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A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells
Analytical Biochemistry 492 (2016) 49e55
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A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells Yoshikatsu Ogawa a, *, Mutsuo Tanaka b a Biomedical Research Institute and DBTeAIST International Laboratory for Advanced Biomedicine (DAILAB), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan b Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2015 Received in revised form 14 August 2015 Accepted 3 September 2015 Available online 11 September 2015
Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin eDarby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane. © 2015 Elsevier Inc. All rights reserved.
Keywords: Chemical probe Live-cell imaging Microscopy Sterol Detergent-resistant membrane
Cholesterol is an essential component of mammalian cell membranes, and it plays various roles in cell homeostasis. Free (non-esterified) cholesterol (Fig. 1a) is a major component of the cell membranes and is believed to influence the packing density of the lipid bilayer. Cholesterol is non-uniformly distributed among various cell membranes, and it is precisely controlled. Previous findings, including those of biochemical analyses, have indicated that 80e90% of total cellular cholesterol resides in the plasma membrane [1,2]. In addition, cholesterol is particularly abundant in detergent-resistant fractions of cell membranes (DRMs) [3,4].
Abbreviations: DRM, detergent-resistant membrane; DHE, dehydroergosterol; 22-NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24bisnor-5-cholen-3b-ol; 25-NBD-cholesterol, 25-[N-[(7-nitro-2-1,3-benzoxadiazol4-yl)methyl]amino]-27-norcholesterol; BODIPY-cholesterol, 23-(dipyrrometheneboron difluoride)-24-norcholesterol; R-Chol, 2-[3-dimethylamino-6dimethylazaniumylidenexanthen-9-yl]-5-[2-[(5-cholestene-3b-yl)carbamoyl]ethylcarbamoyl]benzoate; NMR, nuclear magnetic resonance; MDCK, MadineDarby canine kidney; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; WST, water-soluble tetrazolium; DIC, differential interference contrast; PBS, phosphate-buffered saline; UV, ultraviolet; TRITC, tetramethylrhodamine B isothiocyanate; PEG-cholesterol, poly(ethylene glycol)-derivatized cholesterol. * Corresponding author. E-mail address: [email protected] (Y. Ogawa). http://dx.doi.org/10.1016/j.ab.2015.09.003 0003-2697/© 2015 Elsevier Inc. All rights reserved.
The localization and dynamics of intracellular free cholesterol has been investigated using fluorescent probes such as dehydroergosterol (DHE), filipin, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (22-NBD-cholesterol), 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27norcholesterol (25-NBD-cholesterol), and 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol), each of which has specific advantages and disadvantages (Table 1) [5,6]. Two types of commercially available NBD-labeled cholesterol analogues have been widely used to investigate the intracellular distribution of free cholesterol [7,8]. The fluorescent NBD group is attached to cholesterol at C-22 (yielding 22-NBD-cholesterol) or C25 (25-NBD-cholesterol) (Fig. 1e and f). However, fluorescence microscopy has shown that exogenous NBD-cholesterol is scarcely observed at cellular plasma membranes [9]. Here, we converted the hydroxyl group of cholesterol into an amino group to synthesize 3b-amino-5-cholestene and use it as a template to develop a new fluorescent cholesterol analogue for investigating the localization and dynamics of free cholesterol in the plasma membrane of live cells. We then generated the new fluorescent cholesterol analogue, 2-[3-dimethylamino-6dimethylazaniumylidenexanthen-9-yl]-5-[2-[(5-cholestene-3b-yl) carbamoyl]ethylcarbamoyl]benzoate (R-Chol; Fig. 1b) by crosslinking a fluorescent group with 3b-amino-5-cholestene through
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Fig.1. Chemical structures of cholesterol and fluorescent probes. Chemical structures of free cholesterol (a), R-Chol (b), DHE (c), filipin (d), 22-NBD-cholesterol (e), 25-NBDcholesterol (f), and BODIPY-cholesterol (g) are shown.
an amide bond using a technique similar to that described by Peterson's group [10,11]. Our approach involved modifying the hydrophilic part of cholesterol, which was coupled with the fluorescent group. Accordingly, the sterane backbone in the fluorescent cholesterol analogue remained intact (Fig. 1b). The hydroxyl group of cholesterol is considered important for molecular recognition through forming hydrogen bonds with proximal lipids and proteins located in the membrane [12e14]. We expected that the amide group of R-Chol would function in a manner similar to the hydroxyl group of free cholesterol. We stained live cells with the new fluorescent cholesterol analogue R-Chol and investigated the intracellular distribution of fluorescent signals emitted by the analogue. The behavior of R-Chol in the live cells was compared with that of NBD-cholesterol using microscopy. Like free cholesterol, R-Chol was detected mostly at the plasma membrane, and because it was essentially non-cytotoxic, it should contribute to the study of free cholesterol in the plasma membrane of live cells. Materials and methods Organic synthesis of fluorescent cholesterol analogue We synthesized 3b-amino-5-cholestene from cholesterol in four steps by modifying published methods [15,16]. The fluorescent cholesterol analogue, R-Chol, was synthesized in three steps from 3b-amino-5-cholestene via standard methods of peptide synthesis (Fig. 1b). The structure of the product was confirmed using a model MCP500 1H NMR (nuclear magnetic resonance) spectrometer (JEOL, Tokyo, Japan) and a model JMS-700V high-resolution mass spectrometer (JEOL). Details of the procedures involved in the synthesis are provided in online supplementary material.
Cell culture and influence of fluorescent cholesterol analogues on cell growth Media and reagents for cell culture were purchased from Life Technologies (Paisley, Scotland, UK). 22-NBD-cholesterol was purchased from Life Technologies (Eugene, OR, USA), and 25-NBDcholesterol was purchased from Avanti Polar Lipids (Birmingham, AL, USA). MDCK (MadineDarby canine kidney) cells were cultured in 96-well plates (Falcon, Franklin Lakes, NJ, USA), in glass-bottom dishes (Iwaki, Chiba, Japan), or in plastic flasks (Corning, Corning, NY, USA) containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 C under a humidified 5% CO2 atmosphere. The fluorescent probe was dissolved in ethanol (5.0 mmol/ml), 10 ml of which was supplemented with 50 ml of 10% Pluronic F127 (Life Technologies) and 9.94 ml of DMEM and then vortex-mixed (final concentration of fluorescent probe: 5.0 mM) for use in the following experiments. The influence of fluorescent cholesterol analogues on cell viability was investigated as follows. MDCK cells (4 0 103/well) were cultured overnight in 96-well plates, washed twice with DMEM (100 ml/well), incubated with the fluorescent probe (100 ml/well) at 37 C for 10 min, washed twice with DMEM (100 ml/well), and then incubated with DMEM containing 10% FBS (100 ml/well) at 37 C for 6, 24, and 48 h. Next, 10 ml of the water-soluble tetrazolium salt WST-8 (Dojindo, Kumamoto, Japan) was added to each well, and the incubation was continued at 37 C under a 5% CO2 atmosphere for 1 h. WST-8 is reduced by cellular dehydrogenase to produce WST-8 formazan dye, which is used as an index of cell viability. Absorbance at 450/ 655 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA) to quantify the generated WST-8 formazan.
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Table 1 Comparison of fluorescent cholesterol probes for study of intracellular cholesterol distributions. Probe
DHE Filipin 22-NBD-cholesterol 25-NBD-cholesterol BODIPY-cholesterol TRITC-PEG-cholesterol R-Chol
Wavelength (nm) Excitation
Emission
328 340 469 450 475 550 550
378 480 537 510 510 580 580
Expected subcellular localization
Reference(s) and application
PM, periNM PM, periNM periNM periNM PM, periNM PM PM, periNM
[28,29], live cell [32], fixed cell [7], live cell [9], live cell [33], live cell [35,36], live cell This study, live cell
Note. PM, plasma membrane; periNM, perinuclear membranes.
The influence of R-Chol on doubling time of MDCK cells was investigated, and the experimental procedures involved in the analysis are described in supplementary material. We examined the effect of R-Chol on the cell motility using wound healing assay as described by Takahashi and coworkers [17]. The procedures of wound healing assay are also provided in the supplementary material.
Imaging of fluorescent cholesterol analogues in cells All solutions used for cell culture and washing were prewarmed at 37 C. The MDCK cells (1e5 0 105/dish) were cultured overnight on 35-mm glass-bottom dishes (Iwaki), washed three times with 1.0 ml of DMEM, and supplemented with 1.0 ml of 5.0 mM fluorescent probe prepared as described above. After an additional 10 min of incubation at 37 C, the MDCK cells were washed three times with 1.0 ml of DMEM and supplemented with 1.5 ml of DMEM containing 10% FBS. Cells were visualized as differential interference contrast (DIC) and fluorescence images using an IX70 microscope (Olympus, Tokyo, Japan) with standard UPlanSApo (200 magnification, 0.75 N.A.) and water immersion UPlanApo (600 magnification, 1.20 N.A.) objectives. Fluorescent signals emitted by NBD and tetramethylrhodamine were detected using U-MWIB3 and U-MWG2 cubes with 460- to 495-nm and 510- to 550-nm bandpass excitation filters and 510- and 570-nm longpass emission filters (Olympus) respectively. Digital images were captured using an Orca ER charge-coupled device (CCD) camera system (Hamamatsu Photonics, Hamamatsu, Japan) and processed using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Pulse chase imaging of the fluorescent cholesterol analogues in live MDCK cells proceeded as follows. The cells (1e5 0 105/dish) were stained with a fluorescent probe as described above, washed with DMEM, incubated in 1.5 ml of DMEM supplemented with 10% FBS at 37 C for 10 or 60 min, and visualized as described above.
Analysis of detergent-resistant membrane fraction of MDCK cells stained with R-Chol We analyzed the accumulation of R-Chol in the DRM fraction of cells using floatation assays as described by Hanwell and coworkers [18]. Confluent cells in 10-cm dishes were washed twice with 10 ml of DMEM and incubated with 5 ml of 5 mM R-Chol in DMEM at 37 C for 10 min. The stained cells were washed three times with 10 ml of DMEM, once with 10 ml of ice-cold phosphate-buffered saline (PBS), suspended in 660 ml of ice-cold TN (25 mM TriseHCl [pH 7.5], 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, one proteinase inhibitor cocktail tablet per 30 ml [Roche, Mannheim, Germany], and 1% Triton X-100) and placed in a tube on ice for 30 min. Thereafter, the cell suspension (330 ml) was mixed with 670 ml of ice-cold 60% OptiPrep (AxiseShield, Oslo, Norway).
The resulting suspension (1.0 ml) was layered on the top of a stepwise gradient comprising 1.5 ml of 35%, 1.5 ml of 30%, and 1.0 ml of 5% OptiPrep in TN and separated by centrifugation at 41,000 rpm in an SW55Ti rotor using an Optima L-80 XP centrifuge (Beckman Coulter, Brea, CA, USA) for 5 h at 4 C. Fractions (700 ml) collected from the top of the centrifuge tubes were vortex-mixed with 175 ml of 100% (w/v) trichloroacetic acid and placed on ice for 15 min. The mixtures were then separated by centrifugation at 15,000 rpm for 15 min at 4 C, and the supernatants were discarded. The precipitates were washed twice with ethanol (500 ml) and once with acetone (500 ml), dried in air at room temperature, dissolved in 20 ml of loading buffer, and resolved by sodium dodecyl sulfateepolyacrylamide gel electrophoresis. Polypeptides separated on gels were analyzed by immunoblotting with antibodies against caveolin-1 (BD Biosciences, San Jose, CA, USA) and transferrin receptor (Abcam, Cambridge, UK). We analyzed the distribution of the fluorescent probe across fractions as follows. Fractions (700 ml) were sonicated in 20% Triton X-100 (300 ml), and then sonicates (200 ml) were transferred to black 96-well plates. Fluorescent intensity was measured at excitation and emission wavelengths of 550 and 580 nm, respectively. Values for fluorescent intensity were normalized against the sum of all fractions from each centrifuge tube. Results Synthesis of fluorescent cholesterol analogue Fig. 1b shows the chemical structure of R-Chol obtained in three steps from 3b-amino-5-cholestene with a 47.2% yield. The structure was confirmed by 1H NMR and high-resolution mass spectrometry. Influence of fluorescent cholesterol analogues on MDCK cell growth The cytotoxicity of R-Chol was examined within the concentration range for cell staining as follows. The cells were pulsed at 37 C for 10 min with R-Chol or with an NBD-cholesterol, washed with fresh DMEM, and incubated at 37 C for various periods in medium containing 10% FBS. Cell viability was evaluated as the generation of formazan in response to intracellular dehydrogenase activities in the WST-8 assay. The viability of cells incubated with 0.5e50 mM R-Chol for 10 min was identical to that of controls within an experimental error range at any incubation time point (Fig. 2A). The morphology of the cells incubated with the fluorescent cholesterol analogues determined by microscopy did not differ from that of control cells. These findings indicated that R-Chol did not affect the viability of MDCK cells like NBD-cholesterol (Fig. 2B and C). In addition, the doubling time of the cells incubated with R-Chol (final concentration: 0.5, 1, or 5 mM) did not differ from that of control cells (see Fig. S1 in supplementary material), indicating that R-Chol did not affect the growth of MDCK cells. To investigate the
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Fig.2. Cell viability assays. The generation of WST-8 formazan, an index of cell viability, was measured as absorbance at 450/655 nm using a microplate reader. The vertical axis shows relative absorbance of stained cells to that of control cells (100%). Values are shown as means ± standard deviations (n ¼ 5). The horizontal axis shows fluorophore concentrations (A: R-Chol; B: 22-NBD-cholesterol; C: 25-NBD-cholesterol) applied during incubations for 6 h (open circles), 24 h (closed circles), or 48 h (open triangles). Viability of cells incubated with R-Chol was essentially identical to that of control cells within the experimental range of error at all incubation time points.
cytotoxicity of R-Chol, the effect of R-Chol on the cell motility was also examined by wound healing assay. The data indicated that the motility of the cells incubated with 1e10 mM R-Chol did not differ from that of the cells incubated without R-Chol (Fig. S2), showing that R-Chol did not affect the motility of MDCK cells. Localization of fluorescent cholesterol analogues in cells To investigate the localization of R-Chol, MDCK cells were stained with R-Chol or NBD-cholesterol for 10 min and assessed by fluorescent microscopy immediately after removing the fluorophores by washing with DMEM. The NBD-cholesterols emitted intense fluorescent signals at perinuclear membranes, possibly the Golgi bodies and endosomes, but very little at the plasma membrane (Fig. 3B and C). These findings agreed with those of a previous study [9]. In contrast, R-Chol signals were emitted not only at the perinuclear membranes but also at the entire plasma membrane (Fig. 3A), and thus the shape of the cells was clearly defined. A comparison of fluorescence and DIC images confirmed that R-Chol was localized at the plasma membrane in all cells in the entire visual field under microscopy. Cultured MDCK cells form monolayers with tight junctions, which is a feature of epithelial cells. R-Chol signals were intense at the contact surface of confluent MDCK cells, which yielded mesh-work images when stained with R-Chol (Fig. 3A). These findings indicated that R-Chol localized to the plasma membrane immediately after staining. Time course of intracellular distribution of fluorophores The fluorophores were removed from MDCK cells by washing with DMEM after a 10-min incubation with R-Chol or NBDcholesterols. Next, the cells were incubated with DMEM with 10% FBS for 10 or 60 min and then were visualized. The NBD-cholesterol signals were slight or absent at the plasma membrane at all time points (Fig. 4B and C). Most NBD-cholesterol signals were found at the perinuclear membranes immediately after staining, but the intensity gradually decreased. Although R-Chol signals were detected at the plasma membrane at 10 and 60 min after staining, those in perinuclear membranes significantly increased over time (Fig. 4A). The result is consistent with the pathway of intracellular cholesterol transport proposed by previous studies [19e21]. Distribution of R-Chol in DRM fractions Mammalian cell membranes involve microdomains with abundant sphingolipids and free cholesterol; lipids at such
microdomains form an ordered liquid crystalline phase, and then the domains segregate from the surrounding region of a relatively disordered lipid phase [4]. In addition, such microdomains can be isolated from the membranes of various mammalian cells using non-ionic detergents such as Triton X-100 [22]. We used floatation assays to determine whether the distribution behavior of R-Chol is similar to that of free cholesterol in DRM fractions. Stained MDCK cells with R-Chol were treated with Triton X-100 on ice and were fractionated by density gradient centrifugation into DRM and non-DRM fractions. The DRM fractions contained 80% of the fluorescent signals generated by R-Chol (Fig. 5). Approximately 10% of the total fluorescent signal was located in the boundary between DRM and non-DRM fractions, with a few signals being located in non-DRM fractions. Discussion The hydroxyl group is believed to be important for the molecular recognition of cholesterol through hydrogen bonds with proximal molecules and also for the formation of microdomains [23]. For example, the probe cholesteryl BODIPY FL C12 (Life Technologies), which is synthesized by esterification of the hydroxyl group of cholesterol with a fluorescent group, localizes at cellular lipid bodies but not at the plasma membrane [24,25]. In contrast, the fluorescent cholesterol analogue, R-Chol, localized mostly at the plasma membrane like free cholesterol (Fig. 3A) and was found mostly in DRM fractions (Fig. 5). The hydroxyl group of cholesterol is believed to play important roles for the localization of cholesterol [12,23], whereas the amide group of R-Chol could conceivably play similar roles. The amide group of R-Chol exists between the sterane backbone and the zwitterionic fluorescent group (Fig. 1b). One of the characteristics that is common to hydroxyl group and amide group is to possibly act as hydrogen-donating and/or hydrogenaccepting group to form an intermolecular hydrogen bond [12]. This would explain the localization of R-Chol at the plasma membrane. The intracellular localization of cholesterol has often been investigated using NBD-cholesterol (Fig. 1e and f), which might be problematic because fluorescent signals would not be observed at the plasma membrane, where most intracellular free cholesterol is localized [7,9]. This suggests that chemical modification at the alkyl chain of cholesterol influences its intracellular localization. The current and previous findings [9,26,27] imply that both hydrophobic interaction through the alkyl chain and hydrogen bonding through the hydroxyl group would play important roles in the localization of free cholesterol at the plasma membrane. The sterol DHE (Fig. 1c) has also been applied as a fluorescent probe in studies of cellular cholesterol using microscopy because it
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Fig.3. Live MDCK cells stained with fluorescent cholesterol analogues. MDCK cells were observed using DIC and fluorescence microscopy immediately after incubation with fluorescent cholesterol analogues R-Chol (A), 22-NBD-cholesterol (B), and 25-NBD-cholesterol (C). Signals were found mostly at the plasma membrane of MDCK cells incubated with R-Chol (FL, A). In contrast, signals were found mostly at the perinuclear membranes of MDCK cells incubated with 22-NBD-cholesterol (FL, B) or 25-NBD-cholesterol (FL, C). DIC, differential interference contrast microscopy; FL, fluorescence microscopy. Panels D and E show autofluorescence emitted from non-stained cells under the same conditions as those shown in panel A for R-Chol and in panels B and C for NBD-cholesterols. Bar ¼ 50 mm.
Fig.4. Pulse chase images of fluorescent cholesterol analogues in live MDCK cells. Cells incubated with fluorescent cholesterol analogues R-Chol (A), 22-NBD-cholesterol (B), and 25NBD-cholesterol (C) examined by fluorescence microscopy at 10 and 60 min after analogue removal are shown. Typical high-magnification images of cells incubated with R-Chol (D), 22-NBD-cholesterol (E), and 25-NBD-cholesterol (F) at 10 and 60 min after analogue removal are also shown. Signal distribution changed from plasma to perinuclear membranes in the cells incubated with R-Chol (A,D) but not in those incubated with 22-NBD-cholesterol (B,E) or 25-NBD-cholesterol (C,F). Bar ¼ 50 mm.
accurately mimics the distribution of cholesterol in mammalian cells, probably because of its structural similarity to cholesterol [9,28,29]. DHE occurs naturally in yeasts, and it is an intrinsically fluorescent lipid. However, its excitation wavelength is in the ultraviolet (UV) region (300e350 nm), and thus special attachments are required that are normally not included with conventional microscopes to detect fluorescent signals emitted by DHE. Furthermore, close attention must be paid when using DHE in experimental studies because UV irradiation can damage live cells.
On the other hand, R-Chol signals were detectable by conventional fluorescence microscopy. Thus, R-Chol should be more readily applicable to observation using fluorescent microscopy with exciting the fluorophore by less damaging visible light. The fluorescent macrocyclic compound filipin (Fig. 1d) isolated from the actinomycete, Streptomyces filipinensis, forms a specific complex with free cholesterol [30,31]. Thus, filipin has served as a fluorescent probe for visualizing free cholesterol in cells [32]. Because its excitation wavelength is in the UV region, filipin is
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interior and returns to the plasma membrane [37], and the endocytic recycling compartment contains high levels of cholesterol [27]. The hydroxyl group of free cholesterol is esterified in the interior of cells via a lipid storage system [37,38], but R-Chol lacks a hydroxyl group and cannot be esterified (Fig. 1b), which might explain R-Chol accumulation at perinuclear membranes. Free cholesterol accumulates in the perinuclear membranes of a mutant cell line that has defective cholesterol esterification [38]. R-Chol signals were detectable at the plasma membrane within 1 h of cell staining (Fig. 4A and D). Because the fluorescent group of R-Chol (rhodamine) is comparatively photostable with relatively bright fluorescence, R-Chol should confer an advantage for continuous observation under microscopy. In addition, R-Chol was abundant in DRM fractions like free cholesterol (Fig. 5). Therefore, we consider that R-Chol could serve as a useful tool for investigating the dynamics of free cholesterol in the plasma membranes of live cells. Conclusions The novel fluorescent cholesterol analogue, R-Chol, did not influence the growth of MDCK cells in vitro. The chemical modification of cholesterol required to generate R-Chol scarcely affected cholesterol localization mostly at the plasma membrane and in DRM fractions of live cells. Acknowledgments Fig.5. Distribution of R-Chol in fractions determined using floatation assays. Assay procedures are described in detail in Materials and Methods. (A) Fluorescence intensity of R-Chol was measured at excitation and emission of 550 and 580 nm, respectively. Vertical axis: Ratios (%) of fluorescence intensity versus total fluorescence (sum of signals from all fractions). Values are shown as means ± standard deviations of five assays. Fraction numbers are shown on the horizontal axis from top of centrifuge tube. (B) Western blotting identified transferrin receptor, a non-DRM marker, in fraction 7 and identified caveolin 1, a DRM marker, in fractions 2 and 3.
readily applicable to studies of fixed cells but perhaps not to studies of live cells. BODIPY-cholesterol (Fig. 1g) has also been used as a fluorescent probe for investigations into the intracellular localization of free cholesterol, and it is commercially available as TopFluor cholesterol. The distribution of BODIPY-cholesterol signals in live cells is essentially the same as that of DHE [33]. On the other hand, BODIPY-cholesterol diffuses far more slowly in the lipid bilayer than free cholesterol [34]. One possible reason for this is that the size of the fluorophore, which is comparable to that of the sterane backbone, is linked to the hydrophobic portion of cholesterol (Fig. 1g). However, the hydrophobic portions of R-Chol and cholesterol are structurally identical (Fig. 1b), and therefore R-Chol and free cholesterol should diffuse throughout the lipid bilayer at similar rates. TRITC-PEG-cholesterol (tetramethylrhodamine B isothiocyanate [TRITC]-labeled poly(ethylene glycol) [PEG]-cholesterol) was reported to distribute in the outer plasma membrane leaflet and to be enriched in microdomains [35,36]. It is believed that TRITC-PEGcholesterol does not transverse lipid bilayer because of the bulky size of PEG group. Because the molecular size of TRITC-PEGcholesterol is much larger than that of R-Chol, the dynamics of TRITC-PEG-cholesterol in the plasma membrane should be different from that of R-Chol and cholesterol. The results of the pulse chase experiment using MDCK cells stained with R-Chol showed that R-Chol signals were observed in the perinuclear membranes within 1 h of staining (Fig. 4A and D). Free cholesterol in the plasma membrane moves into the cell
The authors thank Y. Onishi (National Institute of Advanced Industrial Science and Technology [AIST], Japan) for input on the cell viability assay, T. Mizuno (AIST) for providing the MDCK cell line and for advice on cell imaging, T. Yoko-o (AIST) for advice on floating assays, and T. Shimizu (AIST) for suggestions and discussion. This work was supported by a Grant-in-Aid for Scientific Research (C, 17550140) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ab.2015.09.003. References [1] Y. Lange, Disposition of intracellular cholesterol in human fibroblasts, J. Lipid Res. 32 (1991) 329e339. [2] D.E. Warnock, C. Roberts, M.S. Lutz, W.A. Blackburn, W.W. Young Jr., J.U. Baenziger, Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography, J. Biol. Chem. 268 (1993) 10145e10153. [3] R. Schroeder, E. London, D. Brown, Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 12130e12134. [4] K. Simons, E. Ikonen, Functional rafts in cell membranes, Nature 387 (1997) 569e572. [5] G. Gimpl, K. Gehrig-Burger, Cholesterol reporter molecules, Biosci. Rep. 27 (2007) 335e358. [6] D. Wünstner, Fluorescent sterols as tools in membrane biophysics and cell biology, Chem. Phys. Lipids 146 (2007) 1e25. [7] I.F. Craig, D.P. Via, W.W. Mantulin, H.J. Pownall, A.M. Gotto Jr., L.C. Smith, Low density lipoproteins reconstituted with steroids containing the nitrobenzoxadiazole fluorophore, J. Lipid Res. 22 (1981) 687e696. [8] A. Chattopadhyay, E. London, Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spinlabeled phospholipids, Biochemistry 26 (1987) 39e45. [9] S. Mukherjee, X. Zha, I. Tabas, F.R. Maxfield, Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog, Biophys. J. 75 (1998) 1915e1925. [10] S.L. Hussey, B.R. Peterson, Efficient delivery of streptavidin to mammalian cells: Clathrin-mediated endocytosis regulated by a synthetic ligand, J. Am. Chem. Soc. 124 (2002) 6265e6273.
Y. Ogawa, M. Tanaka / Analytical Biochemistry 492 (2016) 49e55 [11] S. Boonyarattanakalin, S.E. Martin, S.A. Dykstra, B.R. Peterson, Synthetic mimics of small mammalian cell surface receptors, J. Am. Chem. Soc. 126 (2004) 16379e16386. [12] J.M. Boggs, Lipid intermolecular hydrogen bonding: influence on structure organization and membrane function, Biochem. Biophys. Acta 906 (1987) 353e404. [13] Y.J. Im, S. Raychaudhuri, W.A. Prinz, J.H. Hurley, Structural mechanism for sterol sensing and transport by OSBP-related proteins, Nature 437 (2005) 154e158. [14] J. Fantini, F.J. Barrantes, How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains, Front. Physiol. 4 (2013), http://dx.doi.org/10.3389/ fphys.2013.00031. [15] P.L. Julian, A. Magnani, E.W. Meyer, W. Cole, Sterols: V. The i-cholesterylamines, J. Am. Chem. Soc. 70 (1948) 1834e1837. [16] E. Kosower, S. Winstein, Neighboring carbon and hydrogen: XXII. Homoallylic systems: the preparation and behavior of certain 3,5-cyclosteroids, J. Am. Chem. Soc. 78 (1956) 4347e4354. [17] M. Takahashi, M. Furihata, N. Akimitsu, M. Watanabe, S. Kaul, N. Yumoto, T. Okada, A highly bone marrow metastatic murine breast cancer model established through in vivo selection exhibits enhanced anchorageindependent growth and cell migration mediated by ICAM-1, Clin. Exp. Metastasis 25 (2008) 517e529. [18] D. Hanwell, T. Ishikawa, R. Saleki, D. Rotin, Trafficking and cell surface stability of the epithelial Naþ channel expressed in epitherial MadineDarby canine kidney cells, J. Biol. Chem. 277 (2002) 9772e9779. [19] J.D. Butler, J. Blanchette-Mackie, E. Goldin, R.R. O'Neill, G. Carstea, C.F. Roff, M.C. Patterson, S. Patel, M.E. Comly, A. Cooney, M.T. Vanier, R.O. Brady, P.G. Pentchev, Progesterone blocks cholesterol translocation from lysosomes, J. Biol. Chem. 267 (1992) 23797e23805. [20] L. Liscum, N.J. Munn, Intracellular cholesterol transport, Biochem. Biophys. Acta 1438 (1999) 19e37. € bius, E. van Donselaar, Y. Ohno-Iwashita, Y. Shimada, H.F.G. Heijnen, [21] W. Mo J.W. Slot, H.J. Geuze, Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway, Traffic 4 (2003) 222e231. [22] K. Fiedler, T. Kobayashi, T.V. Kurzchalia, K. Simons, Glycosphingolipidenriched, detergent-insoluble complexes in protein sorting in epitherial cells, Biochemistry 32 (1993) 6365e6373. € nen, I. Vattulainen, Atom-scale molecular interactions [23] P.S. Niemel€ a, M.T. Hyvo in lipid raft mixtures, Biochem. Biophys. Acta 1788 (2009) 122e135. [24] C.L. Bisgaier, L.L. Minton, A.D. Essenburg, A. White, R. Homan, Use of fluorescent cholesteryl ester microemulsions in cholesteryl ester transfer protein assays, J. Lipid Res. 34 (1993) 1625e1634. [25] E. Reaven, L. Tsai, S. Azhar, Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters, J. Biol. Chem. 271 (1996) 16208e16217.
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[26] H.A. Scheidt, P. Müller, A. Herrmann, D. Huster, The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol, J. Biol. Chem. 278 (2003) 45563e45569. [27] R.F. Epand, A. Thomas, R. Brasseur, S.A. Vishwanathan, E. Hunter, R.M. Epand, Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains, Biochemistry 45 (2006) 6105e6114. [28] M. Hao, S.X. Lin, O.J. Karylowski, D. Wüstner, T.E. McGraw, F.R. Maxfield, Vesicular and non-vesicular sterol transport in living cells, J. Biol. Chem. 277 (2002) 609e617. [29] R. Homan, N. Esmaeil, L. Mendelsohn, G.J. Kato, A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase, Anal. Biochem. 441 (2013) 80e86. [30] G.B. Whitefield, T.D. Brock, A. Ammann, D. Gottlieb, H.E. Carter, Filipin, an antifungal antibiotic: isolation and properties, J. Am. Chem. Soc. 77 (1955) 4799e4801. [31] M.A.R.B. Castanho, A. Coutinho, M.J.E. Prieto, Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol, J. Biol. Chem. 267 (1992) 204e209. [32] J.D. Butler, M.E. Comly, H.S. Kruth, M. Vanier, M. Filling-Katz, J. Fink, N. Barton, H. Weintroub, J.M. Quirk, T. Tokoro, D.C. Marshall, R.O. Brady, P.G. Pentchev, NiemanePick variant disorder: comparison of errors of cellular cholesterol homeostasis in group D and group C fibroblasts, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 556e560. [33] D. Wünstner, L. Solanko, E. Sokol, O. Garvik, Z. Li, R. Bittman, T. Korte, A. Herrmann, Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol, Chem. Phys. Lipids 164 (2011) 221e235. [34] F.S. Ariola, Z. Li, C. Cornejo, R. Bittman, A.A. Heikal, Membrane fluidity and lipid order in ternary giant unilamellar vesicles using a new Bodipycholesterol derivative, Biophys. J. 96 (2009) 2696e2708. [35] S.B. Sato, K. Ishii, A. Makino, K. Iwabuchi, A. Yamaji-Hasegawa, Y. Senoh, I. Nagaoka, H. Sakuraba, T. Kobayashi, Distribution and transport of cholesterol-rich membrane domains monitored by a membrane-impermeant fluorescent polyethylene glycol-derivatized cholesterol, J. Biol. Chem. 279 (2004) 23790e23796. [36] M. Takahashi, M. Murate, M. Fukuda, S.B. Sato, A. Ohta, T. Kobayashi, Cholesterol controls lipid endocytosis through Rab11, Mol. Biol. Cell 18 (2007) 2667e2677. [37] Y. Lange, F. Strebel, T.L. Steck, Role of the plasma membrane in cholesterol esterification in rat hepatoma cells, J. Biol. Chem. 268 (1993) 13838e13843. [38] P.G. Pentchev, M.E. Comly, H.S. Kruth, M.T. Vanier, D.A. Wenger, S. Patel, R.O. Brady, A defect in cholesterol esterification in NiemannePick disease (type C) patients, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 8247e8251.
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A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells Yoshikatsu Ogawa a, *, Mutsuo Tanaka b a Biomedical Research Institute and DBTeAIST International Laboratory for Advanced Biomedicine (DAILAB), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan b Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2015 Received in revised form 14 August 2015 Accepted 3 September 2015 Available online 11 September 2015
Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin eDarby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane. © 2015 Elsevier Inc. All rights reserved.
Keywords: Chemical probe Live-cell imaging Microscopy Sterol Detergent-resistant membrane
Cholesterol is an essential component of mammalian cell membranes, and it plays various roles in cell homeostasis. Free (non-esterified) cholesterol (Fig. 1a) is a major component of the cell membranes and is believed to influence the packing density of the lipid bilayer. Cholesterol is non-uniformly distributed among various cell membranes, and it is precisely controlled. Previous findings, including those of biochemical analyses, have indicated that 80e90% of total cellular cholesterol resides in the plasma membrane [1,2]. In addition, cholesterol is particularly abundant in detergent-resistant fractions of cell membranes (DRMs) [3,4].
Abbreviations: DRM, detergent-resistant membrane; DHE, dehydroergosterol; 22-NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24bisnor-5-cholen-3b-ol; 25-NBD-cholesterol, 25-[N-[(7-nitro-2-1,3-benzoxadiazol4-yl)methyl]amino]-27-norcholesterol; BODIPY-cholesterol, 23-(dipyrrometheneboron difluoride)-24-norcholesterol; R-Chol, 2-[3-dimethylamino-6dimethylazaniumylidenexanthen-9-yl]-5-[2-[(5-cholestene-3b-yl)carbamoyl]ethylcarbamoyl]benzoate; NMR, nuclear magnetic resonance; MDCK, MadineDarby canine kidney; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; WST, water-soluble tetrazolium; DIC, differential interference contrast; PBS, phosphate-buffered saline; UV, ultraviolet; TRITC, tetramethylrhodamine B isothiocyanate; PEG-cholesterol, poly(ethylene glycol)-derivatized cholesterol. * Corresponding author. E-mail address: [email protected] (Y. Ogawa). http://dx.doi.org/10.1016/j.ab.2015.09.003 0003-2697/© 2015 Elsevier Inc. All rights reserved.
The localization and dynamics of intracellular free cholesterol has been investigated using fluorescent probes such as dehydroergosterol (DHE), filipin, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (22-NBD-cholesterol), 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27norcholesterol (25-NBD-cholesterol), and 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol), each of which has specific advantages and disadvantages (Table 1) [5,6]. Two types of commercially available NBD-labeled cholesterol analogues have been widely used to investigate the intracellular distribution of free cholesterol [7,8]. The fluorescent NBD group is attached to cholesterol at C-22 (yielding 22-NBD-cholesterol) or C25 (25-NBD-cholesterol) (Fig. 1e and f). However, fluorescence microscopy has shown that exogenous NBD-cholesterol is scarcely observed at cellular plasma membranes [9]. Here, we converted the hydroxyl group of cholesterol into an amino group to synthesize 3b-amino-5-cholestene and use it as a template to develop a new fluorescent cholesterol analogue for investigating the localization and dynamics of free cholesterol in the plasma membrane of live cells. We then generated the new fluorescent cholesterol analogue, 2-[3-dimethylamino-6dimethylazaniumylidenexanthen-9-yl]-5-[2-[(5-cholestene-3b-yl) carbamoyl]ethylcarbamoyl]benzoate (R-Chol; Fig. 1b) by crosslinking a fluorescent group with 3b-amino-5-cholestene through
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Fig.1. Chemical structures of cholesterol and fluorescent probes. Chemical structures of free cholesterol (a), R-Chol (b), DHE (c), filipin (d), 22-NBD-cholesterol (e), 25-NBDcholesterol (f), and BODIPY-cholesterol (g) are shown.
an amide bond using a technique similar to that described by Peterson's group [10,11]. Our approach involved modifying the hydrophilic part of cholesterol, which was coupled with the fluorescent group. Accordingly, the sterane backbone in the fluorescent cholesterol analogue remained intact (Fig. 1b). The hydroxyl group of cholesterol is considered important for molecular recognition through forming hydrogen bonds with proximal lipids and proteins located in the membrane [12e14]. We expected that the amide group of R-Chol would function in a manner similar to the hydroxyl group of free cholesterol. We stained live cells with the new fluorescent cholesterol analogue R-Chol and investigated the intracellular distribution of fluorescent signals emitted by the analogue. The behavior of R-Chol in the live cells was compared with that of NBD-cholesterol using microscopy. Like free cholesterol, R-Chol was detected mostly at the plasma membrane, and because it was essentially non-cytotoxic, it should contribute to the study of free cholesterol in the plasma membrane of live cells. Materials and methods Organic synthesis of fluorescent cholesterol analogue We synthesized 3b-amino-5-cholestene from cholesterol in four steps by modifying published methods [15,16]. The fluorescent cholesterol analogue, R-Chol, was synthesized in three steps from 3b-amino-5-cholestene via standard methods of peptide synthesis (Fig. 1b). The structure of the product was confirmed using a model MCP500 1H NMR (nuclear magnetic resonance) spectrometer (JEOL, Tokyo, Japan) and a model JMS-700V high-resolution mass spectrometer (JEOL). Details of the procedures involved in the synthesis are provided in online supplementary material.
Cell culture and influence of fluorescent cholesterol analogues on cell growth Media and reagents for cell culture were purchased from Life Technologies (Paisley, Scotland, UK). 22-NBD-cholesterol was purchased from Life Technologies (Eugene, OR, USA), and 25-NBDcholesterol was purchased from Avanti Polar Lipids (Birmingham, AL, USA). MDCK (MadineDarby canine kidney) cells were cultured in 96-well plates (Falcon, Franklin Lakes, NJ, USA), in glass-bottom dishes (Iwaki, Chiba, Japan), or in plastic flasks (Corning, Corning, NY, USA) containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 C under a humidified 5% CO2 atmosphere. The fluorescent probe was dissolved in ethanol (5.0 mmol/ml), 10 ml of which was supplemented with 50 ml of 10% Pluronic F127 (Life Technologies) and 9.94 ml of DMEM and then vortex-mixed (final concentration of fluorescent probe: 5.0 mM) for use in the following experiments. The influence of fluorescent cholesterol analogues on cell viability was investigated as follows. MDCK cells (4 0 103/well) were cultured overnight in 96-well plates, washed twice with DMEM (100 ml/well), incubated with the fluorescent probe (100 ml/well) at 37 C for 10 min, washed twice with DMEM (100 ml/well), and then incubated with DMEM containing 10% FBS (100 ml/well) at 37 C for 6, 24, and 48 h. Next, 10 ml of the water-soluble tetrazolium salt WST-8 (Dojindo, Kumamoto, Japan) was added to each well, and the incubation was continued at 37 C under a 5% CO2 atmosphere for 1 h. WST-8 is reduced by cellular dehydrogenase to produce WST-8 formazan dye, which is used as an index of cell viability. Absorbance at 450/ 655 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA) to quantify the generated WST-8 formazan.
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Table 1 Comparison of fluorescent cholesterol probes for study of intracellular cholesterol distributions. Probe
DHE Filipin 22-NBD-cholesterol 25-NBD-cholesterol BODIPY-cholesterol TRITC-PEG-cholesterol R-Chol
Wavelength (nm) Excitation
Emission
328 340 469 450 475 550 550
378 480 537 510 510 580 580
Expected subcellular localization
Reference(s) and application
PM, periNM PM, periNM periNM periNM PM, periNM PM PM, periNM
[28,29], live cell [32], fixed cell [7], live cell [9], live cell [33], live cell [35,36], live cell This study, live cell
Note. PM, plasma membrane; periNM, perinuclear membranes.
The influence of R-Chol on doubling time of MDCK cells was investigated, and the experimental procedures involved in the analysis are described in supplementary material. We examined the effect of R-Chol on the cell motility using wound healing assay as described by Takahashi and coworkers [17]. The procedures of wound healing assay are also provided in the supplementary material.
Imaging of fluorescent cholesterol analogues in cells All solutions used for cell culture and washing were prewarmed at 37 C. The MDCK cells (1e5 0 105/dish) were cultured overnight on 35-mm glass-bottom dishes (Iwaki), washed three times with 1.0 ml of DMEM, and supplemented with 1.0 ml of 5.0 mM fluorescent probe prepared as described above. After an additional 10 min of incubation at 37 C, the MDCK cells were washed three times with 1.0 ml of DMEM and supplemented with 1.5 ml of DMEM containing 10% FBS. Cells were visualized as differential interference contrast (DIC) and fluorescence images using an IX70 microscope (Olympus, Tokyo, Japan) with standard UPlanSApo (200 magnification, 0.75 N.A.) and water immersion UPlanApo (600 magnification, 1.20 N.A.) objectives. Fluorescent signals emitted by NBD and tetramethylrhodamine were detected using U-MWIB3 and U-MWG2 cubes with 460- to 495-nm and 510- to 550-nm bandpass excitation filters and 510- and 570-nm longpass emission filters (Olympus) respectively. Digital images were captured using an Orca ER charge-coupled device (CCD) camera system (Hamamatsu Photonics, Hamamatsu, Japan) and processed using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Pulse chase imaging of the fluorescent cholesterol analogues in live MDCK cells proceeded as follows. The cells (1e5 0 105/dish) were stained with a fluorescent probe as described above, washed with DMEM, incubated in 1.5 ml of DMEM supplemented with 10% FBS at 37 C for 10 or 60 min, and visualized as described above.
Analysis of detergent-resistant membrane fraction of MDCK cells stained with R-Chol We analyzed the accumulation of R-Chol in the DRM fraction of cells using floatation assays as described by Hanwell and coworkers [18]. Confluent cells in 10-cm dishes were washed twice with 10 ml of DMEM and incubated with 5 ml of 5 mM R-Chol in DMEM at 37 C for 10 min. The stained cells were washed three times with 10 ml of DMEM, once with 10 ml of ice-cold phosphate-buffered saline (PBS), suspended in 660 ml of ice-cold TN (25 mM TriseHCl [pH 7.5], 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, one proteinase inhibitor cocktail tablet per 30 ml [Roche, Mannheim, Germany], and 1% Triton X-100) and placed in a tube on ice for 30 min. Thereafter, the cell suspension (330 ml) was mixed with 670 ml of ice-cold 60% OptiPrep (AxiseShield, Oslo, Norway).
The resulting suspension (1.0 ml) was layered on the top of a stepwise gradient comprising 1.5 ml of 35%, 1.5 ml of 30%, and 1.0 ml of 5% OptiPrep in TN and separated by centrifugation at 41,000 rpm in an SW55Ti rotor using an Optima L-80 XP centrifuge (Beckman Coulter, Brea, CA, USA) for 5 h at 4 C. Fractions (700 ml) collected from the top of the centrifuge tubes were vortex-mixed with 175 ml of 100% (w/v) trichloroacetic acid and placed on ice for 15 min. The mixtures were then separated by centrifugation at 15,000 rpm for 15 min at 4 C, and the supernatants were discarded. The precipitates were washed twice with ethanol (500 ml) and once with acetone (500 ml), dried in air at room temperature, dissolved in 20 ml of loading buffer, and resolved by sodium dodecyl sulfateepolyacrylamide gel electrophoresis. Polypeptides separated on gels were analyzed by immunoblotting with antibodies against caveolin-1 (BD Biosciences, San Jose, CA, USA) and transferrin receptor (Abcam, Cambridge, UK). We analyzed the distribution of the fluorescent probe across fractions as follows. Fractions (700 ml) were sonicated in 20% Triton X-100 (300 ml), and then sonicates (200 ml) were transferred to black 96-well plates. Fluorescent intensity was measured at excitation and emission wavelengths of 550 and 580 nm, respectively. Values for fluorescent intensity were normalized against the sum of all fractions from each centrifuge tube. Results Synthesis of fluorescent cholesterol analogue Fig. 1b shows the chemical structure of R-Chol obtained in three steps from 3b-amino-5-cholestene with a 47.2% yield. The structure was confirmed by 1H NMR and high-resolution mass spectrometry. Influence of fluorescent cholesterol analogues on MDCK cell growth The cytotoxicity of R-Chol was examined within the concentration range for cell staining as follows. The cells were pulsed at 37 C for 10 min with R-Chol or with an NBD-cholesterol, washed with fresh DMEM, and incubated at 37 C for various periods in medium containing 10% FBS. Cell viability was evaluated as the generation of formazan in response to intracellular dehydrogenase activities in the WST-8 assay. The viability of cells incubated with 0.5e50 mM R-Chol for 10 min was identical to that of controls within an experimental error range at any incubation time point (Fig. 2A). The morphology of the cells incubated with the fluorescent cholesterol analogues determined by microscopy did not differ from that of control cells. These findings indicated that R-Chol did not affect the viability of MDCK cells like NBD-cholesterol (Fig. 2B and C). In addition, the doubling time of the cells incubated with R-Chol (final concentration: 0.5, 1, or 5 mM) did not differ from that of control cells (see Fig. S1 in supplementary material), indicating that R-Chol did not affect the growth of MDCK cells. To investigate the
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Fig.2. Cell viability assays. The generation of WST-8 formazan, an index of cell viability, was measured as absorbance at 450/655 nm using a microplate reader. The vertical axis shows relative absorbance of stained cells to that of control cells (100%). Values are shown as means ± standard deviations (n ¼ 5). The horizontal axis shows fluorophore concentrations (A: R-Chol; B: 22-NBD-cholesterol; C: 25-NBD-cholesterol) applied during incubations for 6 h (open circles), 24 h (closed circles), or 48 h (open triangles). Viability of cells incubated with R-Chol was essentially identical to that of control cells within the experimental range of error at all incubation time points.
cytotoxicity of R-Chol, the effect of R-Chol on the cell motility was also examined by wound healing assay. The data indicated that the motility of the cells incubated with 1e10 mM R-Chol did not differ from that of the cells incubated without R-Chol (Fig. S2), showing that R-Chol did not affect the motility of MDCK cells. Localization of fluorescent cholesterol analogues in cells To investigate the localization of R-Chol, MDCK cells were stained with R-Chol or NBD-cholesterol for 10 min and assessed by fluorescent microscopy immediately after removing the fluorophores by washing with DMEM. The NBD-cholesterols emitted intense fluorescent signals at perinuclear membranes, possibly the Golgi bodies and endosomes, but very little at the plasma membrane (Fig. 3B and C). These findings agreed with those of a previous study [9]. In contrast, R-Chol signals were emitted not only at the perinuclear membranes but also at the entire plasma membrane (Fig. 3A), and thus the shape of the cells was clearly defined. A comparison of fluorescence and DIC images confirmed that R-Chol was localized at the plasma membrane in all cells in the entire visual field under microscopy. Cultured MDCK cells form monolayers with tight junctions, which is a feature of epithelial cells. R-Chol signals were intense at the contact surface of confluent MDCK cells, which yielded mesh-work images when stained with R-Chol (Fig. 3A). These findings indicated that R-Chol localized to the plasma membrane immediately after staining. Time course of intracellular distribution of fluorophores The fluorophores were removed from MDCK cells by washing with DMEM after a 10-min incubation with R-Chol or NBDcholesterols. Next, the cells were incubated with DMEM with 10% FBS for 10 or 60 min and then were visualized. The NBD-cholesterol signals were slight or absent at the plasma membrane at all time points (Fig. 4B and C). Most NBD-cholesterol signals were found at the perinuclear membranes immediately after staining, but the intensity gradually decreased. Although R-Chol signals were detected at the plasma membrane at 10 and 60 min after staining, those in perinuclear membranes significantly increased over time (Fig. 4A). The result is consistent with the pathway of intracellular cholesterol transport proposed by previous studies [19e21]. Distribution of R-Chol in DRM fractions Mammalian cell membranes involve microdomains with abundant sphingolipids and free cholesterol; lipids at such
microdomains form an ordered liquid crystalline phase, and then the domains segregate from the surrounding region of a relatively disordered lipid phase [4]. In addition, such microdomains can be isolated from the membranes of various mammalian cells using non-ionic detergents such as Triton X-100 [22]. We used floatation assays to determine whether the distribution behavior of R-Chol is similar to that of free cholesterol in DRM fractions. Stained MDCK cells with R-Chol were treated with Triton X-100 on ice and were fractionated by density gradient centrifugation into DRM and non-DRM fractions. The DRM fractions contained 80% of the fluorescent signals generated by R-Chol (Fig. 5). Approximately 10% of the total fluorescent signal was located in the boundary between DRM and non-DRM fractions, with a few signals being located in non-DRM fractions. Discussion The hydroxyl group is believed to be important for the molecular recognition of cholesterol through hydrogen bonds with proximal molecules and also for the formation of microdomains [23]. For example, the probe cholesteryl BODIPY FL C12 (Life Technologies), which is synthesized by esterification of the hydroxyl group of cholesterol with a fluorescent group, localizes at cellular lipid bodies but not at the plasma membrane [24,25]. In contrast, the fluorescent cholesterol analogue, R-Chol, localized mostly at the plasma membrane like free cholesterol (Fig. 3A) and was found mostly in DRM fractions (Fig. 5). The hydroxyl group of cholesterol is believed to play important roles for the localization of cholesterol [12,23], whereas the amide group of R-Chol could conceivably play similar roles. The amide group of R-Chol exists between the sterane backbone and the zwitterionic fluorescent group (Fig. 1b). One of the characteristics that is common to hydroxyl group and amide group is to possibly act as hydrogen-donating and/or hydrogenaccepting group to form an intermolecular hydrogen bond [12]. This would explain the localization of R-Chol at the plasma membrane. The intracellular localization of cholesterol has often been investigated using NBD-cholesterol (Fig. 1e and f), which might be problematic because fluorescent signals would not be observed at the plasma membrane, where most intracellular free cholesterol is localized [7,9]. This suggests that chemical modification at the alkyl chain of cholesterol influences its intracellular localization. The current and previous findings [9,26,27] imply that both hydrophobic interaction through the alkyl chain and hydrogen bonding through the hydroxyl group would play important roles in the localization of free cholesterol at the plasma membrane. The sterol DHE (Fig. 1c) has also been applied as a fluorescent probe in studies of cellular cholesterol using microscopy because it
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Fig.3. Live MDCK cells stained with fluorescent cholesterol analogues. MDCK cells were observed using DIC and fluorescence microscopy immediately after incubation with fluorescent cholesterol analogues R-Chol (A), 22-NBD-cholesterol (B), and 25-NBD-cholesterol (C). Signals were found mostly at the plasma membrane of MDCK cells incubated with R-Chol (FL, A). In contrast, signals were found mostly at the perinuclear membranes of MDCK cells incubated with 22-NBD-cholesterol (FL, B) or 25-NBD-cholesterol (FL, C). DIC, differential interference contrast microscopy; FL, fluorescence microscopy. Panels D and E show autofluorescence emitted from non-stained cells under the same conditions as those shown in panel A for R-Chol and in panels B and C for NBD-cholesterols. Bar ¼ 50 mm.
Fig.4. Pulse chase images of fluorescent cholesterol analogues in live MDCK cells. Cells incubated with fluorescent cholesterol analogues R-Chol (A), 22-NBD-cholesterol (B), and 25NBD-cholesterol (C) examined by fluorescence microscopy at 10 and 60 min after analogue removal are shown. Typical high-magnification images of cells incubated with R-Chol (D), 22-NBD-cholesterol (E), and 25-NBD-cholesterol (F) at 10 and 60 min after analogue removal are also shown. Signal distribution changed from plasma to perinuclear membranes in the cells incubated with R-Chol (A,D) but not in those incubated with 22-NBD-cholesterol (B,E) or 25-NBD-cholesterol (C,F). Bar ¼ 50 mm.
accurately mimics the distribution of cholesterol in mammalian cells, probably because of its structural similarity to cholesterol [9,28,29]. DHE occurs naturally in yeasts, and it is an intrinsically fluorescent lipid. However, its excitation wavelength is in the ultraviolet (UV) region (300e350 nm), and thus special attachments are required that are normally not included with conventional microscopes to detect fluorescent signals emitted by DHE. Furthermore, close attention must be paid when using DHE in experimental studies because UV irradiation can damage live cells.
On the other hand, R-Chol signals were detectable by conventional fluorescence microscopy. Thus, R-Chol should be more readily applicable to observation using fluorescent microscopy with exciting the fluorophore by less damaging visible light. The fluorescent macrocyclic compound filipin (Fig. 1d) isolated from the actinomycete, Streptomyces filipinensis, forms a specific complex with free cholesterol [30,31]. Thus, filipin has served as a fluorescent probe for visualizing free cholesterol in cells [32]. Because its excitation wavelength is in the UV region, filipin is
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interior and returns to the plasma membrane [37], and the endocytic recycling compartment contains high levels of cholesterol [27]. The hydroxyl group of free cholesterol is esterified in the interior of cells via a lipid storage system [37,38], but R-Chol lacks a hydroxyl group and cannot be esterified (Fig. 1b), which might explain R-Chol accumulation at perinuclear membranes. Free cholesterol accumulates in the perinuclear membranes of a mutant cell line that has defective cholesterol esterification [38]. R-Chol signals were detectable at the plasma membrane within 1 h of cell staining (Fig. 4A and D). Because the fluorescent group of R-Chol (rhodamine) is comparatively photostable with relatively bright fluorescence, R-Chol should confer an advantage for continuous observation under microscopy. In addition, R-Chol was abundant in DRM fractions like free cholesterol (Fig. 5). Therefore, we consider that R-Chol could serve as a useful tool for investigating the dynamics of free cholesterol in the plasma membranes of live cells. Conclusions The novel fluorescent cholesterol analogue, R-Chol, did not influence the growth of MDCK cells in vitro. The chemical modification of cholesterol required to generate R-Chol scarcely affected cholesterol localization mostly at the plasma membrane and in DRM fractions of live cells. Acknowledgments Fig.5. Distribution of R-Chol in fractions determined using floatation assays. Assay procedures are described in detail in Materials and Methods. (A) Fluorescence intensity of R-Chol was measured at excitation and emission of 550 and 580 nm, respectively. Vertical axis: Ratios (%) of fluorescence intensity versus total fluorescence (sum of signals from all fractions). Values are shown as means ± standard deviations of five assays. Fraction numbers are shown on the horizontal axis from top of centrifuge tube. (B) Western blotting identified transferrin receptor, a non-DRM marker, in fraction 7 and identified caveolin 1, a DRM marker, in fractions 2 and 3.
readily applicable to studies of fixed cells but perhaps not to studies of live cells. BODIPY-cholesterol (Fig. 1g) has also been used as a fluorescent probe for investigations into the intracellular localization of free cholesterol, and it is commercially available as TopFluor cholesterol. The distribution of BODIPY-cholesterol signals in live cells is essentially the same as that of DHE [33]. On the other hand, BODIPY-cholesterol diffuses far more slowly in the lipid bilayer than free cholesterol [34]. One possible reason for this is that the size of the fluorophore, which is comparable to that of the sterane backbone, is linked to the hydrophobic portion of cholesterol (Fig. 1g). However, the hydrophobic portions of R-Chol and cholesterol are structurally identical (Fig. 1b), and therefore R-Chol and free cholesterol should diffuse throughout the lipid bilayer at similar rates. TRITC-PEG-cholesterol (tetramethylrhodamine B isothiocyanate [TRITC]-labeled poly(ethylene glycol) [PEG]-cholesterol) was reported to distribute in the outer plasma membrane leaflet and to be enriched in microdomains [35,36]. It is believed that TRITC-PEGcholesterol does not transverse lipid bilayer because of the bulky size of PEG group. Because the molecular size of TRITC-PEGcholesterol is much larger than that of R-Chol, the dynamics of TRITC-PEG-cholesterol in the plasma membrane should be different from that of R-Chol and cholesterol. The results of the pulse chase experiment using MDCK cells stained with R-Chol showed that R-Chol signals were observed in the perinuclear membranes within 1 h of staining (Fig. 4A and D). Free cholesterol in the plasma membrane moves into the cell
The authors thank Y. Onishi (National Institute of Advanced Industrial Science and Technology [AIST], Japan) for input on the cell viability assay, T. Mizuno (AIST) for providing the MDCK cell line and for advice on cell imaging, T. Yoko-o (AIST) for advice on floating assays, and T. Shimizu (AIST) for suggestions and discussion. This work was supported by a Grant-in-Aid for Scientific Research (C, 17550140) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ab.2015.09.003. References [1] Y. Lange, Disposition of intracellular cholesterol in human fibroblasts, J. Lipid Res. 32 (1991) 329e339. [2] D.E. Warnock, C. Roberts, M.S. Lutz, W.A. Blackburn, W.W. Young Jr., J.U. Baenziger, Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography, J. Biol. Chem. 268 (1993) 10145e10153. [3] R. Schroeder, E. London, D. Brown, Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 12130e12134. [4] K. Simons, E. Ikonen, Functional rafts in cell membranes, Nature 387 (1997) 569e572. [5] G. Gimpl, K. Gehrig-Burger, Cholesterol reporter molecules, Biosci. Rep. 27 (2007) 335e358. [6] D. Wünstner, Fluorescent sterols as tools in membrane biophysics and cell biology, Chem. Phys. Lipids 146 (2007) 1e25. [7] I.F. Craig, D.P. Via, W.W. Mantulin, H.J. Pownall, A.M. Gotto Jr., L.C. Smith, Low density lipoproteins reconstituted with steroids containing the nitrobenzoxadiazole fluorophore, J. Lipid Res. 22 (1981) 687e696. [8] A. Chattopadhyay, E. London, Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spinlabeled phospholipids, Biochemistry 26 (1987) 39e45. [9] S. Mukherjee, X. Zha, I. Tabas, F.R. Maxfield, Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog, Biophys. J. 75 (1998) 1915e1925. [10] S.L. Hussey, B.R. Peterson, Efficient delivery of streptavidin to mammalian cells: Clathrin-mediated endocytosis regulated by a synthetic ligand, J. Am. Chem. Soc. 124 (2002) 6265e6273.
Y. Ogawa, M. Tanaka / Analytical Biochemistry 492 (2016) 49e55 [11] S. Boonyarattanakalin, S.E. Martin, S.A. Dykstra, B.R. Peterson, Synthetic mimics of small mammalian cell surface receptors, J. Am. Chem. Soc. 126 (2004) 16379e16386. [12] J.M. Boggs, Lipid intermolecular hydrogen bonding: influence on structure organization and membrane function, Biochem. Biophys. Acta 906 (1987) 353e404. [13] Y.J. Im, S. Raychaudhuri, W.A. Prinz, J.H. Hurley, Structural mechanism for sterol sensing and transport by OSBP-related proteins, Nature 437 (2005) 154e158. [14] J. Fantini, F.J. Barrantes, How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains, Front. Physiol. 4 (2013), http://dx.doi.org/10.3389/ fphys.2013.00031. [15] P.L. Julian, A. Magnani, E.W. Meyer, W. Cole, Sterols: V. The i-cholesterylamines, J. Am. Chem. Soc. 70 (1948) 1834e1837. [16] E. Kosower, S. Winstein, Neighboring carbon and hydrogen: XXII. Homoallylic systems: the preparation and behavior of certain 3,5-cyclosteroids, J. Am. Chem. Soc. 78 (1956) 4347e4354. [17] M. Takahashi, M. Furihata, N. Akimitsu, M. Watanabe, S. Kaul, N. Yumoto, T. Okada, A highly bone marrow metastatic murine breast cancer model established through in vivo selection exhibits enhanced anchorageindependent growth and cell migration mediated by ICAM-1, Clin. Exp. Metastasis 25 (2008) 517e529. [18] D. Hanwell, T. Ishikawa, R. Saleki, D. Rotin, Trafficking and cell surface stability of the epithelial Naþ channel expressed in epitherial MadineDarby canine kidney cells, J. Biol. Chem. 277 (2002) 9772e9779. [19] J.D. Butler, J. Blanchette-Mackie, E. Goldin, R.R. O'Neill, G. Carstea, C.F. Roff, M.C. Patterson, S. Patel, M.E. Comly, A. Cooney, M.T. Vanier, R.O. Brady, P.G. Pentchev, Progesterone blocks cholesterol translocation from lysosomes, J. Biol. Chem. 267 (1992) 23797e23805. [20] L. Liscum, N.J. Munn, Intracellular cholesterol transport, Biochem. Biophys. Acta 1438 (1999) 19e37. € bius, E. van Donselaar, Y. Ohno-Iwashita, Y. Shimada, H.F.G. Heijnen, [21] W. Mo J.W. Slot, H.J. Geuze, Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway, Traffic 4 (2003) 222e231. [22] K. Fiedler, T. Kobayashi, T.V. Kurzchalia, K. Simons, Glycosphingolipidenriched, detergent-insoluble complexes in protein sorting in epitherial cells, Biochemistry 32 (1993) 6365e6373. € nen, I. Vattulainen, Atom-scale molecular interactions [23] P.S. Niemel€ a, M.T. Hyvo in lipid raft mixtures, Biochem. Biophys. Acta 1788 (2009) 122e135. [24] C.L. Bisgaier, L.L. Minton, A.D. Essenburg, A. White, R. Homan, Use of fluorescent cholesteryl ester microemulsions in cholesteryl ester transfer protein assays, J. Lipid Res. 34 (1993) 1625e1634. [25] E. Reaven, L. Tsai, S. Azhar, Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters, J. Biol. Chem. 271 (1996) 16208e16217.
55
[26] H.A. Scheidt, P. Müller, A. Herrmann, D. Huster, The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol, J. Biol. Chem. 278 (2003) 45563e45569. [27] R.F. Epand, A. Thomas, R. Brasseur, S.A. Vishwanathan, E. Hunter, R.M. Epand, Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains, Biochemistry 45 (2006) 6105e6114. [28] M. Hao, S.X. Lin, O.J. Karylowski, D. Wüstner, T.E. McGraw, F.R. Maxfield, Vesicular and non-vesicular sterol transport in living cells, J. Biol. Chem. 277 (2002) 609e617. [29] R. Homan, N. Esmaeil, L. Mendelsohn, G.J. Kato, A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase, Anal. Biochem. 441 (2013) 80e86. [30] G.B. Whitefield, T.D. Brock, A. Ammann, D. Gottlieb, H.E. Carter, Filipin, an antifungal antibiotic: isolation and properties, J. Am. Chem. Soc. 77 (1955) 4799e4801. [31] M.A.R.B. Castanho, A. Coutinho, M.J.E. Prieto, Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol, J. Biol. Chem. 267 (1992) 204e209. [32] J.D. Butler, M.E. Comly, H.S. Kruth, M. Vanier, M. Filling-Katz, J. Fink, N. Barton, H. Weintroub, J.M. Quirk, T. Tokoro, D.C. Marshall, R.O. Brady, P.G. Pentchev, NiemanePick variant disorder: comparison of errors of cellular cholesterol homeostasis in group D and group C fibroblasts, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 556e560. [33] D. Wünstner, L. Solanko, E. Sokol, O. Garvik, Z. Li, R. Bittman, T. Korte, A. Herrmann, Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol, Chem. Phys. Lipids 164 (2011) 221e235. [34] F.S. Ariola, Z. Li, C. Cornejo, R. Bittman, A.A. Heikal, Membrane fluidity and lipid order in ternary giant unilamellar vesicles using a new Bodipycholesterol derivative, Biophys. J. 96 (2009) 2696e2708. [35] S.B. Sato, K. Ishii, A. Makino, K. Iwabuchi, A. Yamaji-Hasegawa, Y. Senoh, I. Nagaoka, H. Sakuraba, T. Kobayashi, Distribution and transport of cholesterol-rich membrane domains monitored by a membrane-impermeant fluorescent polyethylene glycol-derivatized cholesterol, J. Biol. Chem. 279 (2004) 23790e23796. [36] M. Takahashi, M. Murate, M. Fukuda, S.B. Sato, A. Ohta, T. Kobayashi, Cholesterol controls lipid endocytosis through Rab11, Mol. Biol. Cell 18 (2007) 2667e2677. [37] Y. Lange, F. Strebel, T.L. Steck, Role of the plasma membrane in cholesterol esterification in rat hepatoma cells, J. Biol. Chem. 268 (1993) 13838e13843. [38] P.G. Pentchev, M.E. Comly, H.S. Kruth, M.T. Vanier, D.A. Wenger, S. Patel, R.O. Brady, A defect in cholesterol esterification in NiemannePick disease (type C) patients, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 8247e8251.