DIOC6 STAINING REVEALS ORGANELLE STRUCTURE AND DYNAMICS IN LIVING YEAST-CELLS

When present at low concentrations, the fluorescent lipophilic dye, Di OC6, Stains mitochondria in living yeast cells [Pringle et al.: Method s in Cell Biol. 31:357-435, 1989; Weisman et al.: Proc. Natl. Acad. Sc i. U.S.A. 87:1076-1080, 1990]. However, we found that the nuclear enve lope and endoplasmic reticulum were specifically stained if the dye co ncentration was increased or if certain respiratory-deficient yeast st rains were examined. The quality of nuclear envelope staining with DiO C6 was sufficiently sensitive to reveal alterations in the nuclear env elope known as karmellae. These membranes were previously apparent onl y by electron microscopy. At the high dye concentrations required to s tain 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 aff ect 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 y east cells stained with DiOC6. These in vivo observations correlated v ery well with previous electron microscopic studies, including analyse s of mitochondria, karmellae, and mitosis. For example, cycles of mito chondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time-l apse studies of living DiOC6-stained cells. This technique also reveal ed new aspects of nuclear disposition and interactions with other orga nelles. For example, the nucleus and vacuole appeared to form a struct urally coupled unit that could undergo coordinated movements. Furtherm ore, unlike the general view that nuclear movements occur only in asso ciation with division, the nucleus/vacuole underwent dramatic migratio ns 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 forc es of the spindle on the nuclear envelope during mitosis. This techniq ue should have broad application in analyses of yeast cell structure a nd function.