TY - JOUR
AU - Palcic, Monica, M.
AB - Abstract Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, α-d-Glc(1→2)α-d-Glc(1→3)α-d-Glc-O(CH2)8CONHCH2-CH2NH-COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing α-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate. in vivo hydrolysis, α-glucosidase, capillary electrophoresis, laser induced fluorescence, spheroplast single cell analysis Introduction 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 a.l, 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 glycosidases 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 of 8 × 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 α-glucosidase activity in single yeast cells employing a synthetic triglucoside substrate. Results and discussion Figure 1 shows typical photographs from confocal laser scanning microscopy of yeast cells that were incubated with TMR-triglucoside, α-d-Glc(1→2)α-d-Glc(1→3)α-d-Glc-TMR, for 5 min, 1 h, and 24 h. By 1 h of incubation many cells are fluorescent and some show intense fluorescence. The difference in fluorescence intensity is attributed to heterogeneity of the cells; for instance, uptake may vary with the age and the viability of the cells, which are different in a cell population. Further incubation to 2, 3, 4, 5, and 24 h results in the increased uptake of the fluorescent substrate. Intense fluorescence from the cells after 24 h of incubation is shown in Figure 1. Control experiments where the cells were incubated without substrate did not show detectable fluorescence. 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 1, 24 h/2 µm), indicating that the TMR analogue can pass through the cell wall and membrane, and accumulate inside the cell. The resolution from the confocal scanning system does not provide details of organelle localization, therefore organelle-specific accumulation of the substrate could not be interpreted from the photographs. However, the design of the substrate described below eliminates a requirement for exact cellular localization in monitoring in vivo transformation since its hydrolysis is controlled by α-glucosidase I, found only in the endoplasmic reticulum. 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 α-d-Glc(1→2)α-d-Glc(1→3)α-d-Glc-TMR by α-glucosidase I gives TMR-disaccharide, α-d-Glc(1→3)α-d-Glc-TMR. The disaccharide can either be sequentially hydrolyzed to monosaccharide α-d-Glc-TMR and the linking arm by α-glucosidase II and/or other α-glucosidases or converted directly to the linking arm by endo-glucosidases. Figure 2a shows an electropherogram obtained from the analysis of a standard mixture of substrate and all potential hydrolytic products where TMR-trisaccharide (T) substrate is clearly well resolved from the expected products, TMR-disaccharide (D), TMR-monosaccharide (M), and the TMR-linker arm (L). The baseline separation is achieved by using micelles (SDS) as well as borate and phenylboronic acid in the running buffer solution. The use of borate and phenylboronic acid enhances resolution because their complexation with sugar hydroxyl groups results in electrophoretic differences between the saccharides (Honda et al., 1989; Hoffstetter-Kuhn et al., 1991; Le et al., 1997). The detection limit for the TMR labeled saccharides by using the CE/LIF system is ∼8 × 10−23 mol (or 50 molecules) (Le et al., 1995). The sensitive detection and resolution make the present technique suitable for the determination of substrate and enzyme reaction products in single cells. Figure 2b,c shows electropherograms of the supernatant of the incubation media that contains only unmodified trisaccharide substrate and the final cell-wash solution prior to preparing spheroplasts. The analysis of the last PBS wash solution does not show a detectable level of saccharides. This ensures that results from spheroplast analysis are due only to the compounds present inside the cells. Only background fluorescence was detected in the solution surrounding the spheroplasts. Figure 3a shows electropherograms of the contents of spheroplasts prepared from yeast cells grown in the presence of 50 µM trisaccharide for 24 h. Hydrolysis of substrate produced mainly the linker arm, with minor amounts of monosaccharide and disaccharide intermediates. The peak intensity of the linker arm is normalized against the substrate to give a 0.78 linker arm/trisaccharide (L/T) ratio. This ratio represents the extent of hydrolysis as a function of the initial substrate concentration. The ratio ranged from 0.5 to 0.9 in repeated experiments. This variation is attributed to cell to cell differences in the uptake and localization of substrate in the endoplasmic reticulum. To date no other enzymes besides α-glucosidase I have been found which hydrolyze this trisaccharide substrate. No glucose release was detected in a standard assay (Neverova et al., 1994) when trisaccharide was incubated with commercial brewers yeast α-glucosidase, bakers yeast α-glucosidase, and isolated yeast α-glucosidase II. 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 α-glucosidase 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 α-glucosidase I (Figure 3b). When the concentration of castanospermine was increased to 60 µM, no further reduction of the amount of linker arm was apparent (Figure 3c). As expected, the inhibition of α-glucosidase I activity did not result in the accumulation of di- and monosaccharide. Analysis of the contents of individual spheroplasts introduced into a capillary and monitored by CE/LIF is shown in Figure 4. Each of the three electropherograms was obtained from the analysis of fluorescent molecules in one individual spheroplast. Single spheroplast was introduced, via a micromanipulator set-up (Figure 5), into the CE separation capillary that was filled with running buffer. The nonphysiological buffer containing 10 mM SDS lysed the spheroplast inside the capillary. Subsequent CE/LIF analysis was the result of the lysate from the single spheroplast. As shown in Figure 4, the major fluorescent compounds in the single spheroplasts appear to be the TMR labeled trisaccharide and free linker arm. Approximately 500–1000 TMR labeled trisaccharide and free linker arm molecules are present in a single spheroplast, although the absolute number varies among the individual spheroplasts as indicated by the intensity of the two peaks (T and L) in each of the electropherograms. Fewer than 100 molecules of intermediate TMR labeled di- and monosaccharide were also detected. The overall peak patterns, as governed by the enzymatic hydrolysis of TMR labeled triglucoside, are generally consistent with those previously reported (Le et al., 1995) and the results above in Figure 3, where a population of cells was examined. However, variation between the individual spheroplasts is evident. Fig. 1. Open in new tabDownload slide Photographs obtained from confocal laser scanning microscopy. Yeast cells were incubated with 50 µM of the tetramethylrhodamine (TMR) labeled triglucoside, α-d-Glc(1→2)α-d-Glc(1→3)α-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 (λex = 568 nm and λem = 590 nm). Fig. 1. Open in new tabDownload slide Photographs obtained from confocal laser scanning microscopy. Yeast cells were incubated with 50 µM of the tetramethylrhodamine (TMR) labeled triglucoside, α-d-Glc(1→2)α-d-Glc(1→3)α-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 (λex = 568 nm and λem = 590 nm). 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. Fig. 2. Open in new tabDownload slide Electropherograms obtained from CE/LIF analysis of: (a) standards containing 10−9 M of each of the TMR derivatives: α-d-Glc(1→2)α-d-Glc(1→3)α-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (T); α-d-Glc(1→3)α-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (D); α-D-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (M); and the TMR-linker arm HO(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). Fig. 2. Open in new tabDownload slide Electropherograms obtained from CE/LIF analysis of: (a) standards containing 10−9 M of each of the TMR derivatives: α-d-Glc(1→2)α-d-Glc(1→3)α-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (T); α-d-Glc(1→3)α-d-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (D); α-D-Glc-O(CH2)8CONHCH2CH2NHCO-TMR (M); and the TMR-linker arm HO(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). 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. Materials and methods 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. Fig. 3. Open in new tabDownload slide Intracellular inhibition of yeast α-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. Fig. 3. Open in new tabDownload slide Intracellular inhibition of yeast α-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. Incubation of yeast cells with trisaccharide, α-d-Glc(1→2)α-d-Glc(1→3)α-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, α-d-Glc(1→2)α-d-Glc(1→3)α-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. Fig. 4. Open in new tabDownload slide 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. Fig. 4. Open in new tabDownload slide 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. 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 α-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, λ = 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 5 shows a schematic diagram for the introduction of a single spheroplast into the capillary for electrophoresis analysis. Approximately 1 cm of coating from one end of the separation capillary (40–50 cm) was removed. This end was then etched by using hydrofluoric acid (39%) for 10–15 min. The capillary was filled with aqueous electrophoresis buffer prior to the etching and periodically flushed with the buffer during the etching, in order to minimize the damage of the inner wall and to maximize the etching of the outer wall of the capillary. The etched, sharp tip (∼1–5 µm wall thickness) of the capillary was arranged to be at the injection end, and the other end of the capillary was inserted into the sheath flow cuvette. The etched, injection end of the capillary was held to a coarse micromanipulator MX100R and a hydraulic-controlled fine micromanipulator MX630R (Newport/Klinger, Mississauga, Canada), both of which could be used to move the capillary in three dimensions. A spheroplast suspension (2–5 µl) was placed on the center of a depressed glass slide. An inverted fluorescence microscope IMT-2 (Olympus, Lake Success, NY) was used to view the spheroplasts and the injection end of the capillary. When an appropriate spheroplast was located, the capillary tip was moved close to the spheroplast, with the aid of the micromanipulators. Using an airtight syringe or a syringe pump a gentle suction from the detection end of the capillary was created. A single spheroplast along with minimum surrounding solution was introduced into the capillary under the microscope view. Immediately after the spheroplast was introduced into the capillary, suction was stopped, the injection end of the capillary was removed from the glass slide and placed into an electrophoresis buffer vial. A high voltage (20,000–25,000 V) was applied for 10 s to drive the electrophoresis buffer flowing into the capillary. The high voltage was stopped for 1 min before being reapplied. At this moment, the electrophoresis buffer, containing 10 mM each of phosphate, tetraborate, phenylboronic acid, and sodium dodecyl sulfate (SDS), surrounds the spheroplast. The single spheroplast was lysed inside the capillary due to the nonphysiological buffer and to the presence of SDS. The intracellular substrate and enzyme products from a single spheroplast were electrophoretically separated in the capillary and were detected by laser induced fluorescence. A similar amount of solution surrounding the spheroplasts in the cell suspension was also injected separately and analyzed in a similar manner, confirming undetectable fluorescence in the solution. Fig. 5. Open in new tabDownload slide A schematic diagram showing the introduction of a single cell into a capillary for subsequent analysis by capillary electrophoresis with laser induced fluorescence detection. Fig. 5. Open in new tabDownload slide A schematic diagram showing the introduction of a single cell into a capillary for subsequent analysis by capillary electrophoresis with laser induced fluorescence detection. Acknowledgements 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. Abbreviations Abbreviations CBQCA 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde CE capillary electrophoresis CE/LIF capillary electrophoresis with laser induced fluorescence detection TMR tetramethylrhodamine References Almers W. , Neher E. . The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading , FEBS Lett. , 1985 , vol. 192 (pg. 13 - 18 ) Google Scholar Crossref Search ADS PubMed WorldCat Chiu D.T. , Lillard S.J. , Scheller R.H. , Zare R.N. , Rodriguez-Cruz S.E. , Williams E.R. , Orwar O. , Sandberg M. , Lundqvist J.A. . 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TI - Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast
JO - Glycobiology
DO - 10.1093/glycob/9.3.219
DA - 1999-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/single-cell-studies-of-enzymatic-hydrolysis-of-a-tetramethylrhodamine-Xkrc2QIqv4
SP - 219
EP - 225
VL - 9
IS - 3
DP - DeepDyve
ER -