 hours. The abundance of glycosylated HC-3 increased by 0.8-fold for RBCs treated with 0.01 μg/ml cortisol for 8 hours. A variation in HC-3 protein abundance was observed for freshly isolated RBCs. The subcellular localization and fluorescent intensity (arbitrary units) of the HC-3 protein were analyzed via scanning laser confocal microscopy and immunocytochemistry using ImageJ software. Perinuclear localization of the HC-3 protein was oberved in RBCs exposed to 0.1 and 1.0 μg/ml cortisol for 4 hours and membrane localization for RBCs exposed to 0.1 μg/ml cortisol for 8 hours. Fluorescent analysis of RBCs exposed to 0.01 and 1.0 μg/ml cortisol for 8 hours exhibit enhanced HC-3 intensity in the cytosol compared to control RBCs. Therefore, there is a potential correlation between cryoprotective glycerol, freeze tolerance, and the role of cortisol in regulating HC-3 protein expression and subcellular localization.

Permission Statement

This item is protected by copyright law (Title 17, U.S. Code) and may only be used for noncommercial, educational, and scholarly purposes

Disciplines

Biology


Included in

Biology Commons

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