Microtubule-associated movement of mitochondria and small particles in Acanthamoeba castellanii

Using video-enhanced differential interference microscopy and digital image processing, we have observed organelle motility in Acanthamoeba castellanii. In amoebae taken from cultures in rapid growth phase, mitochondria and small particles moved over distances of several microns and at an average velocity of approximately 2 microns/s. Mitochondrial motility was verified by intensified fluorescence microscopy of cells that were labeled in vivo with the DNA-binding dye DAPI or the mitochondria-specific dye MitoTracker. We further studied the role of microtubules (MTs) in the translocation of cell organelles. Double-labeling of fixed cells with mitochondrial markers (anti-F1 beta antibody, MitoTracker) and cytoskeletal markers (anti-tubulin antibody, rhodamine-phalloidin) demonstrate that the mitochondria colocalize with MTs in the subcortical cell area and are excluded from the F-actin-rich cell cortex. Colchicine treatment resulted in an almost complete depolymerization of MTs and an inhibition of organelle motility. Moreover, we have directly visualized MTs in vivo in flattened amoebae. Mitochondria and small particles moved along the MTs in a bidirectional mode at an average velocity of approximately 1 micron/s. We conclude that the observed movement of mitochondria and small particles in Acanthamoeba castellanii mainly occurs via microtubules and associated motor proteins.