3-3--dihexyl-2-2--oxacarbocyanine and 2-(4-(dimethylamino)styryl)-1-methylpyridinium

This chapter describes labeling methods and optical approaches for live-cell imaging of the cytoskeleton and of a specific organelle-cytoskeleton interaction in budding yeast.

Topics: Actins; Carbocyanines; Coloring Agents; Cytoskeleton; Fluorescent Antibody Technique; Fluorescent Dyes; Green Fluorescent Proteins; Indoles; Membrane Glycoproteins; Microfilament Proteins; Microtubules; Mitochondria; Pyridinium Compounds; Rhodamine 123; Saccharomyces cerevisiae; Staining and Labeling; Tropomyosin

Previous studies have reported that mitochondrial morphology and volume in yeast cells are linked to cellular respiratory capacity. These studies revealed that mitochondrial morphology in glucose-repressed or anaerobically grown cells, which lack or have reduced levels of respiration, is different from that in fully respiring cells. Although both oxygen deprivation and glucose repression decrease the levels of respiratory chain proteins, they decrease the expression of many non-mitochondrial proteins as well, making it difficult to determine whether it is a defect in respiration or something else that effects mitochondrial morphology. To determine whether mitochondrial morphology is dependent on respiration per se, we used a strain with a null mutation in PET100, a nuclear gene that is specifically required for the assembly of cytochrome c oxidase. Although this strain lacks respiration, the mitochondrial morphology and volumes are both comparable to those found in its respiration-proficient parent. These findings indicate that respiration is not involved in the establishment or maintenance of yeast mitochondrial morphology, and that the previously observed effects of oxygen availability and glucose repression on mitochondrial morphology are not exerted through the respiratory chain. By applying the principle of symmorphosis to these findings, we conclude that the shape and size of the mitochondrial reticulum found in respiring yeast cells is maintained for reasons other than respiration.

Topics: Carbocyanines; Electron Transport Complex IV; Fluorescent Dyes; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Oxygen Consumption; Phenotype; Pyridinium Compounds; Saccharomyces cerevisiae; Spectrophotometry