Protein Ice Nucleators (PINs)

Winter survival for many ectotherms depends on the regulation of ice formation; protein ice nucleators are one adaptation that aid in this process. Ice nucleation occurs when specific proteins catalyze the formation of ice at relatively high temperatures in order to protect the organism from damage by intracellular ice formation through extensive supercooling (Figure 2). This is advantageous because it gives the insect time to make metabolic adjustments and it reduces osmotic shock because the equilibrium content of ice will be low (Storey, 1996). Insects often initiate freezing in autumn or early winter at temperatures around -5 to -10˚C. However, there are variations among freeze-tolerant insects. For example, Larvae of Xylophagus species freeze above −10◦C and are able to survive below −40◦C. Also some species lack protein ice nucleators all together and are able to supercool to very low temperatures and still survive freezing (Strathdee, 1998).

Permission from Richard Lee.

The first step in ice nucleation is the aggregation of water molecules to form an embryo. When this cluster reaches critical size, it no longer disperses but grows; a nucleus is formed on which ice crystal can grow. These nuclei can be created by two mechanisms: homogeneous and heterogeneous nucleation. Homogeneous nucleation involves the spontaneous aggregation of water molecules; in this case the probability of reaching critical size increases with decreasing temperature. Heterogeneous nucleation occurs when some other entity, not water, acts as the template for ice formation.  These entities are ice-nucleating agents (INA) that encourage the clustering of water molecules and increase the probability of embryos reaching critical size (Lee, 1998). Once the ice matrix begins to form, an osmotic gradient is established between the intracellular and extracellular space. This causes the dehydration of cells because of the high concentration of solutes in the extracellular space; this in turn lowers the freezing and supercooling point of the intracellular water. Protein ice nucleators act as antifreezes for intracellular water and isolate the formation of ice to the extracellular space. This is very important for insects because intracellular ice formation is often lethal (Duman, 2001).  

There are different classes of ice nucleating proteins, the most well known class includes proteins and lipoproteins found in freeze-tolerant insects.  It has also been suggested that there is a mutualistic relationship between insects and ice-nucleating microorganisms. Certain bacteria have been isolated from the gut of insects with ice-nucleating proteins similar to insects. Researchers suggest that often times proteins found in the cell walls’ of microorganisms function as the nuclei for the initiation of crystal growth. Sometimes these bacteria are essential to the insect’s survival. The temperature at which a specific ice nucleator initiates freezing varies from molecule to molecule. Insects have a wide range of ice nucleators, but only the ice nucleating agent that initiates freezing at the highest temperature catalyzes the ice nucleation event. Because once freezing begins ice continues to grow throughout the insect’s body (Lee, 1998).