DiOC₆ staining reveals organelle structure and dynamics in living yeast cells

Abstract

When present at low concentrations, the fluorescent lipophilic dye, DiOC6, stains mitochondria in living yeast cells [Pringle et al.: Methods in Cell Biol. 31:357-435, 1989; Weisman et al.: Proc. Natl. Acad. Sci. U.S.A. 87:1076-1080, 1990]. However, we found that the nuclear envelope and endoplasmic reticulum were specifically stained if the dye concentration was increased or if certain respiratory-deficient yeast strains were examined. The quality of nuclear envelope staining with DiOC6 was sufficiently sensitive to reveal alterations in the nuclear envelope known as karmellae. These membranes were previously apparent only by electron microscopy. At the high dye concentrations required to stain the nuclear envelope, wild-type cells could no longer grow on non-fermentable carbon sources. In spite of this effect on mitochondrial function, the presence of high dye concentration did not adversely affect cell viability or general growth characteristics when strains were grown under standard conditions on glucose. Consequently, time-lapse confocal microscopy was used to examine organelle dynamics in living yeast cells stained with DiOC6. These in vivo observations correlated very well with previous electron microscopic studies, including analyses of mitochondria, karmellae, and mitosis. For example, cycles of mitochondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time-lapse studies of living DiOC6-stained cells. This technique also revealed new aspects of nuclear disposition and interactions with other organelles. For example, the nucleus and vacuole appeared to form a structurally coupled unit that could undergo coordinated movements. Furthermore, unlike the general view that nuclear movements occur only in association with division, the nucleus/vacuole underwent dramatic migrations around the cell periphery as cells exited from stationary phase. In addition to the large migrations or rotations of the nucleus/vacuole, DiOC6 staining also revealed more subtle dynamics, including the forces of the spindle on the nuclear envelope during mitosis. This technique should have broad application in analyses of yeast cell structure and function.

Mitochondrial Protein Sorting

“…Aliquots of each strain were taken and analyzed for mitochondrial morphology. Either yeast strains had been transformed with an expression vector encoding a mitochondria-targeted version of the enhanced green fluorescent protein b 2 ⌬-EGFP (34) and selected on minimal medium (60) or mitochondria were stained with 150 nM 3,3Јdihexyloxacarbocyanine iodide (DiOC 6 ; Molecular Probes) for 15 min in growth medium (61,62). Cells were centrifuged at 400 ϫ g for 5 min, washed, resuspended in 10 mM HEPES, pH 7.4, and directly mounted in 0.5% (w/v) low melting agarose (FMC Bioproducts) on a microscope slide.…”

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

“…DiOC 6 staining was performed as described previously (Koning et al, 1993). Cells in log phase growth were stained with 10 g/ml DiOC 6 (Kodak, Rochester, NY) per 10 7 cells using a 1 mg/ml ethanolic stock.…”

Proliferation of the endoplasmic reticulum occurs normally in cells that lack a functional unfolded protein response

“…(Koning et al, 1993(Koning et al, , 1996Profant et al, 2000;Wright et al, 1988) The lack of a growth assay has slowed searches for genes involved in karmellae assembly, since the only way to detect their presence is by direct microscopic examination. The discovery of a strain background (ire1) in which the presence of karmellae conferred a death phenotype was extremely exciting to us (Cox et al, 1997).…”

“…If this were true, the cells we saw growing in our serial dilution assays may not actually be producing karmellae. To determine whether ire1 mutants were capable of generating normal levels of karmellae, ire1D:: KanMX6 cells from the different genetic backgrounds were stained with DiOC 6 and the percentage of karmellae in the population was determined by fluorescence microscopy (Koning et al, 1993). In the JRY527 wild-type background, karmellae are present in about 30-60% of the cells, depending on the promoter used to express Hmg1p.…”

“…Both strains were grown at 30uC for 14 h before samples were removed to score karmellae assembly. The vital dye, 3,3k-dihexyloxacarbocyanine iodide (DiOC 6 ), was used to visualize karmellae in living cells (Koning et al, 1993). The person who scored the samples did so in a blind manner, without knowledge of the identity of the culture.…”