Atomic force microscopy of isolated mitochondria.

This chapter describes methods for isolating and imaging metabolically and toxicologically challenged mitochondria with atomic force microscopy. Mitochondria were isolated from rat dorsal root ganglia or brain and exposed to glucose or dinitrobenzene (DNB) to simulate the cellular environment of a diabetic animal that has been exposed to excess glucose or to DNB. It is one of only a few articles to present images of membrane structures, such as voltage-dependent, anion-selective channel pores, on intact organelles. The purpose of the chapter is not to report on the metabolic or toxic effects, but to communicate in more detail than a typical journal paper allows the methods used to image isolated organelles. We also provide a series images revealing the outer membrane and outer membrane pores. An image of an isolated nucleus as well as a set of notes written to avoid common pitfalls in isolation, labeling, and imaging is also included.

Dehydration-induced expression of a 31-kDa dehydrin in Polypodium polypodioides (Polypodiaceae) may enable large, reversible deformation of cell walls.

Current and predicted climate changes caused by global warming compel greater understanding of the molecular mechanisms that plants use to survive drought. The desiccation-tolerant fern Polypodium polypodioides exhibits extensive cell wall folding when dried to less than 15% relative water content (RWC) and rapidly (within 24 h) rehydrates when exposed to water and high humidity. A 31-kDa putative dehydrin polypeptide expressed in partially and fully dry tissues detected via western blotting was present only during drying and rapidly dissipated (within 24 h) upon tissue rehydration. Immunostaining indicates the presence of dehydrin near the cell wall of partially and fully dried tissues. Atomic force microscopy of tracheal scalariform perforations indicates that dry vascular tissue does not undergo significant strain. Additionally, environmental scanning electron microscopy reveals differential hydrophilicity between the abaxial and adaxial leaf surfaces as well as large, reversible deformation. The ability to avoid cell wall damage in some desiccation-tolerant species may be partially attributed to cell wall localization of dehydrins enabling reversible, large cell-wall deformation. Thus, the de novo synthesis of dehydrin proteins and potential localization to the cell walls of these desiccation-tolerant species may play a role in avoiding mechanical failure during drought.