Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product

Abstract

Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5-(and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail. The uptake rate of the prefluorochromes increased in direct proportion to the concentration and was not saturable, which suggests that transport occurs via a passive diffusion process. The permeability coefficient for cFDA was 1.3 ؋ 10 ؊8 m s ؊1 . Once inside the cell, the esters were hydrolyzed by intracellular esterases and their fluorescent products accumulated. FDA hydrolysis (at 40؇C) in cell extracts could be described by first-order reaction kinetics, and a rate constant (K) of 0.33 s ؊1 was calculated. Hydrolysis of cFDA (at 40؇C) in cell extracts was described by Michaelis-Menten kinetics with an apparent V max and K m of 12.3 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 and 0.29 mM, respectively. Accumulation of fluorescein was most likely limited by the esterase activity, since transport of FDA was faster than the hydrolysis rate. In contrast, accumulation of carboxyfluorescein was limited by the much slower transport of cFDA through the cell envelope. A simple mathematical model was developed to describe the fluorescence staining. The implications for optimal staining of yeast cells with FDA and cFDA are discussed.

Confocal Imaging with a Fluorescent Bile Acid Analogue Closely Mimicking Hepatic Taurocholate Disposition

“…It has previously been demonstrated that after passive diffusion into hepatocytes, the nonfluorescent CDFDA is rapidly hydrolyzed by intracellular esterases to the fluorescent dye CDF, which is subsequently actively transported into the canalicular compartments by Mrp2. 22,23 The overlay of confocal and transmission images of SCRH (Fig. 6) showed that CDF is indeed predominantly located in the canalicular networks after 15 min of incubation, confirming the functional activity of canalicular transport in SCRH.…”

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

“…For viability staining, cells were resuspended in phosphate-bu¡ered saline (PBS) containing 10 Wg ml 31 £uorescein diacetate (FDA) and 25 Wg ml 31 PI. FDA is taken up by live cells and converted to its £uorescent derivative £uorescein by cellular esterases [17]. PI is taken up by dead cells because of a loss of membrane impermeability [18].…”

Development of flow cytometry‐based algal bioassays for assessing toxicity of copper in natural waters

“…More recently, flow cytometry has been applied to physiological studies with algae, bacteria, and yeasts. Examples include cell viability analysis using fluorescein diacetate (FDA) to measure esterase activity [8] and membrane potential analysis using the cationic dye 3,3Ј-dihexyloxacarbocyanine (DiOC 6 [3]) [9,10]. Flow cytometric techniques have important advantages over conventional biochemical assays, because cell functions can be quickly determined at conditions close to the in vivo state [9].…”