13.1 The Processes and Challenges of Freezing and Thawing [in progress]

Freezing is lethal to most organisms, but remarkably there are some organisms that are freeze-tolerant and survive internal ice formation. To understand freeze tolerance, it is important to think through all of the changes that can occur when a cell is cooled, frozen, and then thawed (Figure 13.2). Many types of organisms can survive freezing, including some plants and animals. In this chapter section, we will focus on what happens to animals and their cells during cooling, freezing, and thawing. Each of these processes has its own associated stresses, which we will cover in the subsequent sections. A review of Chapters 8 – 12 will provide a good basis for understanding these stresses in the context of freezing.

Figure 13.2 Summary of the processes and challenges associated with cooling, freezing, and thawing over time in a freeze-tolerant cricket under laboratory-controlled temperature exposure. Cell temperature generally follows the environmental temperature, which was manipulated by the researcher. A single cell is shown, along with the impacts of extracellular ice (blue hexagons), although ice can also form intracellularly (not shown). Stressors are highlighted in yellow shapes, and the explanation of each stressor will be expanded on in the chapter sections that follow.

Cooling (and supercooling)

As environmental temperatures drop, the organism and its cell begin to cool. At low temperatures, proteins become less flexible, chemical reaction rates decrease, membranes become less fluid, and the viscosity of liquids (e.g., the cytosol, circulatory fluids) increases. These changes associated with low temperature stress usually impair organismal function and cause energy stress, even before freezing occurs. Animals such as insects lose the ability to move due to the impact of low temperatures on their neurons and muscles, and they enter chill coma – a (usually reversible) paralysis induced by chilling. Oxidative stress can also occur at low temperatures due to the reduced efficacy of antioxidant enzymes at low temperatures.

The fluids in and around cells will remain liquid until ice begins to freeze in or around the organism. The temperature at which this happens differs among species and among individuals within a species. Every solution (including the fluids in an organism’s body) has a melting point – the temperature at which a solid changes phase into a liquid. For example, pure water has a melting point of 0°C; we usually also think of this temperature as the freezing point of water (but that is not necessarily correct, as explained below). If there are solutes dissolved in water, the melting point decreases. So most biological fluids (water + stuff dissolved in water) have a melting point at temperatures below 0°C, and will freeze at temperatures below 0°C. In addition, many biological fluids will actually supercool – that is, they will remain liquid at temperature below the melting point. Another way to phrase that is that freezing occurs below the melting point – cool!!

Ice nucleation and propagation

When temperatures get cold enough, ice formation begins in or around cells. The temperature at which ice formation begins is called the supercooling point (for reasons related to the previous section). The process that initiates ice formation is called ice nucleation. Here, nucleation refers to a central point (nucleus) around which water molecules start to organize themselves into the crystalline structure of ice (Figure 13.3). This ice nucleation can occur in pure water when enough water molecules spontaneously form an ice crystal, and is called homogenous (“homo” = same) nucleation. Molecules other than water (e.g., some proteins) can act as ice nucleating agents (INAs) that help organize water molecules into ice. When molecules other than water initiate ice nucleation, we call that heterogenous ice nucleation. Regardless of the type of ice nucleation, as ice forms it expands in volume because there is more space between the water molecules (Figure 13.3). Ice nucleation is followed by ice propagation – the growth of ice crystals as more and more water molecules join the growing crystalline structure. Ice propagation tends to occur fastest at lower temperatures, and is slower at warmer (closer to 0°C) temperatures. The amount of ice that forms depends on temperature (lower temperatures = more ice) and the osmolarity of the biological fluids (lower osmolarity = more ice).

Figure 13.3. As water freezes, the molecules orient into a crystalline structure. This forces molecules farther apart, resulting in expansion. (Image from https://www.sciencefacts.net/why-does-water-expand-when-it-freezes.html) [replace or check copyright]

Ice may nucleate inside or outside of cells, with different impacts on the cell. Extracellular ice can also propagate into cells if propagation is rapid. If ice forms inside cells or propagates into cells, this is usually lethal for the cell due to mechanical stress associated with the ice (see Chapter 13.3 for additional detail). Therefore, we will focus our remaining discussion on extracellular ice formation that does not penetrate into cells. When ice nucleation is extracellular, as the ice propagates the osmotic environment around the cell also changes. This is because ice crystals include only water molecules, and not the solutes that were dissolved in the water. Therefore, as more ice forms and less liquid water is available, the solute concentration (and osmotic pressure) in the remaining extracellular fluid increases. This solute concentration will continue to increase until the ice content stabilizes (i.e., ice crystals are no longer growing). In some organisms, up to 80% of water is converted to ice! The hyperosmotic extracellular environment during freezing can cause an osmotic stress for the cells. Cells will lose water to their environment via osmosis, and will continue to do so until the osmotic pressure is equal inside and outside of the cell. Usually, cell volume decreases during this process, causing the cell to shrink.

The frozen state

Once ice content has reached an equilibrium (no more ice is forming), cells that survive freezing must endure staying in the frozen state until thawing occurs. As you might imagine, processes like whole organism movement, feeding, and the functions of various physiological systems (circulatory system, excretory system, etc.) are inhibited when the organism is frozen. The inability to feed and the slowdown of metabolic processes in the frozen state can cause energy stress. However, some cellular processes can still occur. There is evidence that some metabolism such as cryoprotectant synthesis (more on that later) can still occur in freeze-tolerant organisms. However, metabolism that relies on oxygen may be inhibited in the frozen state because ice is fairly impermeable to gases. Therefore, when cells or organisms are surrounded by ice, they are oxygen-limited and likely experience hypoxia, further contributing to energy stress.

Thawing

When temperatures increase, the frozen fluids in or around cells should thaw. If the ice was extracellular, thawing of the ice results in a decrease in extracellular osmolarity as the water dilutes the existing solutes. This can cause osmotic stress for cells, as they will now regain water via osmosis until intracellular and extracellular osmolality equilibrate. This is particularly stressful if the thawing is rapid, as the cells have less time to adjust to changing osmotic concentrations than under slower thawing. In addition, if cells were in a hypoxic state due to encasement in ice, thawing causes a reintroduction of oxygen to cells that can cause oxidative stress via formation of reactive oxygen species (ROS). After thawing, many organisms require some time before they can fully restore homeostasis and normal function. This suggests that many freeze-tolerant organisms must expend energy (potentially contributing to additional energy stress) during a recovery period, likely to repair damage that occurred during cooling, freezing, the frozen state, or thawing.

Strategies and mechanisms of cold tolerance

As discussed above, freezing and thawing involve exposure to multiple stressors, including low temperature stress, mechanical stress (due to ice), osmotic stress (as ice forms and melts), hypoxia (while frozen), energy stress (while cold and frozen), and oxidative stress (during thawing). To survive temperatures below zero, organisms usually use one of two strategies: freeze avoidance or freeze tolerance.

Freeze-avoidant organisms (also called freeze-intolerant) avoid freezing, usually by accumulating molecules that decrease their supercooling point. Thus, freeze-avoidant organisms can supercool to (often very) low temperatures, and must survive the stresses associated with low temperatures but not ice, freezing, or thawing. To supercool effectively, freeze-avoidant organisms accumulate compatible osmolytes that decrease the melting point of their fluids. These compatible osmolytes are also often cryoprotectant molecules (e.g., glycerol, proline, trehalose) that can help protect cellular components from the stress associated with low temperatures. In addition, many freeze-avoidant organisms produce antifreeze proteins – proteins that actually inhibit the growth of ice crystals.

Freeze-tolerant organisms survive internal ice formation, and must use various mechanisms to combat all of the stresses listed above. Briefly, these mechanisms can be organized into five categories: controlling ice formation and propagation, decreasing ice content, stabilizing cells and macromolecules, altering or decreasing metabolism, and coordinating repair and recovery post-thaw. Not all freeze-tolerant organisms use all of these mechanisms, but it is hypothesized that freeze-tolerant organisms likely need to use multiple mechanisms to survive the wide range of challenges associated with cooling, freezing, and thawing. The rest of this chapter will focus on the stresses and mechanisms associated with freeze tolerance.