Isolation of mitochondria with cubic membrane morphology reveals specific ionic requirements for the preservation of membrane structure.

Biological membranes with cubic symmetry are a hallmark of virus-infected or diseased cells. The mechanisms of formation and specific cellular functions of cubic membranes, however, are unclear. The best-documented cubic membrane formation occurs in the free-living giant amoeba Chaos carolinense. In that system, mitochondrial inner membranes undergo a reversible structural change from tubular to cubic membrane organization upon starvation of the organism. As a prerequisite to further analyze the structural and functional features of cubic membranes, we adapted protocols for the isolation of mitochondria from starved amoeba and have identified buffer conditions that preserve cubic membrane morphology in vitro. The requirement for high concentration of ion-chelating agents in the isolation media supports the importance of a balanced ion milieu in establishing and maintaining cubic membranes in vivo.