Aquaglyceroporin expression and regulation in erythrocytes from freeze tolerant Cope's gray treefrog, Hyla chrysoscelis

Date of Award


Degree Name

Ph.D. in Biology


Department of Biology


Advisor: Carissa M. Krane


Cope's gray treefrog, H. chrysoscelis is a freeze tolerant anuran that accumulates glycerol as a cryoprotectant during cold acclimation. Aquaporins, members of the major intrinsic protein (MIP) family of transmembrane water pores, may play an important role in the mechanism of freeze tolerance by mediating glycerol and water transport across cell membranes. Thus, we hypothesize that HC-3, an ortholog of mammalian aquaglyceroporin AQP3, enhances membrane permeability to glycerol, facilitating the cellular response to osmotic gradients formed when extracellular water freezes. To address the hypothesis, erythrocytes from H. chrysoscelis were used as an in vitro cell culture model to study the regulation of HC-3 protein expression. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs showed abundant HC-3 protein and enhanced plasma membrane localization of HC-3. Erythrocytes, regardless of the original acclimation state, exhibited time and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Likewise, erythrocytes cultured in glycerol-containing media consistently expressed relatively more glycosylated HC-3 than erythrocytes cultured in normal cell culture media. Thus, part of the regulation of HC-3 expression that occurs naturally during cold-acclimation is cell-based. Erythrocyte protein, when subjected to deglycosylation resulted in downward shift of high molecular weight HC-3 protein, demonstrating that HC-3 is post-translationally modified by N-linked glycosylation. In the absence of genomic knockout tools, a novel method of antisense HC-3 morpholino delivered in to cultured suspension erythrocytes via a peptide mediated Endo-Porter was developed where HC-3 protein expression was reduced by 94% in morpholino targeted cells (as assessed by Western blotting) as compared to controls. In addition, immunocytochemistry revealed a substantial decrease in HC-3 membrane expression in >65% of erythrocytes, with an additional 30% of erythrocytes showing no HC-3 expression, indicating that the method efficiently knocked down expression in >95% of cells. Furthermore, erythrocytes cultured for 48 hrs in media made hyperosmotic (400 mOsM) through the addition of 150 mM glycerol or urea showed enhanced membrane localization of HC-3 compared with those cultured in control media or media made hypertonic by addition of 150 mM sorbitol or 75 mM NaCl. The degree of hypotonic- induced cell shape changes were less for erythrocytes cultured in glycerol containing media compared to erythrocytes cultured in control media. In addition cell lysis data indicate that 60 % of erythrocytes cultured in glycerol or urea were still intact after 15 minutes as compared to controls, which were completely lysed within 15 minutes of challenge. Taken together, these studies strongly implicate a role for HC-3 in freeze tolerance in H. chrysoscelis.


Aquaporins Effect of cold on, Histones Effect of cold on, Erythrocytes Effect of cold on, Hylidae Effect of cold on, Thermal tolerance (Physiology), Biochemistry; biology; cellular biology; molecular biology; physiology; aquaglyceroporins; aquaporins; cold acclimation; freeze tolerance; Hyla chrysoscelis; HC-3 expression in erythrocytes; post translational modifications

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