Key words: in vivo hydrolysis/[alpha]-glucosidase/capillary electrophoresis/laser induced fluorescence/spheroplastsingle cell analysis
In 1953, Edstrom used fine silk fibers of 15 µm diameter and 1-2 cm length for the electrophoretic determination of a hundred picograms of RNA contained within a single cell (Edstrom, 1953). Since then, the study of the chemical contents of individual biological cells has been of interest. This arises from both fundamental interest in cell heterogeneity and in potential applications of single cell assays to clinical diagnosis and pharmaceutical research. There has been much activity in the analysis of neurotransmitters contained within individual neurons because of interest in neurochemistry and also the availability of sensitive electrochemical detectors for neuroactive amines (Kennedy et al., 1989; Ewing et al., 1992). Much of this work has relied on the use of large (200 µm) ganglia from snails. There are a number of other single cell analytical techniques. Neher and coworkers have used microinjection to load a cell with a calcium-sensitive fluorescence dye and subsequently used fluorescence microscopy to measure the calcium concentration in single rat peritoneal mast cells (Almers and Neher, 1985). They further developed patch-clamping techniques, which can provide information, such as ion channels and signaling networks, within individual cells (Neher and Sakmann, 1992). Flow cytometry also provides a rapid method for counting cells and sorting normal and abnormal cell populations (Steinkamp, 1984). Yeung and colleagues (Yeung, 1994; Rosenzweig and Yeung, 1994; Xue and Yeung, 1994) have assayed lactate dehydrogenase and glucose-6-phosphate dehydrogenase in single human erythrocytes using capillary electrophoresis with laser-induced fluorescence detection (CE/LIF), demonstrating the utility of CE/LIF for single cell analysis. More recently Zare and coworkers (Chiu et al., 1998) have combined CE/LIF with optical trapping to study single secretory vesicles from the atrial gland of the gastopod mollusk Aplysia californica. They identified taurine, a possible neuromodulator or hormone, as one of the most abundant molecules present in atrial gland vesicles. Capillary electrophoresis techniques for the analysis of single cells have been recently reviewed (Lillard and Yeung, 1997; Swanek et al., 1997).
Glycosyltransferases and glycosidases are the enzymes responsible for the formation and hydrolysis of oligosaccharides, respectively. Assay of the activity of these enzymes is essential to understanding their roles in biology. There have been several reports on the detection of glycosidase activity in single cells using nonfluorescent substrates that are enzymatically hydrolyzed to yield the detectable fluorophores (Rotman, 1961; Yashphe and Halvorson, 1976; Luyten et al., 1985; Jain and Magrath, 1991). These assays require 4000 molecules of hydrolase, yielding ~1 fmol of product, which is detected within the cell (Jain and Magrath, 1991). Some of these assays further require that lipophilic substrates pass through a cell membrane. As a result, there is ambiguity in the assays because the fluorescence signal is related to both the uptake of the substrate by the cell and the enzyme activity within the cell.
Several reports have demonstrated the use of capillary electrophoresis separation and analysis of labeled oligosaccharides. Honda et al. (1989) derivatized monosaccharides to N-2-pyridylglycamines and analyzed these derivatives using capillary electrophoresis with UV detection. They obtained a detection limit of 10 pmol. Novotny's group pioneered the use of CBQCA as a fluorogenic reagent to label oligosaccharides (Liu et al., 1991, 1992; Novotny and Sudor, 1993; Stefansson and Novotny, 1994). They achieved detection limits of 0.5 amol (300,000 molecules) of labeled monosaccharides using on-column laser induced fluorescence detection. It is not clear if these derivatized monosaccharides would act as substrates for enzymes. Lee et al. (1992) developed an electrophoresis-based assay for glycosyltransferases. Using capillary electrophoresis separation and laser induced fluorescence detection of sugar-fluorescent conjugates (7-amino-1,3-naphthalene-disulfonic acid), they obtained a detection limit of 80 fmol.
We have previously developed ultrasensitive assays for glyco-sidases and glycosyltransferases, using capillary electrophoresis separation with laser induced fluorescence detection (Zhao et al., 1994; Le et al., 1995; Zhang et al., 1995). The CE/LIF technique can resolve isomeric oligosaccharides at a detection limit of8 × 10-23 mol (or 50 molecules) of tetramethylrhodamine (TMR) labeled saccharides. The conversion of a fluorescent substrate to more than one product can also be monitored. Our techniques are several orders of magnitude more sensitive than the most sensitive assay previously reported for these enzymes (Lee et al., 1992). The limiting enzyme activity by the action of either competing or sequential enzymes can be measured, because of the efficient separation and sensitive detection of the fluorescent substrate and products. We report here the utilization of CE/LIF to monitor [alpha]-glucosidase activity in single yeast cells employing a synthetic triglucoside substrate.
Figure
Figure 1. Photographs obtained from confocal laser scanning microscopy. Yeast cells were incubated with 50 µM of the tetramethylrhodamine (TMR) labeled triglucoside, [alpha]-d-Glc(1->2)[alpha]-d-Glc(1->3)[alpha]-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR, at 25°C for 5 min, 1 h, and 24 h at different magnifications. The scale bars represent 10 and 2 µm. The intensity of red color corresponds to the intensity of fluorescence from TMR ([lambda]ex = 568 nm and [lambda]em = 590 nm).
If the adherence of the dye to the cell surface contributes substantially to the fluorescence of the cell, an incubation of the cells with the dye for as short as several min would result in cell fluorescence. This is clearly not the case, since cells incubated for 5 min with the TMR labeled trisaccharide shows no detectable fluorescence. It is expected that adsorption of the dye by the cell surface would be much faster than the uptake process. Thus, the fluorescence observed in the yeast cells following 1 h and 24 h incubation is primarily due to the uptake of the TMR labeled triglucoside by the cells and not due to the adsorption of the dye by the cell surface.
A detailed examination of a single yeast cell shows that the fluorescence is localized throughout the cell (Figure
Confocal laser scanning microscopy can provide information on the uptake of fluorescent compounds. Since this technique does not identify fluorescent species, CE/LIF analysis was employed to characterize the fluorescent substrate and any conversion products from enzymatic transformation within the cells. Hydrolysis of [alpha]-d-Glc(1->2)[alpha]-d-Glc(1->3)[alpha]-d-Glc-TMR by [alpha]-glucosidase I gives TMR-disaccharide, [alpha]-d-Glc(1->3)[alpha]-d-Glc-TMR. The disaccharide can either be sequentially hydrolyzed to monosaccharide [alpha]-d-Glc-TMR and the linking arm by [alpha]-glucosidase II and/or other [alpha]-glucosidases or converted directly to the linking arm by endo-glucosidases. Figure
Figure 2. Electropherograms obtained from CE/LIF analysis of:(a) standards containing 10-9 M of each of the TMR derivatives: [alpha]-d-Glc(1->2)[alpha]-d-Glc(1->3)[alpha]-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (T); [alpha]-d-Glc(1->3)[alpha]-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (D); [alpha]-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (M); and the TMR-linker arm [EEgr]O(CH2)8CONHCH2CH2NHCO-TMR (L). Approximately 6 pl of the solution was injected for the analysis. (b) supernatant from the incubation media. (c) final cell wash solution. A 50 cm capillary (10 µm i.d.) was used for electrophoretic separation under 20,000 V. The electrophoresis buffer contained 10 mM each of phosphate, tetraborate, phenylboronic acid, and sodium dodecyl sulfate (SDS).
Figure
Figure
Figure 3. Intracellular inhibition of yeast [alpha]-glucosidase I by castanospermine. (a) Hydrolysis of 50 µM TMR-trisaccharide. (b) Hydrolysis of TMR-trisaccharide in the presence of 12 µM inhibitor; (c) Hydrolysis of TMR-trisaccharide in the presence of 60 µM inhibitor. A 40 cm capillary was used for electrophoretic separation under 16,000 V. The electrophoresis buffer contained 10 mM each of phosphate, tetraborate, phenylboronic acid, and SDS.
To confirm that hydrolysis of TMR-trisaccharide to the linker arm occurred in a stepwise fashion with initial conversion to TMR-disaccharide, the cells were incubated with 50 µM trisaccharide substrate and 12 µM castanospermine. Castanospermine is a competitive inhibitor of purified yeast [alpha]-gluco-sidase I with a Ki of 12 µM. The production of the linker arm was reduced approximately 6-fold as a result of the inhibition of [alpha]-glucosidase I (Figure
Analysis of the contents of individual spheroplasts introduced into a capillary and monitored by CE/LIF is shown in Figure
Figure 4. Electropherograms of the contents of three individual yeast spheroplasts separately introduced in a capillary. A 40-50 cm capillary (10 µm i.d.) was used for electrophoretic separation under 20,000 V. The electrophoresis buffer contained 10 mM each of phosphate, tetraborate, phenylboronic acid, and SDS. Each spheroplast was injected into the buffer filled capillary. The spheroplast was lysed inside the capillary by the nonphysiological buffer and SDS and the lysate from the single spheroplast was analyzed with CE/LIF. For clarity, the electropherograms have been manually shifted.
Figure 5. A schematic diagram showing the introduction of a single cell into a capillary for subsequent analysis by capillary electrophoresis with laser induced fluorescence detection.
Additional CE/LIF analysis of other individual spheroplasts from the same cell suspension also showed differences in fluorescence intensity and in the levels of substrate and intermediate products within different cells. These differences probably reflect the heterogeneous nature of the cellular population, such as different stages of maturity.
The present methodology can be expended to assay virtually any class of enzymes for which a specific fluorescent substrate can be synthesized. The assay of enzyme activity on a cell-by-cell basis allows the use of the distribution of activity in the cellular population as a diagnostic and prognostic indicator of cancer and other diseases. The assay may also be used in the pharmaceutical industry as a tool in the development and in vivo evaluation of novel enzyme inhibitors.
TMR-hydroxysuccinimide ester was obtained from Molecular Probes (Eugene, OR). Fluorescently labeled oligosaccharides were prepared as described previously (Zhang et al., 1995; Scaman et al., 1996). Stock solutions including 0.1 M Na2HPO4 (Fisher), 0.1 M tetraborate (Fisher), 0.1 M sodium dodecyl sulfate (BDH), and 0.1 M phenylboronic acid (Sigma) were prepared in deionized water (Barnstead NANO pure system) and filtered with 0.2 µm pore size disposable filter (Nalgene). The electrophoresis running buffer was prepared by mixing these stock solutions to final concentrations of 10 mM Na2HPO4, 10 mM tetraborate, 10 mM sodium dodecyl sulfate, and 10 mM phenylboronic acid (pH 9.3). Unless otherwise indicated, all reagents were of analytical reagent grade. Incubation of yeast cells with trisaccharide, [alpha]-d-Glc(1->2)[alpha]-d-Glc(1->3)[alpha]-d-Glc-O(CH2)8CO-NHCH2CH2NHCO-TMR
Saccharomyces cerevisiae (baker's yeast, Fleischman) was grown on Sabouraud dextrose agar plates (Difco) at 37°C, and then stored at 4°C. A typical colony was inoculated into 1 ml sterile Sabouraud dextrose media and grown over night at 25°C with shaking. A 200 µl aliquot was transferred to a sterile micro-centrifuge tube and pelleted by centrifugation at 14,000 r.p.m. for 2 min. Old media was removed and fresh media (1 ml) was added to the pelleted cells along with sterile filtered TMR-trisaccharide, [alpha]-d-Glc(1->2)[alpha]-d-Glc(1->3)[alpha]-d-Glc-O(CH2)8CO-NHCH2CH2NHCO-TMR, from a 5 mM stock solution. The final concentration of the labeled trisaccharide was 50 µM. The cell suspension was incubated at 25°C with shaking. At incubation intervals of 5 min and 1 h, 2, 3, 4, 5, and 24 h, 100 µl of the cell suspension sample was withdrawn and washed thoroughly with phosphate-buffered saline (PBS). The cells were then subject to confocal laser scanning microscopy analysis to study the uptake of the TMR labeled trisaccharide by the yeast cells. A parallel control containing the same amount of the yeast cells and media but without the TMR-triglucoside substrate was carried out under identical conditions. Generation of spheroplasts
Cells from parallel 1 ml incubations were transferred to the surface of a 0.45 µm 47 mm HVLP filter (Millipore) and washed under vacuum with PBS, pH 6.0 containing 2% sucrose. A final fraction of the filtrate was collected for analysis by capillary electrophoresis, ensuring that extracellular substrate was completely washed out. Cells were then washed from the membrane into a test tube with the same buffer, pelleted, and washed with 200 µl of 25 mM Tris-HCl, pH 7.5, and 2 M sorbitol. Spheroplasts were generated from the cells by incubating in 25 mM Tris-HCl, pH 7.5, and 2 M sorbitol containing 770 U/100 µl lyticase (Arthrobacter luteus, Sigma) for 2 h at 25°C. Confocal laser scanning microscopy
A 20 µl aliquot of each cell sample was examined by a model 2001 confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA). An argon/krypton gas laser was used as the excitation source at 568 nm selected for TMR. The fluorescence was collected using a 100× objective with oil immersion. The fluorescent intensity of TMR was measured at 590 nm. Data were digitized using the Image Space 3.1 software of the model 2001 confocal microscope. Inhibition of substrate hydrolysis
Castanospermine, a competitive inhibitor of [alpha]-glucosidase I, was used to inhibit the hydrolysis of the trisaccharide substrate. S.cerevisiae was grown on Sabouraud dextrose broth at 25°C with shaking for 72 h in an Erlenmeyer flask. The cell density of the culture was measured at between 32.0 and 37.0 OD600 after 72 h incubation. A 2 ml aliquot of the 72 h culture was then transferred into a 10 ml glass tube. The cells were spun and media replaced with 1 ml of fresh solution containing 12 or 60 µM castanospermine (Boehringer Mannheim). Following 24 h of exposure to castanospermine, media was again replaced, and the same concentration of the inhibitor as well as 50 µM trisaccharide substrate was added. The cells were grown for another 24 h prior to processing and sampling. Control experiments were carried out in parallel with only the addition of trisaccharide substrate. The cells were washed thoroughly and subjected to a 17 h lyticase treatment as described above to facilitate cell lysis. The lysate was filtered through a 0.45 µm PVDF filter (Millipore) to remove cell debris. The filter membrane was then washed with 2 ml of methanol to remove bound dye-labeled substrate or product. The methanol filtrate and the filtrate from the lysate were combined for lyophilization. The dried pellet was resuspended in electrophoresis buffer as described above. Capillary electrophoresis laser induced fluorescence (CE/LIF)
All CE/LIF analyses reported in this study were carried out by using a locally constructed, instrument as described previously (Le et al., 1995). Briefly, the electrophoresis was driven by a CZE1000R high voltage power supply (Spellman, Plainview, NY). Separation was carried out in a 40-50 cm long, 10 µm or 30 µm inner diameter fused silica capillary (Polymicro, Phoenix, AZ) at an electric field of 400-500 V/cm. The aqueous electrophoresis buffer contained 10 mM each of phosphate, tetraborate, phenylboronic acid, and sodium dodecyl sulfate (SDS), at pH 9.3. The sheath fluid was identical to the running buffer and was gravity fed from a 250 ml wash bottle. A 1.0 mW helium-neon laser (Melles Griot, Nepean, Canada) beam, [lambda] = 543.5 nm was focused into a post-column sheath flow cuvette. Fluorescence was collected at a right angle with a high numerical aperture (0.7 N.A.) microscope objective (60×) (Universe Kogaku model 60X-LWD, Oyster Bay, NY), spectrally filtered with a bandpass filter (580DF40) (Omega Optical, Brattleboro, VT), imaged onto one end of a SELFOC fiber collimator (p-type, NSG America, Somerset, NJ), and detected at the other end of the fiber collimator with a R1477 photomultiplier tube (Hamamatsu, Bridgewater, NJ). Data was digitized by a NB-MIO-16× data acquisition board (National Instruments, Austin, TX) in a Macintosh Quadra 650 computer. Single spheroplast introduction
Figure
We thank Dr. Rakesh Bhatnagar for technical assistance on confocal laser scanning microscopy. This work was supported by a Strategic Grant (STR 149003 to O.H., N.J.D. and M.M.P.) from the Natural Sciences and Engineering Research Council of Canada. O.H. gratefully acknowledges a Steacie Fellowship from NSERC. N.J.D. gratefully acknowledges a McCalla Professorship from the University of Alberta.
CBQCA, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; CE, capillary electrophoresis; CE/LIF, capillary electrophoresis with laser induced fluorescence detection; TMR, tetramethylrhodamine.
2To whom correspondence should be addressed.Introduction
Results and discussion
Materials and methods
Acknowledgments
Abbreviations
References
3Present address: Department of Food Science, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4
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