Visualization and Quantification of Mitochondrial Dynamics in Living Animal Cells

This chapter focuses on the methods to visualize mitochondria and quantify their dynamic properties in living mammalian cells. The methods described to visualize mitochondria and quantify mitochondrial dynamics in living animal cells will facilitate elucidation of the regulation and molecular mechanisms of these fundamental, largely unexplored areas. There are few cell-permeable fluorescent small molecule probes that can be used to visualize the cytoskeleton in living cells. However, although fluorescent taxol labels microtubules in living cells, it stabilizes microtubules and inhibits microtubule dynamics. Comparing cytoskeletal structure in transfected and non transfected controls by indirect immunofluorescence staining after fixation is necessary to exclude any artifacts due to overexpression. Mitochondria are also highly dynamic organelles; their dynamics have been subdivided into morphological dynamics and motility although several authors suggest that mitochondrial function, morphology, and motility are closely linked. Furthermore, the chapter discusses the methods to investigate the interaction of mitochondria with the cytoskeleton in living animal cells. A comprehensive quantitative description of mitochondrial motility needs to incorporate the relative frequency of directed movements—retrograde and anterograde—and pauses and also needs to describe the characteristics of movements and pauses such as velocity and duration.