- Email: [email protected]
[20] Receptor-ligand internalization
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in living cells and the time course of the interaction cannot be evaluated with satisfactory precisionY A technique is now available that allows the visualization of the interaction between directly fluoresceinated ligands and living cells and enables one to follow their possible internalization. In living adherent cells, such a methodological approach was hindered previously by the relatively poor resolution of the conventional fluorescence microscope and the unfavorable signal-to-noise ratio. As a consequence, the visual information has not been exhaustive and subcellular localization has been limited to main cell compartments. 4 More recently, confocal imaging has provided new insights in the observation of fluorescent specimens. The virtual absence of out-of-focus blurring allows a much better definition of probe localization at the subcellular level together with the possibility of exploiting the three-dimensional reconstruction capability of most confocal systems. We have coupled a self-constructed flow chamber to an inverted confocal scanning laser microscope that allows long-term observation of adherent cells under controlled microenvironmental conditions.5 This method not only provides images of intact, nonfixed cells, but also allows one to change culture conditions and to observe living cell responses directly or to perform two-step staining to identify subcellular structures involved in the observed processes. The reliability of this procedure has been verified on a well-known model of receptor-ligand internalization, that of insulin and insulin receptor. The same technique has also been applied to studying the interactions between natural human antibodies, circulating in healthy subjects, and living human endothelial cells, fibroblasts, and proximal tubular epithelial cells. 6
2 p. Jackson and D. Blythe, in "Immunocytochemistry" (J. E. Beesley, ed.), p. 22. Oxford Univ. Press, Oxford, 1993. 3 p. Monaghan, D. Robertson, and E. J. Beesley, in "Immunocytochemistry" (J. E. Beesley, ed.), p. 47. Oxford Univ. Press, Oxford, 1993. 4 H. Lodish, D. Baltimore, A. Berk, Z. S. Lawrence, P. Matsudaira, and J. Darnell, in "Molecular Cell Biology" (J. Darnell, ed.), 3rd ed. Scientific American Books, New York, 1995. s V. Dall'Asta, R. Gatti, G. Orlandini, P. A. Rossi, B. M. Rotoli, R. Sala, O. Bussolati, and G. C. Gazzola, Exp. Cell Res. 231, 260 (1997). 6 N. Ronda, R. Gatti, G. Orlandini, and A. Borghetti, Clin. Exp. Immunol, 109(1), 211 (1997).
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Materials Flow Chamber
Despite the ever-increasing number of available fluorescent probes for living cells acting as vital, almost real-time indicators for a series of functional parameters, several technical problems arise in trying to exploit fully confocal laser scanning microscopy of viable cell monolayers. This kind of system is extremely sensitive to changes of the focal plane, the distance between lens and specimen must be very short, and high numerical aperture lenses are mandatory for optimal resolution. Moreover, three major general requisites must be satisfied in order to achieve improvement over the techniques previously available: (1) perturbation of culture conditions must be kept at a minimum; (2) the system should allow one to extend observation as long as desired; and (3) the relevant stimuli, used in the experimental protocol, should be applied easily and then washed out easily. The device described in this article is simple and inexpensive but fulfills all of the just described criteria. It was developed to fit a Multiprobe 2001Molecular Dynamics computer scanning laser microscope whose optical "conventional" side is based on a Nikon Diaphot inverted microscope (Sunnyvale, CA). The upper portion of the flow chamber is a transparent polyacetate block (Figs. 1, 2). According to the type of experiment to be carried out a hollow, whose depth can vary from 0.1 to 0.5 mm, has been milled on the bottom. When the cover slide (5 z 2.5 cm) bearing the cell culture is applied to the bottom of the block, the chamber is completed, with the
¢ FIG. 1. Schematic representation of the flow chamber. T h e b o t t o m face of the polyacetate block has to be sealed to the cover slide bearing the cell monolayer.
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AIR- CO 2
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FIG. 2. Schematic cutaway view of the flow chamber lodged in the aluminum stand on the microscope stage.
cover slide and the block serving as the floor and the vault, respectively. According to the depth of the hollow the volume of the flow chamber ranges between 60 and 300 /xl. Two vertical tunnels (diameter 4 mm) opening on the upper face of the block allow the introduction of small sylastic catheters (crossing the lid) for inflow and outflow of medium. The inlet and outlet tubes are usually connected to a micropipette and a vacuum apparatus, respectively, but a peristaltic pump for continuous replacement can also be used. Various sealers, provided they are nontoxic to cells, can be adopted in order to secure the cover slide to the polyacetate block and to prevent medium leakage. In our experience the best results can be achieved with silicon vacuum grease or, better, with a special silicon-based liquid gasket for a high-performance engine (Motorsil D, Arexons, Cernusco, Milano, Italy). In any case, a thin, narrow layer of the sealer must be applied around all the block edges in order to avoid untoward spreading into the chamber once the block itself is pushed on the cover slide. The flow chamber is lodged in a thermostatted stand (Fig. 3), a cylindrical aluminum block (diameter 14 × 2 cm) whose base has been opportunely mill finished so as to adhere tightly to the round opening in the microscope
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FIG. 3. View of the stand in place. Note the small tubes for medium substitution and air-CO2 supply. The cable from the aluminum stand is connected to the temperature control unit. The clear Perspex lid allows field lighting from the above condenser. Microscope stage translation (for field selection) carries about the whole system.
stage. A parallelepipedal niche (5 x 2.5 × 1.4 cm) is carved within the stand in order to contain precisely the flow chamber. The bottom of the niche shows an ellipsoidal slit (3 x 1 cm) devoted to objective apposition, wide enough to allow the observation of a large area of the culture by stage translation. The flow cell niche is closed tightly by a Perspex lid with three openings for a medium inlet and outlet and for atmosphere conditioning, respectively. The pressure of the lid also secures the two parts of the system fixed in place. Temperature in the flow chamber is controlled by a resistance coil embedded in the stand. A thermorelay probe is located at the inner surface of the slot; this configuration grants the widest surface for contact between the radiating element and the flow chamber.
Confocal Microscope The confocal scanning laser microscope employed is a Molecular Dynamics Multiprobe 2001 (inverted) equipped with an argon ion laser. Samples are observed through either a 60 or a 100x oil-immersion objective (Nikon PlanApo, NA 1.4), allowing vertical resolution around 1/zm, and the step size is set accordingly. The confocal aperture (pinhole) is set at 50 and 100/xm for 60 and 100x lenses, respectively. For practical purposes, the first setting is chosen whenever possible, as it provides the best vertical resolution (0.8/zm) but, due to the lower brightness, it requires optimal signal intensity.
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Because of its high quantum yield, fluorescein can be detected with a very low excitation power, which is crucial when imaging living ceils. Settings exceeding 1 mW produce phototoxic events in the culture (such as apoptosis or cell detachment) that are probably related to local energy delivery and consequent heating. Another advantage of using low excitation power is that photobleaching and background noise are negligible. In postfixation experiments with two tracers (see later), a secondary beam splitter is placed after the pinhole aperture and before the barrier filters. Signals from fluorescein and the second chromophore, usually tetramethylrhodamine isothiocyanate (TRITC) or Texas Red, are then acquired concurrently by two different photomultipliers that can be set independently in order to correct the unevenness in the quantum yield of the fluorophores. Once the apparatus is ready for image acquisition, it is important that the field of observation is chosen in bright-field microscopy. This allows one to take time in selecting the best field according to the experimental requirements without delivering useless or even noxious high power. Fresh medium or the relevant study solutions can be replaced without shifting along the x, y, or z axis. At each experimental step, a section series is acquired along the whole thickness of the cell (for endothelial cells, 5-6 sections). Complete scanning yields information about the whole cell, allows one to choose the most representative section, and provides the material for accurate three-dimensional reconstruction. Whenever it is important to give a global representation of an internalization process, the image series are smoothed with a Gaussian 3 x 3 × 3 kernel filter and three-dimensional reconstruction is performed according to a maximum intensity algorithm. In other words, for all pixels of a given x-y coordinate in the series, the one with the highest intensity is chosen for final image rendering. Image processing is performed on a Silicon Graphics Personal Iris workstation (Image Space Software, Molecular Dynamics).
Preparation of Ligand-Fluorescein Conjugates Human insulin (Sigma, St. Louis, MO), purified normal human IgG (pooled normal IgG, Sandoglobulin, Sandoz, Basel, Switzerland), and IgG purified from single healthy donors 6 are coupled to fluorescein isothiocyanate (FITC), 7 purified by chromatography on a Sephadex G-25 column, and dialyzed extensively against phosphate-buffered saline (PBS) using a 3500-kDa cutoff membrane (Spectra/Por, Spectrum Medical Industries, Inc., Los Angeles, CA) to allow total elimination of free FITC, as detected 7 A. Johnstone and R. Thorpe, "Immunochemistry in Practice," p. 258. Blackwell, Oxford, 1982.
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by spectrophotometric analysis of the dialysis medium at 495 nm. Fluoresceinated ligands are finally dialyzed in Dulbecco's modified Eagle's medium (DMEM) containing L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin, and filtered with 0.22-/zm filters into sterile tubes. The FITC to protein ratio is 6.3 for IgG and 5.5 for insulin, and final protein concentrations are 8 and 6 mg/ml, respectively. The ligand concentrations used, as indicated in the description of each experiment, are obtained by diluting ligand solutions with DMEM with the addition of L-glutamine, penicillinstreptomycin, and fetal calf serum at a final concentration of 5% (v/v). The presence of FCS does not modify ligand-cell interactions, as demonstrated by comparison with serum-free experiments, but is associated with better preserved cell morphology and greater adhesion to the cover slide, especially in the case of endothelial cells. Aliquots of the last dialysis medium of ligand-FITC are filtered and saved for incubating with ceils at least 2 hr before the beginning of the experiments to further exclude contamination of the ligand-FITC solutions by free FITC (see later). When indicated, possible effects due to lipopolysaccharide (LPS) contamination of the ligand solutions are excluded by repeating the experiments with the addition of 5 txg/ml polymyxin B to all incubation media. Fluoresceinated ligands are kept sterile at 4° without preservatives for up to 2 months. Cell Culture
Human umbilical cord vein endothelial cells (EC), 8 human fibroblasts, 9 and human proximal tubular epithelial cells (PTEC) I° at the first passage are grown to subconfluence without attachment factors (to reduce background signal) on a glass cover slide fitting the flow chamber. In order to obtain good cell density within 24 hr, endothelial cells (which proliferate slowly on glass and in the absence of attachment and growth factors) are seeded carefully, placing 0.5 ml of cell suspension (105 cells/ml) on the cover slide, letting the cells adhere for 4 hr in the incubator, and finally adding the proper amount of culture medium for incubation. All the experiments are performed 24 hr after seeding the cells. Membrane Binding of Ligands The system described allows one to observe membrane binding of a ligand to living cells. In fact, we have been able to show that purified normal IgG binds to the cell membrane of cultured living fibroblasts and is not 8 S. Oravec, N. Ronda, A. Carayon, J. Milliez, M. D. Kazatchkine, and A. Hornych, Nephrol. Dialys, Transplant. 10, 796 (1995). 9 G. Gazzola, V. Dall'Asta, and G. Guidotti, J. Biol. Chem. 255, 929 (1980). 10 C. J. Detrisac, M. A. Sens, A. J. Garvin, S. S. Spicer, and D. A. Sens, Kidney Int. 25, 383 (1984).
[20]
R E C E P T O R - L I G AINTERNALIZATION ND
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FIG. 4. IgG-FITC membrane binding to a livingfibroblast (left-hand side) and to endothelial cells (right-hand side). Bars: 10 tzm (left) and 5 tzm (right). Reproduced with permission from N. Ronda, R. Gatti, G. Orlandini, and A. Borghetti, Clin. Exp. Immunol. 109(1), 211 (1997). internalized (Fig. 4). Before starting a specific experiment, it is advisable to obtain a basal image of the cells that had been incubated for 2 hr with the dialysis medium saved after the final dialysis of I g G - F I T C in DMEM. The complete absence of a fluorescent signal excludes free FITC contamination of the fluoresceinated ligand. Such a preliminary test should be performed before every experiment described in this article. We then incubate fibroblasts in the confocal flow chamber for 30 rain with I g G - F I T C at 2 rag/ ml in standard culture conditions, wash them with culture medium, and observe the cells at 10- to 15-min intervals for 2 hr. A fluorescent signal is detectable with IgG diluted out to 0.5 mg/ml. We did not observe I g G - F I T C binding to P T E C under the same conditions, even using an IgG concentration of 8 mg/ml. In contrast, normal IgG entered living E C within minutes (see later). In order to show membrane binding of IgG to endothelial cells, we have inhibited cell energy-dependent processes by setting the flow chamber temperature to 27 ° (the lowest temperature tolerated by E C without cell damage in our system) and observed cells after 5 min of incubation with 2 mg/ml of I g G - F I T C (Fig. 4). R e c e p t o r - L i g a n d Internalization Whenever a signal from the ligand under investigation is detected, it is necessary to check its localization within the cell and it is advisable to
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identify the nature of the structure or compartment involved. Moreover, it is important to monitor the morphological evolution of the process. In order to achieve these goals, it is useful to counterstain the cells. This is possible, with no need to change the field of observation, by flushing i tzM calcein AM through the flow chamber medium. This neutral dye is converted by intracellular esterases into a fluorescent anionic compound, optimally excited at 488 nm with a peak emission around 520 nm, which stains the nucleus and the cytoplasm (nucleus/cytoplasm signal intensity ratio, 2:1), except for cationic compartments. Calcein allows one to check cell vitality as the fluorescent signal immediately disappears in the presence of membrane damage, despite residual cell esterase activity. 1: Because the calcein signal often overwhelms that emitted by the internalized ligand (see later), once counterstaining is carried out, possible subsequent changes in the distribution pattern of the internalized ligand cannot be visualized. Therefore, it is possible to program the counterstaining at various times of incubation in different samples or to track the relevant changes in a single microscopic field and to delay counterstaining until appropriate. In order to verify the actual intracellular nature of the signal, when the raw and the counterstained images of the same field are overlapped digitally, usually the intensity of the latter must be reduced evenly. However, comparison of the two separate images also can provide additional useful information, as will be illustrated later. As an example of the information achievable related to morphology, timing, and specificity (i.e., receptor involvement) of the internalization of a ligand, we first describe the visualization of receptor-ligand internalization in a well-known model (insulin and endothelial cells) and then in the case of a previously unknown ligand-cell interaction (IgG in endothelial cells). Confocal observation of living endothelial cells incubated with insulinFITC allows the direct visualization of insulin internalization, the morphology and timing of which are consistent with previous knowledge of the process. Insulin binding and internalization are almost immediate, with a fluorescent cytoplasmic fibrillar signal evident after only 2 min of incubation with 1 mg/ml insulin-FITC followed by washing with culture medium (Fig. 5A, see color insert). The initial fibrillar aspect evolves rapidly, and after 10 min the fluorescence is distributed almost entirely in cytoplasmic granules, some of which are larger bodies of 2-8 tzm in diameter (Fig. 5B, see color insert). The signal from intracellular insulin-FITC is relatively weak as compared to that of calcein and is no longer appreciable after counterstaining 11 p. Moore, I. MacCoubrey, and R. Haughland, J. Cell Biol. l U , 58 (1990).
[9,0]
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(Figs. 5C and 5D, see color insert). For overlapping images, it is necessary to attenuate the calcein signal evenly by 25-35% to show insulin localization together with cytoplasmic staining (Fig. 5E, see color insert). This is particularly evident from the comparison between Fig. 5B and Fig. 5D. For the acquisition of the latter, the sensitivity of the photomultiplier, which was appropriate for calcein, was too weak to detect insulin-FITC. Therefore the large granules containing insulin and excluding calcein appear as negative bodies. The preservation from calcein loading indicates that these are acidic compartments, likely to correspond to late endosomes involved in insulin catabolism 12 (Fig. 5D). As stated earlier, the incubation of living endothelial cells with IgGFITC at 2 mg/ml is followed by the internalization of IgG, detectable after 10-15 min of incubation and most evident after 20-30 min. For experiments excluding the presence of contaminants other than IgG that could be responsible for the intracellular fluorescence, see Ronda et al. 6 The intracellular localization of fluorescence can be demonstrated first through a vertical section of a protrusion of a cell (Fig. 6, see color insert) and by calcein loading, as shown earlier, but without a need to reduce counterstain intensity, as the IgG-FITC signal is higher. The fluorescence pattern is that of a fibrillar network, particularly abundant in peripheral areas of the cytoplasm and in protrusions that apparently connect adjacent cells (Fig. 6, top). After 1 hr, most of the fluorescence is localized in cytoplasmic granules and it is reduced greatly after 2 hr. The time course, morphology, and inhibition by the low temperature of the IgG internalization process in endothelial cells are consistent with a receptor-mediated mechanism rather than with pinocytosis, which is a slow process, with poor quantitative efficiency, leading to a nonspecific uptake of extracellular medium. The demonstration that pinocytosis was not responsible for our observation came from the lack of internalization of fluoresceinated IgG fragments by endothelial cells under the same conditions. Proximal tubular epithelial cells, whose nonspecific reabsorption of proteins from preurine is well known, show cytoplasmic granules of IgG after only 48 hr of incubation with IgG-FITC or fluoresceinated IgG fragments. As compared to insulin-FITC, the process of internalization appears morphologically similar, but insulin internalization is faster and, as noted earlier, requires a higher power of excitation and magnification of the signal. Such a difference in fluorescence intensity is likely to be due mainly to the smaller number of FITC molecules per molecule of the ligand in the case of insulin (5600 molecular weight) as compared to IgG (150,000), rather than determined by differences in cell receptor number or affinity. 12 j. Carpentier, Diabetologia 37, Sl17 (1994).
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Two-Step Staining for Identification of S u b c e l l u l a r S t r u c t u r e s The fibrillar pattern of cytoplasmic fluorescence shortly following internalization of I g G - F I T C and insulin-FITC, together with the well-known function of microtubules in molecular/vesicular intracellular trafficking, suggests the possibility that microtubules are involved in the r e c e p t o r ligand internalization systems. We have thus designed an experimental procedure to demonstrate the possible overlapping of signals from internalized fluoresceinated ligands and microtubules stained with a different probe. As a preliminary test we ensured that the cytoplasmic I g G - F I T C signal remains unmodified after cell fixation with methanol. We then induced internalization of I g G - F I T C in endothelial cells in the confocal flow chamber as described, turned off the thermostat, fixed the cells by flushing 100% methanol at 4 ° through the chamber for 2 min, and washed the cells with PBS at room temperature. We then performed indirect immunofluorescence at room temperature using a mouse monoclonal anti-a-tubulin antibody (Sigma) and an antimouse IgG T R I T C (Aex = 552 nm; Aem = 570 nm)conjugated antibody (Sigma). Images were then acquired using a doublechannel system placing a secondary beam splitter (565 nm) after the pinhole and 535-nm ( _ 15) bandpass and 570-nm long-pass barrier filters before two separate photomultipliers. After acquisition, barrier filters were inverted to check for contamination of fluorescein image by TRITC; indeed the absolute negativity of the field excluded such a possibility. The actual purity of each signal was also enhanced using the "separation enhancement" routine of the software, which subtracts a chosen percentage of the signal of each channel from the other one. The images obtained show a perfect correspondence between internalized ligand-FITC localization and some of the microtubular filaments (Fig. 7, see color insert). It is known that microtubules, intermediate filaments, and parts of the endoplasmic reticulum often colocalize, 13 but the actual involvement of microtubules in the internalization of IgG has been demonstrated by the total inhibition of the process obtained by pretreating endothelial cells for 20 min with 100/zg/ml colchicine before incubation with IgG-FITC. 6 Acknowledgments This work was funded by the Department of Clinical Medicine, Nephrology and Health Sciences and partly by CNR target project "Biotechnology." The confocal apparatus is a facility of the Centro Interfacoltfi Misure of the University of Parma. 13H. Lodish,D. Baltimore, A. Berk, S. L. Zipursky,P. Matsudaira, and J. Darnell, in "Molecular Cell Biology" (J. Darnell, ed.). ScientificAmerican Books, New York, 1995.
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in living cells and the time course of the interaction cannot be evaluated with satisfactory precisionY A technique is now available that allows the visualization of the interaction between directly fluoresceinated ligands and living cells and enables one to follow their possible internalization. In living adherent cells, such a methodological approach was hindered previously by the relatively poor resolution of the conventional fluorescence microscope and the unfavorable signal-to-noise ratio. As a consequence, the visual information has not been exhaustive and subcellular localization has been limited to main cell compartments. 4 More recently, confocal imaging has provided new insights in the observation of fluorescent specimens. The virtual absence of out-of-focus blurring allows a much better definition of probe localization at the subcellular level together with the possibility of exploiting the three-dimensional reconstruction capability of most confocal systems. We have coupled a self-constructed flow chamber to an inverted confocal scanning laser microscope that allows long-term observation of adherent cells under controlled microenvironmental conditions.5 This method not only provides images of intact, nonfixed cells, but also allows one to change culture conditions and to observe living cell responses directly or to perform two-step staining to identify subcellular structures involved in the observed processes. The reliability of this procedure has been verified on a well-known model of receptor-ligand internalization, that of insulin and insulin receptor. The same technique has also been applied to studying the interactions between natural human antibodies, circulating in healthy subjects, and living human endothelial cells, fibroblasts, and proximal tubular epithelial cells. 6
2 p. Jackson and D. Blythe, in "Immunocytochemistry" (J. E. Beesley, ed.), p. 22. Oxford Univ. Press, Oxford, 1993. 3 p. Monaghan, D. Robertson, and E. J. Beesley, in "Immunocytochemistry" (J. E. Beesley, ed.), p. 47. Oxford Univ. Press, Oxford, 1993. 4 H. Lodish, D. Baltimore, A. Berk, Z. S. Lawrence, P. Matsudaira, and J. Darnell, in "Molecular Cell Biology" (J. Darnell, ed.), 3rd ed. Scientific American Books, New York, 1995. s V. Dall'Asta, R. Gatti, G. Orlandini, P. A. Rossi, B. M. Rotoli, R. Sala, O. Bussolati, and G. C. Gazzola, Exp. Cell Res. 231, 260 (1997). 6 N. Ronda, R. Gatti, G. Orlandini, and A. Borghetti, Clin. Exp. Immunol, 109(1), 211 (1997).
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Materials Flow Chamber
Despite the ever-increasing number of available fluorescent probes for living cells acting as vital, almost real-time indicators for a series of functional parameters, several technical problems arise in trying to exploit fully confocal laser scanning microscopy of viable cell monolayers. This kind of system is extremely sensitive to changes of the focal plane, the distance between lens and specimen must be very short, and high numerical aperture lenses are mandatory for optimal resolution. Moreover, three major general requisites must be satisfied in order to achieve improvement over the techniques previously available: (1) perturbation of culture conditions must be kept at a minimum; (2) the system should allow one to extend observation as long as desired; and (3) the relevant stimuli, used in the experimental protocol, should be applied easily and then washed out easily. The device described in this article is simple and inexpensive but fulfills all of the just described criteria. It was developed to fit a Multiprobe 2001Molecular Dynamics computer scanning laser microscope whose optical "conventional" side is based on a Nikon Diaphot inverted microscope (Sunnyvale, CA). The upper portion of the flow chamber is a transparent polyacetate block (Figs. 1, 2). According to the type of experiment to be carried out a hollow, whose depth can vary from 0.1 to 0.5 mm, has been milled on the bottom. When the cover slide (5 z 2.5 cm) bearing the cell culture is applied to the bottom of the block, the chamber is completed, with the
¢ FIG. 1. Schematic representation of the flow chamber. T h e b o t t o m face of the polyacetate block has to be sealed to the cover slide bearing the cell monolayer.
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AIR- CO 2
MEDIUM
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M~l-)n rrd
FIG. 2. Schematic cutaway view of the flow chamber lodged in the aluminum stand on the microscope stage.
cover slide and the block serving as the floor and the vault, respectively. According to the depth of the hollow the volume of the flow chamber ranges between 60 and 300 /xl. Two vertical tunnels (diameter 4 mm) opening on the upper face of the block allow the introduction of small sylastic catheters (crossing the lid) for inflow and outflow of medium. The inlet and outlet tubes are usually connected to a micropipette and a vacuum apparatus, respectively, but a peristaltic pump for continuous replacement can also be used. Various sealers, provided they are nontoxic to cells, can be adopted in order to secure the cover slide to the polyacetate block and to prevent medium leakage. In our experience the best results can be achieved with silicon vacuum grease or, better, with a special silicon-based liquid gasket for a high-performance engine (Motorsil D, Arexons, Cernusco, Milano, Italy). In any case, a thin, narrow layer of the sealer must be applied around all the block edges in order to avoid untoward spreading into the chamber once the block itself is pushed on the cover slide. The flow chamber is lodged in a thermostatted stand (Fig. 3), a cylindrical aluminum block (diameter 14 × 2 cm) whose base has been opportunely mill finished so as to adhere tightly to the round opening in the microscope
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FIG. 3. View of the stand in place. Note the small tubes for medium substitution and air-CO2 supply. The cable from the aluminum stand is connected to the temperature control unit. The clear Perspex lid allows field lighting from the above condenser. Microscope stage translation (for field selection) carries about the whole system.
stage. A parallelepipedal niche (5 x 2.5 × 1.4 cm) is carved within the stand in order to contain precisely the flow chamber. The bottom of the niche shows an ellipsoidal slit (3 x 1 cm) devoted to objective apposition, wide enough to allow the observation of a large area of the culture by stage translation. The flow cell niche is closed tightly by a Perspex lid with three openings for a medium inlet and outlet and for atmosphere conditioning, respectively. The pressure of the lid also secures the two parts of the system fixed in place. Temperature in the flow chamber is controlled by a resistance coil embedded in the stand. A thermorelay probe is located at the inner surface of the slot; this configuration grants the widest surface for contact between the radiating element and the flow chamber.
Confocal Microscope The confocal scanning laser microscope employed is a Molecular Dynamics Multiprobe 2001 (inverted) equipped with an argon ion laser. Samples are observed through either a 60 or a 100x oil-immersion objective (Nikon PlanApo, NA 1.4), allowing vertical resolution around 1/zm, and the step size is set accordingly. The confocal aperture (pinhole) is set at 50 and 100/xm for 60 and 100x lenses, respectively. For practical purposes, the first setting is chosen whenever possible, as it provides the best vertical resolution (0.8/zm) but, due to the lower brightness, it requires optimal signal intensity.
[201
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Because of its high quantum yield, fluorescein can be detected with a very low excitation power, which is crucial when imaging living ceils. Settings exceeding 1 mW produce phototoxic events in the culture (such as apoptosis or cell detachment) that are probably related to local energy delivery and consequent heating. Another advantage of using low excitation power is that photobleaching and background noise are negligible. In postfixation experiments with two tracers (see later), a secondary beam splitter is placed after the pinhole aperture and before the barrier filters. Signals from fluorescein and the second chromophore, usually tetramethylrhodamine isothiocyanate (TRITC) or Texas Red, are then acquired concurrently by two different photomultipliers that can be set independently in order to correct the unevenness in the quantum yield of the fluorophores. Once the apparatus is ready for image acquisition, it is important that the field of observation is chosen in bright-field microscopy. This allows one to take time in selecting the best field according to the experimental requirements without delivering useless or even noxious high power. Fresh medium or the relevant study solutions can be replaced without shifting along the x, y, or z axis. At each experimental step, a section series is acquired along the whole thickness of the cell (for endothelial cells, 5-6 sections). Complete scanning yields information about the whole cell, allows one to choose the most representative section, and provides the material for accurate three-dimensional reconstruction. Whenever it is important to give a global representation of an internalization process, the image series are smoothed with a Gaussian 3 x 3 × 3 kernel filter and three-dimensional reconstruction is performed according to a maximum intensity algorithm. In other words, for all pixels of a given x-y coordinate in the series, the one with the highest intensity is chosen for final image rendering. Image processing is performed on a Silicon Graphics Personal Iris workstation (Image Space Software, Molecular Dynamics).
Preparation of Ligand-Fluorescein Conjugates Human insulin (Sigma, St. Louis, MO), purified normal human IgG (pooled normal IgG, Sandoglobulin, Sandoz, Basel, Switzerland), and IgG purified from single healthy donors 6 are coupled to fluorescein isothiocyanate (FITC), 7 purified by chromatography on a Sephadex G-25 column, and dialyzed extensively against phosphate-buffered saline (PBS) using a 3500-kDa cutoff membrane (Spectra/Por, Spectrum Medical Industries, Inc., Los Angeles, CA) to allow total elimination of free FITC, as detected 7 A. Johnstone and R. Thorpe, "Immunochemistry in Practice," p. 258. Blackwell, Oxford, 1982.
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by spectrophotometric analysis of the dialysis medium at 495 nm. Fluoresceinated ligands are finally dialyzed in Dulbecco's modified Eagle's medium (DMEM) containing L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin, and filtered with 0.22-/zm filters into sterile tubes. The FITC to protein ratio is 6.3 for IgG and 5.5 for insulin, and final protein concentrations are 8 and 6 mg/ml, respectively. The ligand concentrations used, as indicated in the description of each experiment, are obtained by diluting ligand solutions with DMEM with the addition of L-glutamine, penicillinstreptomycin, and fetal calf serum at a final concentration of 5% (v/v). The presence of FCS does not modify ligand-cell interactions, as demonstrated by comparison with serum-free experiments, but is associated with better preserved cell morphology and greater adhesion to the cover slide, especially in the case of endothelial cells. Aliquots of the last dialysis medium of ligand-FITC are filtered and saved for incubating with ceils at least 2 hr before the beginning of the experiments to further exclude contamination of the ligand-FITC solutions by free FITC (see later). When indicated, possible effects due to lipopolysaccharide (LPS) contamination of the ligand solutions are excluded by repeating the experiments with the addition of 5 txg/ml polymyxin B to all incubation media. Fluoresceinated ligands are kept sterile at 4° without preservatives for up to 2 months. Cell Culture
Human umbilical cord vein endothelial cells (EC), 8 human fibroblasts, 9 and human proximal tubular epithelial cells (PTEC) I° at the first passage are grown to subconfluence without attachment factors (to reduce background signal) on a glass cover slide fitting the flow chamber. In order to obtain good cell density within 24 hr, endothelial cells (which proliferate slowly on glass and in the absence of attachment and growth factors) are seeded carefully, placing 0.5 ml of cell suspension (105 cells/ml) on the cover slide, letting the cells adhere for 4 hr in the incubator, and finally adding the proper amount of culture medium for incubation. All the experiments are performed 24 hr after seeding the cells. Membrane Binding of Ligands The system described allows one to observe membrane binding of a ligand to living cells. In fact, we have been able to show that purified normal IgG binds to the cell membrane of cultured living fibroblasts and is not 8 S. Oravec, N. Ronda, A. Carayon, J. Milliez, M. D. Kazatchkine, and A. Hornych, Nephrol. Dialys, Transplant. 10, 796 (1995). 9 G. Gazzola, V. Dall'Asta, and G. Guidotti, J. Biol. Chem. 255, 929 (1980). 10 C. J. Detrisac, M. A. Sens, A. J. Garvin, S. S. Spicer, and D. A. Sens, Kidney Int. 25, 383 (1984).
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FIG. 4. IgG-FITC membrane binding to a livingfibroblast (left-hand side) and to endothelial cells (right-hand side). Bars: 10 tzm (left) and 5 tzm (right). Reproduced with permission from N. Ronda, R. Gatti, G. Orlandini, and A. Borghetti, Clin. Exp. Immunol. 109(1), 211 (1997). internalized (Fig. 4). Before starting a specific experiment, it is advisable to obtain a basal image of the cells that had been incubated for 2 hr with the dialysis medium saved after the final dialysis of I g G - F I T C in DMEM. The complete absence of a fluorescent signal excludes free FITC contamination of the fluoresceinated ligand. Such a preliminary test should be performed before every experiment described in this article. We then incubate fibroblasts in the confocal flow chamber for 30 rain with I g G - F I T C at 2 rag/ ml in standard culture conditions, wash them with culture medium, and observe the cells at 10- to 15-min intervals for 2 hr. A fluorescent signal is detectable with IgG diluted out to 0.5 mg/ml. We did not observe I g G - F I T C binding to P T E C under the same conditions, even using an IgG concentration of 8 mg/ml. In contrast, normal IgG entered living E C within minutes (see later). In order to show membrane binding of IgG to endothelial cells, we have inhibited cell energy-dependent processes by setting the flow chamber temperature to 27 ° (the lowest temperature tolerated by E C without cell damage in our system) and observed cells after 5 min of incubation with 2 mg/ml of I g G - F I T C (Fig. 4). R e c e p t o r - L i g a n d Internalization Whenever a signal from the ligand under investigation is detected, it is necessary to check its localization within the cell and it is advisable to
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identify the nature of the structure or compartment involved. Moreover, it is important to monitor the morphological evolution of the process. In order to achieve these goals, it is useful to counterstain the cells. This is possible, with no need to change the field of observation, by flushing i tzM calcein AM through the flow chamber medium. This neutral dye is converted by intracellular esterases into a fluorescent anionic compound, optimally excited at 488 nm with a peak emission around 520 nm, which stains the nucleus and the cytoplasm (nucleus/cytoplasm signal intensity ratio, 2:1), except for cationic compartments. Calcein allows one to check cell vitality as the fluorescent signal immediately disappears in the presence of membrane damage, despite residual cell esterase activity. 1: Because the calcein signal often overwhelms that emitted by the internalized ligand (see later), once counterstaining is carried out, possible subsequent changes in the distribution pattern of the internalized ligand cannot be visualized. Therefore, it is possible to program the counterstaining at various times of incubation in different samples or to track the relevant changes in a single microscopic field and to delay counterstaining until appropriate. In order to verify the actual intracellular nature of the signal, when the raw and the counterstained images of the same field are overlapped digitally, usually the intensity of the latter must be reduced evenly. However, comparison of the two separate images also can provide additional useful information, as will be illustrated later. As an example of the information achievable related to morphology, timing, and specificity (i.e., receptor involvement) of the internalization of a ligand, we first describe the visualization of receptor-ligand internalization in a well-known model (insulin and endothelial cells) and then in the case of a previously unknown ligand-cell interaction (IgG in endothelial cells). Confocal observation of living endothelial cells incubated with insulinFITC allows the direct visualization of insulin internalization, the morphology and timing of which are consistent with previous knowledge of the process. Insulin binding and internalization are almost immediate, with a fluorescent cytoplasmic fibrillar signal evident after only 2 min of incubation with 1 mg/ml insulin-FITC followed by washing with culture medium (Fig. 5A, see color insert). The initial fibrillar aspect evolves rapidly, and after 10 min the fluorescence is distributed almost entirely in cytoplasmic granules, some of which are larger bodies of 2-8 tzm in diameter (Fig. 5B, see color insert). The signal from intracellular insulin-FITC is relatively weak as compared to that of calcein and is no longer appreciable after counterstaining 11 p. Moore, I. MacCoubrey, and R. Haughland, J. Cell Biol. l U , 58 (1990).
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(Figs. 5C and 5D, see color insert). For overlapping images, it is necessary to attenuate the calcein signal evenly by 25-35% to show insulin localization together with cytoplasmic staining (Fig. 5E, see color insert). This is particularly evident from the comparison between Fig. 5B and Fig. 5D. For the acquisition of the latter, the sensitivity of the photomultiplier, which was appropriate for calcein, was too weak to detect insulin-FITC. Therefore the large granules containing insulin and excluding calcein appear as negative bodies. The preservation from calcein loading indicates that these are acidic compartments, likely to correspond to late endosomes involved in insulin catabolism 12 (Fig. 5D). As stated earlier, the incubation of living endothelial cells with IgGFITC at 2 mg/ml is followed by the internalization of IgG, detectable after 10-15 min of incubation and most evident after 20-30 min. For experiments excluding the presence of contaminants other than IgG that could be responsible for the intracellular fluorescence, see Ronda et al. 6 The intracellular localization of fluorescence can be demonstrated first through a vertical section of a protrusion of a cell (Fig. 6, see color insert) and by calcein loading, as shown earlier, but without a need to reduce counterstain intensity, as the IgG-FITC signal is higher. The fluorescence pattern is that of a fibrillar network, particularly abundant in peripheral areas of the cytoplasm and in protrusions that apparently connect adjacent cells (Fig. 6, top). After 1 hr, most of the fluorescence is localized in cytoplasmic granules and it is reduced greatly after 2 hr. The time course, morphology, and inhibition by the low temperature of the IgG internalization process in endothelial cells are consistent with a receptor-mediated mechanism rather than with pinocytosis, which is a slow process, with poor quantitative efficiency, leading to a nonspecific uptake of extracellular medium. The demonstration that pinocytosis was not responsible for our observation came from the lack of internalization of fluoresceinated IgG fragments by endothelial cells under the same conditions. Proximal tubular epithelial cells, whose nonspecific reabsorption of proteins from preurine is well known, show cytoplasmic granules of IgG after only 48 hr of incubation with IgG-FITC or fluoresceinated IgG fragments. As compared to insulin-FITC, the process of internalization appears morphologically similar, but insulin internalization is faster and, as noted earlier, requires a higher power of excitation and magnification of the signal. Such a difference in fluorescence intensity is likely to be due mainly to the smaller number of FITC molecules per molecule of the ligand in the case of insulin (5600 molecular weight) as compared to IgG (150,000), rather than determined by differences in cell receptor number or affinity. 12 j. Carpentier, Diabetologia 37, Sl17 (1994).
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Two-Step Staining for Identification of S u b c e l l u l a r S t r u c t u r e s The fibrillar pattern of cytoplasmic fluorescence shortly following internalization of I g G - F I T C and insulin-FITC, together with the well-known function of microtubules in molecular/vesicular intracellular trafficking, suggests the possibility that microtubules are involved in the r e c e p t o r ligand internalization systems. We have thus designed an experimental procedure to demonstrate the possible overlapping of signals from internalized fluoresceinated ligands and microtubules stained with a different probe. As a preliminary test we ensured that the cytoplasmic I g G - F I T C signal remains unmodified after cell fixation with methanol. We then induced internalization of I g G - F I T C in endothelial cells in the confocal flow chamber as described, turned off the thermostat, fixed the cells by flushing 100% methanol at 4 ° through the chamber for 2 min, and washed the cells with PBS at room temperature. We then performed indirect immunofluorescence at room temperature using a mouse monoclonal anti-a-tubulin antibody (Sigma) and an antimouse IgG T R I T C (Aex = 552 nm; Aem = 570 nm)conjugated antibody (Sigma). Images were then acquired using a doublechannel system placing a secondary beam splitter (565 nm) after the pinhole and 535-nm ( _ 15) bandpass and 570-nm long-pass barrier filters before two separate photomultipliers. After acquisition, barrier filters were inverted to check for contamination of fluorescein image by TRITC; indeed the absolute negativity of the field excluded such a possibility. The actual purity of each signal was also enhanced using the "separation enhancement" routine of the software, which subtracts a chosen percentage of the signal of each channel from the other one. The images obtained show a perfect correspondence between internalized ligand-FITC localization and some of the microtubular filaments (Fig. 7, see color insert). It is known that microtubules, intermediate filaments, and parts of the endoplasmic reticulum often colocalize, 13 but the actual involvement of microtubules in the internalization of IgG has been demonstrated by the total inhibition of the process obtained by pretreating endothelial cells for 20 min with 100/zg/ml colchicine before incubation with IgG-FITC. 6 Acknowledgments This work was funded by the Department of Clinical Medicine, Nephrology and Health Sciences and partly by CNR target project "Biotechnology." The confocal apparatus is a facility of the Centro Interfacoltfi Misure of the University of Parma. 13H. Lodish,D. Baltimore, A. Berk, S. L. Zipursky,P. Matsudaira, and J. Darnell, in "Molecular Cell Biology" (J. Darnell, ed.). ScientificAmerican Books, New York, 1995.