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Molecular Biology of Freezing Tolerance

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Abstract

Winter survival for many kinds of animals involves freeze tolerance, the ability to endure the conversion of about 65% of total body water into extracellular ice and the consequences that freezing imposes including interruption of vital processes (e.g., heartbeat and breathing), cell shrinkage, elevated osmolality, anoxia/ischemia, and potential physical damage from ice. Freeze‐tolerant animals include various terrestrially hibernating amphibians and reptiles, many species of insects, and numerous other invertebrates inhabiting both terrestrial and intertidal environments. Well‐known strategies of freezing survival include accumulation of low molecular mass carbohydrate cryoprotectants (e.g., glycerol), use of ice nucleating agents/proteins for controlled triggering of ice growth and of antifreeze proteins that inhibit ice recrystallization, and good tolerance of anoxia and dehydration. The present article focuses on more recent advances in our knowledge of the genes and proteins that support freeze tolerance and the metabolic regulatory mechanisms involved. Important roles have been identified for aquaporins and transmembrane channels that move cryoprotectants, heat shock proteins and other chaperones, antioxidant defenses, and metabolic rate depression. Genome and proteome screening has revealed many new potential targets that respond to freezing, in particular implicating cytoskeleton remodeling as a necessary facet of low temperature and/or cell volume adaptation. Key regulatory mechanisms include reversible phosphorylation control of metabolic enzymes and microRNA control of gene transcript expression. These help to remodel metabolism to preserve core functions while suppressing energy expensive metabolic activities such as the cell cycle. All of these advances are providing a much more complete picture of life in the frozen state. © 2013 American Physiological Society. Compr Physiol 3:1283‐1308, 2013.

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Figure 1. Figure 1.

Cell responses to freezing: consequences and defenses. If undefended, ice nucleation in the extracellular space leads to a rapid growth of ice. Ice crystals exclude solutes and the osmolality of remaining extracellular fluid rises rapidly placing an osmotic stress on cells that causes massive water outflow and cells shrink. If a critical minimum cell volume is exceeded, permanent damage can result causing loss of integrity of the plasma membrane when thawed. Transmembrane nucleation can also occur so that intracellular ice forms. Freeze‐tolerant organisms protect against these forms of damage with a variety of defenses. Ice nucleating proteins (INPs) or agents trigger crystal growth close to the equilibrium FP of body fluids so that ice growth is slow and controlled. Antifreeze proteins (AFPs) provide inhibition of recrystallization so that ice crystal size stays small. Low molecular weight carbohydrates are proliferated such as glycerol (g) that limit cell volume reduction by colligative effects whereas sugars such as trehalose act as membrane protectants (MP) to stabilize bilayer structure. Membrane transporters (AQPs) including aquaporins, aquaglyceroporins, and transporters for other cryoprotectants ensure high flux movements of water and cryoprotectants across the membranes.

Figure 2. Figure 2.

Cryoprotectant acquisition by freeze‐tolerant animals. (A) Seasonal synthesis of glycerol and sorbitol by larvae of the goldenrod gall fly, Eurosta solidaginis, and corresponding consumption of larval glycogen reserves. (B) Freeze responsive accumulation of glucose by wood frogs, Rana sylvatica, frozen at −3°C. Ice nucleation on the skin triggers a nearly instant activation of glycogenolysis in liver and glucose is rapidly produced, exported into the blood and distributed to all other organs. Thawing reverses the process to restore glucose into liver glycogen but occurs over a longer time frame. (C) E. solidaginis larva within an opened gall and two galls on a goldenrod stem. (D) R. sylvatica, unfrozen and frozen. Photos, with permission, by J.M. Storey.

Figure 3. Figure 3.

Cryoprotectant acquisition by freeze‐tolerant animals. (A) Seasonal synthesis of glycerol and sorbitol by larvae of the goldenrod gall fly, Eurosta solidaginis, and corresponding consumption of larval glycogen reserves. (B) Freeze responsive accumulation of glucose by wood frogs, Rana sylvatica, frozen at −3°C. Ice nucleation on the skin triggers a nearly instant activation of glycogenolysis in liver and glucose is rapidly produced, exported into the blood and distributed to all other organs. Thawing reverses the process to restore glucose into liver glycogen but occurs over a longer time frame. (C) E. solidaginis larva within an opened gall and two galls on a goldenrod stem. (D) R. sylvatica, unfrozen and frozen. Photos, with permission, by J.M. Storey.

Figure 4. Figure 4.

Relative expression levels of heat shock proteins Hsp110, Hsp70, Hsp60, and Hsp40 from September to April in freeze‐tolerant Eurosta solidaginis larvae. Data are means ± SEM, n = 4. “a” shows values that are significantly different from the corresponding September value, P < 0.05. Modified, with permission, from Zhang et al. ().

Figure 5. Figure 5.

Na+K+‐ATPase and sarco(endo)plasmic Ca2+‐ATPase (SERCA) in larvae of freeze‐tolerant Eurosta solidaginis. (A and C) Seasonal changes in Na+K+‐ATPase and SERCA activities in larvae sampled in the second week of each month. (B and D) Effect of in vitro incubations to stimulate endogenous protein kinases (PKA, PKG, or PKC) or the addition of exogenous calf‐intestinal alkaline phosphatase (CIAP) on ATPase activities in extracts from 15°C‐acclimated larvae. Data are means ± SEM, n = 3‐5. * shows values that are significantly different from corresponding Sep/Oct or control values, P < 0.05; ** shows values that are different from all other months. Modified, with permission, from McMullen and Storey ().

Figure 6. Figure 6.

(A) Synthesis of microRNA. Primary transcripts are transcribed by RNA polymerase II and processed by riboendonucleases (Drosha, Dicer) into single‐stranded mature microRNAs. These then join a microRNA‐induced silencing complex (miRISC) and bind to mRNA transcripts at their 3′‐UTR to repress translation. (B) The freeze‐tolerant marine intertidal snail, Littorina littorea. (C) Relative expression levels of six miRNA species and Dicer protein levels in foot muscle showing effects of freezing (24 h at −6°C) or anoxia (24 h under a N2 gas atmosphere at 10°C), as compared with 10°C controls. Expression levels of miRNAs are normalized against 5S rRNA expression from the same samples. Data are mean ± SEM, n = 3‐4. “a” shows values that are significantly different from the corresponding control (P < 0.05); “b” shows values that are significantly different from corresponding freezing group. Modified, with permission, from Biggar et al. (). Photo by JM Storey.

Figure 7. Figure 7.

(A) Synthesis of microRNA. Primary transcripts are transcribed by RNA polymerase II and processed by riboendonucleases (Drosha, Dicer) into single‐stranded mature microRNAs. These then join a microRNA‐induced silencing complex (miRISC) and bind to mRNA transcripts at their 3′‐UTR to repress translation. (B) The freeze‐tolerant marine intertidal snail, Littorina littorea. (C) Relative expression levels of six miRNA species and Dicer protein levels in foot muscle showing effects of freezing (24 h at −6°C) or anoxia (24 h under a N2 gas atmosphere at 10°C), as compared with 10°C controls. Expression levels of miRNAs are normalized against 5S rRNA expression from the same samples. Data are mean ± SEM, n = 3‐4. “a” shows values that are significantly different from the corresponding control (P < 0.05); “b” shows values that are significantly different from corresponding freezing group. Modified, with permission, from Biggar et al. (). Photo by JM Storey.

Figure 8. Figure 8.

The four stages of the cell cycle showing the Cdk and cyclin pairs that regulate each stage. The kinase activity of the cyclin:Cdk complex activates substrates that regulate the progression and completion of each phase.



Figure 1.

Cell responses to freezing: consequences and defenses. If undefended, ice nucleation in the extracellular space leads to a rapid growth of ice. Ice crystals exclude solutes and the osmolality of remaining extracellular fluid rises rapidly placing an osmotic stress on cells that causes massive water outflow and cells shrink. If a critical minimum cell volume is exceeded, permanent damage can result causing loss of integrity of the plasma membrane when thawed. Transmembrane nucleation can also occur so that intracellular ice forms. Freeze‐tolerant organisms protect against these forms of damage with a variety of defenses. Ice nucleating proteins (INPs) or agents trigger crystal growth close to the equilibrium FP of body fluids so that ice growth is slow and controlled. Antifreeze proteins (AFPs) provide inhibition of recrystallization so that ice crystal size stays small. Low molecular weight carbohydrates are proliferated such as glycerol (g) that limit cell volume reduction by colligative effects whereas sugars such as trehalose act as membrane protectants (MP) to stabilize bilayer structure. Membrane transporters (AQPs) including aquaporins, aquaglyceroporins, and transporters for other cryoprotectants ensure high flux movements of water and cryoprotectants across the membranes.



Figure 2.

Cryoprotectant acquisition by freeze‐tolerant animals. (A) Seasonal synthesis of glycerol and sorbitol by larvae of the goldenrod gall fly, Eurosta solidaginis, and corresponding consumption of larval glycogen reserves. (B) Freeze responsive accumulation of glucose by wood frogs, Rana sylvatica, frozen at −3°C. Ice nucleation on the skin triggers a nearly instant activation of glycogenolysis in liver and glucose is rapidly produced, exported into the blood and distributed to all other organs. Thawing reverses the process to restore glucose into liver glycogen but occurs over a longer time frame. (C) E. solidaginis larva within an opened gall and two galls on a goldenrod stem. (D) R. sylvatica, unfrozen and frozen. Photos, with permission, by J.M. Storey.



Figure 3.

Cryoprotectant acquisition by freeze‐tolerant animals. (A) Seasonal synthesis of glycerol and sorbitol by larvae of the goldenrod gall fly, Eurosta solidaginis, and corresponding consumption of larval glycogen reserves. (B) Freeze responsive accumulation of glucose by wood frogs, Rana sylvatica, frozen at −3°C. Ice nucleation on the skin triggers a nearly instant activation of glycogenolysis in liver and glucose is rapidly produced, exported into the blood and distributed to all other organs. Thawing reverses the process to restore glucose into liver glycogen but occurs over a longer time frame. (C) E. solidaginis larva within an opened gall and two galls on a goldenrod stem. (D) R. sylvatica, unfrozen and frozen. Photos, with permission, by J.M. Storey.



Figure 4.

Relative expression levels of heat shock proteins Hsp110, Hsp70, Hsp60, and Hsp40 from September to April in freeze‐tolerant Eurosta solidaginis larvae. Data are means ± SEM, n = 4. “a” shows values that are significantly different from the corresponding September value, P < 0.05. Modified, with permission, from Zhang et al. ().



Figure 5.

Na+K+‐ATPase and sarco(endo)plasmic Ca2+‐ATPase (SERCA) in larvae of freeze‐tolerant Eurosta solidaginis. (A and C) Seasonal changes in Na+K+‐ATPase and SERCA activities in larvae sampled in the second week of each month. (B and D) Effect of in vitro incubations to stimulate endogenous protein kinases (PKA, PKG, or PKC) or the addition of exogenous calf‐intestinal alkaline phosphatase (CIAP) on ATPase activities in extracts from 15°C‐acclimated larvae. Data are means ± SEM, n = 3‐5. * shows values that are significantly different from corresponding Sep/Oct or control values, P < 0.05; ** shows values that are different from all other months. Modified, with permission, from McMullen and Storey ().



Figure 6.

(A) Synthesis of microRNA. Primary transcripts are transcribed by RNA polymerase II and processed by riboendonucleases (Drosha, Dicer) into single‐stranded mature microRNAs. These then join a microRNA‐induced silencing complex (miRISC) and bind to mRNA transcripts at their 3′‐UTR to repress translation. (B) The freeze‐tolerant marine intertidal snail, Littorina littorea. (C) Relative expression levels of six miRNA species and Dicer protein levels in foot muscle showing effects of freezing (24 h at −6°C) or anoxia (24 h under a N2 gas atmosphere at 10°C), as compared with 10°C controls. Expression levels of miRNAs are normalized against 5S rRNA expression from the same samples. Data are mean ± SEM, n = 3‐4. “a” shows values that are significantly different from the corresponding control (P < 0.05); “b” shows values that are significantly different from corresponding freezing group. Modified, with permission, from Biggar et al. (). Photo by JM Storey.



Figure 7.

(A) Synthesis of microRNA. Primary transcripts are transcribed by RNA polymerase II and processed by riboendonucleases (Drosha, Dicer) into single‐stranded mature microRNAs. These then join a microRNA‐induced silencing complex (miRISC) and bind to mRNA transcripts at their 3′‐UTR to repress translation. (B) The freeze‐tolerant marine intertidal snail, Littorina littorea. (C) Relative expression levels of six miRNA species and Dicer protein levels in foot muscle showing effects of freezing (24 h at −6°C) or anoxia (24 h under a N2 gas atmosphere at 10°C), as compared with 10°C controls. Expression levels of miRNAs are normalized against 5S rRNA expression from the same samples. Data are mean ± SEM, n = 3‐4. “a” shows values that are significantly different from the corresponding control (P < 0.05); “b” shows values that are significantly different from corresponding freezing group. Modified, with permission, from Biggar et al. (). Photo by JM Storey.



Figure 8.

The four stages of the cell cycle showing the Cdk and cyclin pairs that regulate each stage. The kinase activity of the cyclin:Cdk complex activates substrates that regulate the progression and completion of each phase.

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Kenneth B. Storey, Janet M. Storey. Molecular Biology of Freezing Tolerance. Compr Physiol 2013, 3: 1283-1308. doi: 10.1002/cphy.c130007