Intercellular ice propagation: Experimental evidence for ice growth through membrane pores

ropagation of intracellular ice between cells significantly increases the p revalence of intracellular ice in confluent monolayers and tissues. It has been proposed that gap junctions facilitate ice propagation between cells. This study develops an equation for capillary freezing-point depression to determine the effect of temperature on the equilibrium radius of an ice cry stal sufficiently small to grow through gap junctions. Convection cryomicro scopy and video image analysis were used to examine the incidence and patte rn of intracellular ice formation (IIF) in the confluent monolayers of cell lines that do (MDCK) and do not (V-79W) form gap junctions. The effect of gap junctions on intracellular ice propagation was strongly temperature-dep endent. For cells with gap junctions, IIF occurred in a directed wave-like pattern in 100% of the cells below -3 degreesC. At temperatures above -3 de greesC, there was a marked drop in the incidence of IIF, with isolated indi vidual cells initially freezing randomly throughout the sample. This random pattern of IIF was also observed in the V-79W monolayers; and in MDCK mono layers treated to prevent gap junction formation. The significant change in the low temperature behavior of confluent MDCK monolayers at -3 degreesC i s likely the result of the inhibition of gap junction-facilitated ice propa gation, and supports the theory that gap junctions facilitate ice nucleatio n between cells.