DIOC6 STAINING REVEALS ORGANELLE STRUCTURE AND DYNAMICS IN LIVING YEAST-CELLS
DIOC6 STAINING REVEALS ORGANELLE STRUCTURE AND DYNAMICS IN LIVING YEAST-CELLS
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DOI：
10.1002/cm.970250202
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发表时间：
1993-01-01
影响因子：
--
通讯作者：
WRIGHT, R
中科院分区：
文献类型：
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作者：
KONING, AJ;LUM, PY;WRIGHT, R
当低浓度存在时，荧光亲脂性染料 DiOC6 会染色活酵母细胞中的线粒体 [Pringle 等人：细胞生物学方法。 31：357-435，1989；韦斯曼等人：Proc。国家。阿卡德。科学。美国 87：1076-1080，1990]。然而，我们发现，如果增加染料浓度或检查某些呼吸缺陷酵母菌株，核膜和内质网会被特异性染色。 DiOC6 核膜染色的质量足够灵敏，足以揭示核膜（称为卡梅拉）的变化。这些膜以前只能通过电子显微镜才能看到。在核膜染色所需的高染料浓度下，野生型细胞无法再在不可发酵的碳源上生长。尽管对线粒体功能有这种影响，但当菌株在标准条件下用葡萄糖生长时，高浓度染料的存在不会对细胞活力或一般生长特征产生不利影响。因此，使用延时共焦显微镜检查用 DiOC6 染色的活酵母细胞中的细胞器动态。这些体内观察结果与之前的电子显微镜研究非常相关，包括线粒体、卡米拉和有丝分裂的分析。例如，线粒体融合和分裂的周期，以及有丝分裂过程中发生的核形状和位置的变化，在活体 DiOC6 染色细胞的延时研究中很容易成像。这项技术还揭示了核配置以及与其他细胞器相互作用的新方面。例如，细胞核和液泡似乎形成了一个结构耦合的单元，可以进行协调运动。此外，与核运动仅与分裂相关的一般观点不同，当细胞退出静止期时，细胞核/液泡在细胞外围经历了剧烈的迁移。除了细胞核/液泡的大幅迁移或旋转之外，DiOC6 染色还揭示了更微妙的动态，包括有丝分裂期间纺锤体对核膜的作用力。该技术应该在酵母细胞结构和功能的分析中具有广泛的应用。
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.