Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells

Abstract

GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.

Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP)

“…This pattern of fluorescence is similar to that described in other cell types for mitochondria [7,8], though the density of these organelles is, as expected, much higher in hepatocytes than in other cells. The identification of the fluorescent organelles as mitochondria was confirmed by double labeling with rhodamine 123 (data not shown).…”

“…After incubation for 3 h at 37°C in an atmosphere of 5% CO2, 95% air, the cultures were rinsed twice with prewarmed, HEPES (10 mM) buffered, Williams' medium E. Complete Williams' medium E was renewed, and the cells incubated overnight. Following overnight culture, hepatocytes were washed as above and placed in fresh complete Williams' medium E. For the transient expression of GFP the cells were transfected with mtGFP expression plasmid described by Rizzuto et al [7] by the cationic-liposome (DOTAP) method, basically following the manufacturer's instructions. Briefly, DNA and the reagent were mixed in a ratio of 1:7 (w/w) and incubated for 15 min at room temperature.…”

“…lc). Unlike rhodamine, which undergoes rapid photobleaching, GFP fluorescence is very stable even under continuous illumination and allows a prolonged observation of the sample [7]. In addition, GFP fluorescence is unaffected by membrane potential, thus allowing the visualization of mitochondrial morphology also under conditions of reduced membrane potential.…”

“…(i) transfecting the cells with a chimeric plasmid encoding for mitochondrial targeted GFP (mtGFP) [7]; and (ii) staining with the cationic dye rhodamine 123 accumulated in the matrix driven by membrane potential [8].…”

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

“…The LTD-green fluorescent protein (GFP) fusion protein was transiently expressed in protoplasts 30 , and GFP fluorescence exclusively co-localized with chloroplastic chlorophyll (Fig. 2a).…”