Meeting Abstract

P2.13  Saturday, Jan. 5  Ice Nucleation Protein Reduces Cryogenic Injury in Eukaryotic Cells SAMARAJEEWA, DA*; HARDER, A; TONER, M; CHAKRABORTY, N; MENZE, MA; Eastern Illinois University; Harvard University; University of Michigan at Dearborn mmenze@eiu.edu

The bacterium Pseudomonas syringae synthesizes under certain conditions an extracellular ice nucleating protein (INP) protein (Swiss-Prot: O30611) capable of promoting ice-formation of supercooled water. INPs serve as organization platforms in the orderly formation of water molecules during freezing. Intracellular ice formation at supercooled conditions (below the freezing temperature of ice) is considered to be lethal in eukaryotic cells. However, we hypothesized that slow and regulated intracellular ice formation at higher temperatures (without significant supercooling) will be less injurious to the cells. We transgenically expressed part of the highly repetitive central domain of the protein O30611, composed of 530 amino acids (PsINP), in Escherichia coli and purified the protein by affinity chromatography. Addition of purified PsINP to buffer solutions at 0.075 mg/ml substantially raised the ice nucleation temperature to 4.1 ºC. PsINP was then expressed in cells derived from Spodoptera frugiperda (Sf-21) or hepatocellular carcinoma (HepG2). After freezing at 1 ºC min^-1, PsINP expressing Sf-21 cells showed an increase in membrane integrity compared to control cells (60.1 ± 3.3% control vs. 71.6 ± 3.4% Sf-21-PsINP; n = 6; ±SE). Standard cryomicroscopy demonstrated that HepG2 cells expressing a green fluorescent protein labeled variant of PsINP (HepG2-PsINP-GFP) showed intracellular ice-formation at higher temperature than control cells (ΔT = 17.63 ± 1.16 ºC; n = 3; ±SE), and maintained membrane integrity following freezing and thawing. Our results suggest that induction of orderly intracellular ice formation can reduce cell injury during freezing.