Double staining of mitochondria and mitochondrial nucleoids in the living yeast during the life cycle.

Abstract

Mitochondria and mitochondrial nucleoids (mt-nucleoids) of the yeast, Saccharomyces cerevisiae were vitally double-stained with two fluorochromes, 3, 3'-dihexyloxacarbocyanine iodide [ Di0C6(3) ] and 4', 6-diamidino-2-phenylindole (DAPI), respectively in different types of cells during the life cycle. Budded cells at logarithmic phase had a single, branched, giant mitochondrion, a part of which migrated into the daughter cell prior to nuclear division. Mt-nucleoids appeared as a string-of-beads within a giant mitochondrion. Single cells at stationary phase had numerous fragmented mitochondria with dot-like mt-nucleoids. When stationaryphase cells were transferred to the sporulation medium, fragmented mitochondria fused with each other and a network of mitochondrion changed its configuration as the meiotic division proceeded. Finally, mitochondria formed four rings which enclosed each daughter nucleus in premature spores. Mitochondria were hardly stained with Di0C6 (3) in mature spores. Fused mitochondria were clearly observed again in cells at germination and outgrowth. When cells of opposite mating type were mixed in mating medium, zygote formation and first bud emergence occurred in 4 h. Just after mating, string-like and fragmented mitochondria transmitted from each parent cell were separately visible on both sides of a dumbbell-shaped zygote. In contrast, extensive fusion of mitochondria occurred in zygotes with a first bud.

Morphology of mitochondrial nucleoids, mitochondria, and nuclei during meiosis and sporulation of the yeast Saccharomycodes ludwigii

“…2G, arrow). In contrast to the strings-of-beads appearance of mt-nucleoids in sporulating cells of S. cerevisiae (Miyakawa et al, , 1994, mt-nucleoids in S. ludwigii constantly appeared as discrete dots throughout the sporulation culture.…”

“…The contour of tubular mitochondria that assembled around nuclei became obscure in S. ludwigii at the stage of fi rst and second meiotic divisions (Fig. 3DE), whereas tubular mitochondria were clearly visible throughout meiotic division of S. cerevisiae (Miyakawa et al, , 1994. As tubular mitochondria were clearly visible in cells that Fig.…”

“…Mitochondria move to be located close to the dividing nuclei, and fi nally each of four circular mitochondria is distributed into four spores (Gorsich and Shaw, 2004;Miyakawa et al, 1984Miyakawa et al, , 1994. In S. ludwigii, fusion of mitochondria is also facilitated during meiotic prophase to form long tubular mitochondria that surround the periphery of cells, as well as mitochondria of S. cerevisiae.…”

“…After completion of the second meiotic division, almost all mitochondria were incorporated inside the spore wall. The presence of circular mitochondria that encompassed the nucleus, which were seen just before spore formation of S. cerevisiae, was not distinct in S. ludwigii (Miyakawa et al, , 1994 Fig. 3F).…”

“…Stock solution of DiOC 6 (3) (10 μg/ml) was added at a fi nal concentration of 1 μg/ml and incubated for 5 min at 30 C. Mitochondria were observed under B excitation. Double staining with DAPI and DiOC 6 (3) was carried out as follows: To sporulating cells suspended in NS buffer were added DiOC 6 (3) and DAPI, each at a fi nal concentration of 1 μg/ml, which were left for 5 min at 30 C. Mitochondrial nucleoids and nuclei were observed under UV excitation and mitochondria were observed under B excitation (Miyakawa and Yanagamizu, 1998;Miyakawa et al, 1994).…”