Stimulation of Fluorescence in a Small Contact Region between Rat

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Jun 10, 2016 - ment of IgE fluorescence in the field of the evanescent wave. Microscopic ... was undertaken to probe the distribution of fluorescein-la- beled IgE .... Monolayers in the Presence of Specific ZgE-Monolayers containing. DPPC, DSPC ... in triplicate. Total Internal Reflection Experiments-The 488-nm line of an.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 11, Issue of June 10, pp. 6440-6445, 1982 Printed in U.S A .

Stimulation of Fluorescence in a Small Contact Region between Rat Basophil Leukemia Cells and Planar Lipid Membrane Targets by Coherent Evanescent Radiation* (Received for publication, November 18,

1981)

Robert M. Weis+, Krishna Balakrishnan+& BartonA. Smithy, and Harden M. McConnell+ll From the +Department of Chemistry, Stanford University, Stanford, California94305 and the YIBM Research Laboratory, Sun Jose, California 95193

We have included a lipid hapten in lipid monolayers In recent work it has been shown that liposomes having coated on alkylated quartz microscopeslides to serve various lipid compositions and physical properties can stimuas specific antibody-dependenttargets for rat basophil late specific IgE- and lipid hapten-dependent degranulation leukemia cells. At room temperature basophils armed of rat basophil leukemia cells (1, 2). The degranulation trigwith specific monoclonal IgE antibodiesadhere tightly gered by these liposomal membranes was quite similar to to the monolayer only when the monolayers contain control studies in which the degranulation was triggered by lipid hapten and only in the presence of Mg2+ and/or hapten-derivatized bovine serum albumin or anti-IgE antiCa2+.The basophils undergo specific IgE- andhapten- body.Triggering by thelatter substances is typical of a dependent immunologicdegranulation under these number of molecular cross-linkingagents that are expected to conditions. lead to Fc receptor dimerization and oligomerization on the Fluoresceinated IgE antibodies bound Fc toreceptors cell surface (3-7). These andsimilar results, together with the on the basophil and located within -100 nm of the observations that Fc receptors are monomeric (8) and that monolayer target fluoresce when excited by laser raanti-Fc receptor antibodies can trigger degranulation in the diation undergoing total internal reflection within the quartz slide. An optical interference pattern produced absence of IgE (5),have led to theproposal that “unit signals” by two focused intersecting coherent laser beams hav- for basophil triggering are provided by pairs as well as larger ing total internal reflection within the quartz slide oligomers of Fc receptors. As discussed in an accompanying paper (2), we have found serves as a control to ensure that all of the emitted fluorescence radiation from the IgE moleculesis due t o it difficult to interpret thedegranulation of RBL’ cells by lipid stimulation by evanescent radiation and not by scat- hapten-containing membranes, especially fluid target memtered radiation. The interference pattern is also used branes, in terms of molecular cross-linking or cross-bridging for pattern photobleaching experiments to determine of Fc receptors. We have, therefore, suggested that the suffithe rates of lateral diffusion of IgE molecules bound to cient conditions for the triggering of RBL cells may be more the monolayers, in the absence of cells. general, so that triggering by molecular cross-linking of Fc Specific antibody-dependent adherence of basophils receptors is a special case. The present experimental study t o the monolayer target results in a marked enhance- was undertaken to probe the distribution of fluorescein-lament of IgE fluorescencein the field of the evanescent beled IgE antibodies bound to Fc receptors in a narrow spatial wave. Microscopic examination of the fluorescence of region of contact between the plasma membrane of RBL cells the fluoresceinated IgE moleculesin the region of ba- and a planar lipid monolayer containing lipid hapten. sophilmembrane-monolayermembranecontact reIn previous work it has been shown that lipid monolayers veals an extremely complex pattern, presumably due can be coated on glass or quartz microscope slides and that to cellular filopodia-like structures that are anchored these monolayers can be used as specific antibody-dependent t o the target membrane via IgE molecules bound si- targets to study cellular (macrophage) binding and triggering multaneously to Fc receptors and lipid haptens. These (9). Our method forexamining a narrow spatial region of structures are not observable with conventional epiflucontact between the plasma membrane and the monolayer orescence microscopy due t o the fluorescence of IgE the basophils in regions otherthan membrane makes use of the technique of total internal reflecmolecules bound to the interface between the monolayer and basophil tion spectroscopy (10) and is somewhat similar to that emmembranes. The concentration of IgE moleculesin the ployed by Axelrodto study fluorescently labeled cells on glass contact region appears to be high and could give rise surfaces ( 11). Interference patterns produced by intersecting beams of to Fc-Fc receptor complex formationor other redistributions of Fc receptors that provide signals for baso- coherent laser radiation have been used in a number of spectroscopic studies, including measurements of the ratesof phil degranulation. diffusion in liquids and solids (12, 13). As will be seen later, the combination of these interference patterns with internal * The costs of publication of this article were defrayed in part by reflection spectroscopy not onlyprovides a technique for the payment of page charges. This article must therefore be hereby measuring diffusion on the monolayer but also provides a marked “advertisement” in accordance with 18 U.S.C. Section 1734 critical test thatdemonstrates the absence of cellular fluoressolely to indicate this fact. 9 Present address, DNAX Research Institute of Molecular and cence from scattered exciting radiation. Cellular Biology, Palo Alto, CA 94304. 11 Recipient of Grant PCM 80-21993 from the National Science Foundation in support of this work. To whom correspondence concerning this manuscript should be addressed.

’ The abbreviations used are: RBL, rat basophil leukemia; DPPC, dipalmitoylphosphatidylcholine;FITC-IgE, fluorescein-5-isothiocyanate conjugated IgE, DSPC, distearoylphosphatidylcholine; NBDPE, N-4-nitrobenz-2-oxa-1,3-diazole L-a-phosphatidylethanolamine.

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Cells-RBL cells (subline 2H3), a generous gift from Dr. H. Metzger (National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, MD) were maintained and harvested as described by Taurog et al. (17). After harvesting, the cells were incubated with 5 pgof FITC-IgE in 2 ml of complete incubation buffer for no less than 1 h. Unbound FITC-IgE was removed by 3 washes with incubation buffer. The incubation buffer used during washing was either complete or lacking Ca2+and/or Mg" MATERIALS AND METHODS depending on the experiment. The complete incubation buffer contained 135 m~ NaCl, 5 mMKC1, 1n" MgCL, 1.8 mM CaCh, 5.6 mM Preparation of Fluorescent Anti-dinitrophenyl ZgE-Monoclonal mouse IgE against 2,4-dinitrophenyl, the product of a hybridoma glucose, 0.5 mg/ml of gelatin, 10 m~ 4-(2-hydroxyethyl)-l-~iperazinedeveloped by Eshhar et al. (14), was purchased from Miles-Yeda ethanesulfonic acid at pH7.2. Two hundredmicroliters of cells (I X IO7cells/&) were introduced (Elkhart, IN). Five-tenths milligram of IgE in 0.5 ml of phosphatebuffered saline whose pH was adjusted to 9.0 with a 1.0 M solution of under the coverslip of the alkylated monolayer coated microscope sodium carbonate/bicarbonate was stirred with 0.2 mg of FITC (Mo- slide. The solution under the coverslip had been f i s t changed from lecular Probes, Plano, TX) at4 "C for 12 h. Excess FITC was dialyzed distilled water (the solvent used for the air-water interface) to the away against phosphate-buffered saline at 4 "C for 36 h with four incubation buffer. The microscope slide was positioned with the changes of buffer. Before each experiment the FITC-IgE was centri- monolayer head groups pointing up. The cells were incubated on top of the phospholipid monolayer for 5 min at room temperature (25 " c ) fuged at 100,000 X g for 20 min to remove any aggregated IgE. Hapten-The 2,4-dinitrophenyl lipid haptenI was synthesized to allow binding of the cells to the monolayer membranes and then according to Balakrishnan et al. and was a kind gift of Frank Hsu (2). flushed with buffer and clamped on the two long edges of the coverslip Alkylated Quartz Slides-Synthetic fused silica microscope slides with office binder clips (No. 20, small, IDL). The binder clips apply (2 inchesX 1inch X 1m m ) were cleaned and alkylated with octadecyl sufficient pressure to ensure that the spacing between the coverslip trichlorosilane (Petrarch Systems, Bristol, PA) as described by Sagiv and microscope slide is equal to the thickness of the plastic spacers, (15) and von Tscharner and McConnell (16). The slides were boiled 24 pm. The coverslip edges were sealed with silicone vacuum grease for 10 min in a 4-fold aqueous dilution of detergent (Linbro, Ingle- (Dow Coming, Midland, MI) to prevent sample drying. The microwood, CA) followed immediately with sonication for % h. The slides scope slide was inverted and placed on the stage of a Nikon inverted microscope (Nikon Diaphot-TMD). All cells not bound to themonowere rinsed for a t least 2 h with distilled water andwere subsequently dried in an oven at 150 "C for 4 h. The oven-dried slides were layer fell through solution under gravity and came to rest on the transferred to the alkylation bath for 30 min. The alkylation bath coverslip (see Fig. 4). Serotonin Release from RBL Cells Bound to Hapten-containing consisted of freshly mixed octadecyl trichlorosilane at a concentration of Specific ZgE-Monolayers containing of 100 pl/l00 ml (v/v). The bath solvent was 90% hexadecane, 6% Monolayers in the Presence carbon tetrachloride, and 4% chloroform (v/v/v). After removal from DPPC, DSPC, and DSPC-33 mol %cholesterol, either with or without the reaction solution the slides were first rinsed in achloroform bath, 2 mol % hapten I, were taken from an air-water interface in the solid then rinsed in an ethanol bath, and finally cured in an oven at 110 phase and deposited on alkylated round microscope glass coverslips of25 mm diameter. The coverslips were placed at the bottom of "C for 1 h. Preparation of Lipid Monolayers-DPPC monolayers containing individual vials, monolayer up. Approximately IO6 [3H]serotonin la2 mol % lipid hapten I were prepared on an air-water interface using beled RBL cells were added in 250plof the incubation buffer the apparatus and the methods described in von Tscharner and described above, incubated at 37 "C, sometimes with slow swirling McConnell (16). Fifty pl of a 1.5 m~ solution of lipid in a 9 1 hexane following adherence. After 30 min 1.0 ml of ice-cold incubation buffer ethanol solvent was spread onto anair-water interface. A 'h-h period was added to the vials, the contents were shaken, and I-ml aliquots before monolayer compression was begun ensured that thespreading were centrifuged a t 8000 X g for 90 s. Two hundred-microliter aliquots solvent had completely evaporated. Surface pressure was measured of the supernatant were counted for 3H in 4.0 ml of scintiUationfluid. with a Wilhelmy Plate Balance (Roller Smith, Biolar, North Grafton, Samples as well as hapten-negative controls were in triplicate. MA). The area occupied by the monolayer was incrementally deTotal Internal Reflection Experiments-The 488-nm line of an creased over a period of 20 min until the surface pressure attained a Argon Ion laser (model 164-05, Spectra Physics, Mountain View, CA) value between 40 and 45 dynes/cm. At this point the monolayer fitted with an air-spaced etalon (model 58, Spectra Physics) was rested for 2 hduring which period surface pressure decreased slightly directed into the totalinternal reflection apparatus. (The etalon but was never allowed to fall below40 dynes/cm. After 2 h an serves to enhance the coherence of the laser beam and thus the alkylated quartz slide, washed with ethanol and airdried, was placed contrast in the interference pattern). onto and pushed through the air-water interface. The decrease in The beam first passed through the beam splitter-electronic shutter surface area corresponded roughly to the area of the microscope slide. apparatus used previously in studies of rotational and translational Once under the interface care was taken not to expose the monolayer diffusion in membranes (18, 19). After passing through the beam on the quartz slide to air. A glass coverslip (No. 1, 24 X 30 mm, splitter/shutter apparatus, the beam passes through a polarization Corning) was brought to close apposition of the slide by two layers of rotator (model 310A, Spectra Physics). The polarization is important plastic film, 12 pm thick (24 pm total), 1-2 mm wide, and 32-34 mm to ensure proper function of the beam splitter (see Fig. 1). Thebeam long, placed along the long edge of the coverslip. Stripes of adhesive passes through a 50-cm focal length focusing lens and then through tape (3 M Co., St. Paul, MN) were also placed along the long edge a beam-steering device (not shown). At this point the beam is vertimicroscope coverslip on the other side, extending beyond the cover- cally polarized and is then split by a 1:l beam splitter. The reflected slip's and spacer's lengths to fasten the coverslip with the 24-pm portion of the beam is again reflected by a high reflectivity front spacer sandwiched in between the microscope slide and thecoverslip. surface mirror (Newport Research Corporation, Fountain Valley, This narrow spacing is necessary to accommodate the small front CA). The two vertically polarized beams are passed into a specially working distance of the 63 X oil immersion objective (Zeiss) used in constructed high refractive index glass prism (n= 1.8) at an incident the total internal reflection experiments. angle of 10" (see Fig. 1).The incoming beams are reflected down by As a test of membrane integrity, FITC-IgE was incubated with the the prism and refracted at the prism-quartz slide interface. Micromonolayer on the quartz slide under the coverslip for 15 min. The scope immersion oil (Zeiss) is used to make good optical contact excess antibody was flushed away, and the slide was prepared for between the prism and the quartz slide. The beams are totally observation with a 63 X objective lens, using binder clips as described internally reflected inside the quartz slide from the lower and upper below. The fluorescence emission of the membrane-bound FITC-IgG surfaces and are p-polarized at these reflections. The beams are was observed. adjusted relative to themicroscope objective so that they arefocused In addition to monolayers of DPPC, we prepared monolayers over the objective, they intersect over the objective, and they are composed of pure DSPC and DSPCcontaining 33 mol % cholesterol, reflected from the lower surface of the quartz slide over the objective. each containing 2 mol % lipid hapten I. The DSPC and DSPCThe two intersecting beams of coherent laser radiation set up an cholesterol membranes were prepared so we could compare the interference pattern of sinusoidally varying amplitude. In vacuum, triggering by solid and fluid membranes. Membranes composed of the period of this pattern, s, is related to theangle of intersection 6 by DPPC-containing 1%NBD-PE and 2% lipid hapten I were used for the equation, control experiments testing membrane integrity during basophil binding. s = (h/2)(sin6/2)" (1)

As discussed below, our observations are consistent with the view that IgE molecules bound to Fc receptors are bound to lipid haptens on the monolayer membrane in small concentrated patches. These patches are apparently at the ends of filopodia-like extensions from the cell and are involved in basophil binding and triggering.

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where X is the wavelength of the radiation. For X = 488 nm and the value of 8 used in this work, 5.34’, the period s is 5.24 pm. (Note: the wavelength of the radiation in the quartz slide is of course less than 488 nm, and because of refraction, the angle of intersection of the two beams is less than 5.34’; these two effects cancel almost exactly for the geometry in Fig. 1. Equation 1 still gives the correct period.) The incidence angle of the total internally reflected beam was measured to be 70’. The critical angle for quartz (nl = 1.463) and water ( n =~ 1.33) is calculated to be 65.4’ (20). The penetration depth of the evanescent wave intensity is a function of the incidence angle, the wavelength, and therefractive indices of the two media. For total internal reflection the intensity Z of the exciting radiation a vertical distance d away from the monolayer into the aqueous phase is where IOis the intensity at thequartz-monolayer-water interface. The l/e penetration depth isgiven by the formula (11,20).

wave in the aqueous medium below the monolayer, excites FITC-IgE only within distances d of the monolayer membrane (Figs. 2-5). The interference pattern can only be produced by coherent radiation and not scattered radiation. Thus, by focusing the objective lens on the interference pattern one guarantees that the interface is in the focal plane. The absence of fluorescence from the zero intensity regions of the interference pattern provides evidence for the absence of fluorescence from randomly scattered exciting radiation. Blocking one of the two entering beams eliminates the interference pattern, allowing the entire contact region to be observed. Cells were observed under bright field illumination and with fluorescence excitation. Photographs were taken with a Nikon camera attached to the microscope photographic port with ASA lo00 film (2475 recording fdm, Eastman Kodak Co.). By sequentially focusing on the cell-monolayer interface with the fluorescent interference pattern and on the cell equator under bright field illumination it was possible to measure the half-thickness of the cells using the calibrated focusing knob of the Nikon microscope.

do = X/4nnz ([n?/n2]sin2a - I)-’”

RESULTS

I = IOexp(-d/do)

(2)

(3)

=

where X is the wavelength of the radiation in vacuum, n~the refractive index of water, nl the refractive index of quartz, and a the angle of incidence of the internal reflections of the radiation within the quartz slide (Fig. 1). For an angle of incidence of 70“ and 488 nm radiation the l/e decay distance is 111.6 nm. The beams continue beyond the objective, totally internally reflecting in the quartz slide, and then pass into and out of the exit prism. The exit prism permits the exciting laser radiation to exit from the quartz slide with minimal scattering and reflection back into the slide. The lower surface of the quartz microscope slide supports the haptenated monolayer, to which cells are bound (Fig. 1). The totally internally reflected beam, by setting up a rapidly decaying evanescent

RBL cells bind tightly and specifically to monolayers containing 2 mol % hapten I. When the hapten was omitted from the monolayers, the number of cells bindingto themonolayers decreased by a factor of 10, as seen by both bright field and fluorescence microscopyof the monolayer targets. Indeed, it is difficultto shake the cells loose fromthe monolayers when the lipid hapten was present. (Similar observations have been made with specificantibody-dependent adherence of macrophages to hapten-containinglipid monolayers(9).) The release of [3H]serotonin from labeled RBL cells was not as great as observedelswhere (1, 2) withliposomes. However, releasewas always greater in the presence of hapten Top View than in its absence, was always greater for DSPC than for DPPC, and was always greater for DSPC containing 33 mol % cholesterol than for DSPC alone. Examples of release are DSPC + hapten, 20.5%;DPPC + hapten, 18.5%;DSPC alone, 13.4%.In another experiment, DSPC+ hapten, 22.7%;DSPC33 mol % cholesterol + hapten, 27.1%;DSPC-33 mol % cholesterol alone,14.6%.On different days, differenthapten negP? L PI ative monolayer targets were used, includingDPPC, DSPC, DSPC-cholesterol, and a plainglassslide. These negative controls yielded quite similar background release. While the specific release is sometimes small, it is reproducible. The lower release observed with monolayertargets compared to liposomal targets (2) may possiblybe related to some kind of Side View feedbackinhibition of degranulation due to the products FIG. 1. Schematicrepresentation of the total internal reflec- releasedduringdegranulationwhich are doubtless highly tion apparatus. The optical system used to produce evanescent concentrated in limited regionsof membrane-membrane conradiation to observe the fluorescence of FITC-IgE bound to RBL cells when these cells are bound to lipid haptens in a monolayer tact (see later). These results contrast with those observed membrane. The upper and lower frames of this drawing show top and with macrophages, where both liposomes and lipidmonolayers give equally strong triggering of the respiratory burst side views of a quartz microscope slide (QS)placed on the fured stage of an inverted microscope. The lipid monolayer and bound RBL cells (9,21). are attached to the lower surface of the QS. The cell buffer is The following photographs depict the binding properties of contained between the lower surface of the QS and a microscope slide RBL cells to DPPC monolayers containing2 mol % hapten I coverslip (CS),the coverslip being separated from the lower surface or without both M e i b spacers (S). Upper, a horizontally in buffer containingCa2’ and/or of the QS by 24-pm mylar f polarized collimated laser beamexits from a polarization rotator (not and Ca2’ at 25 “C. Fig. 2 fist shows a fluorescence micrograph of FITC-IgE shown) and is focused by a focusing lens to anapproximate diameter of 100 pm directly above the center of the microscope objective lens bound to thehapten-containingmembrane under evanescent (Ob]).After passing through the focusing lens the radiation undergoes wave illumination, in the absence of cells. The evanescent two 90” reflections in a beam steering device (not shown) and is then wave is generated by two beams of focused coherent laser vertically polarized. The beam then hits a 1:l beam splitter (BS),and the reflected portion of the beam is again reflected by a front surface light intersecting and reflecting fromthe lower surfaceof the microscope slide (Fig.1). The period of the interference patmirror (M) so as to intersect the beam not deflected by the beam tern showninFig. 2 is 5.24 pm. The membrane is evenly splitter at an angle of 8 between the two beams. The point of intersection of the beams is over the objective lens of the microscope. fluorescent (apart from the optically generated pattern) indiWith both beams present an interference pattern is created. When cating that themembrane is homogeneous and that hapten is this pattern is not used the beam from M is blocked. Lower, high evenly distributed. The membraneisindeed“solid,” and refractive index (n 1.8) prisms P1 and P2 introduce and remove the is slow ( D 2 10”’ cm2/s). Visual observation with diffusion focused laser beam that undergoes the total internal reflection deone of the two beams after intense illumination with both picted in the drawing. For all experiments described in this paper a beams showsthe persistence of a “photobleached” pattern in 63 X lens requiring immersion oil (IO) were employed.

Me,

-

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the membrane generatedby the evanescent interference pat- lens of the microscope is focused as closely as possible (-0.5 tern during intense illumination. The persistence of the pat- pm) on the monolayer membrane. tern for several minutes is indicative of a solid membrane. Fig. 4 shows a bright field photograph of cells preincubated The diffusion coefficient of a DPPC quartz-supported mono- with FITC-IgE bound to a DPPC monolayer containing 2 mol layer containing 1% of the fluorescent lipid probe NBD-PE % hapten I in the presence of Mg" and Ca2+ at 25 "C. Except has been measured by photobleaching recovery with the in- for the threecells in the lower right-hand corner, thecells are terferencepatterngenerated by the two totallyinternally known to be in contact with the monolayer, based on their reflecting laser beams,like that shownin Fig. 2, and was found fluorescence under evanescent radiation arising from a single to be 2 X 10"" cm2/s (19, 22). Thus it is assured that the focused beam undergoing total internalreflection in the quartz membrane is solid and that the hapten is evenly distributed microscope slide (Fig. 5). The fluorescence arises primarily in the membrane over distances of the order of the stripe from FITC-IgE molecules that areat distances within 100 nm distances. of the monolayer. From arguments given in the following Fig. 3 shows the fluorescence of RBL cell-bound FITC-IgE discussion, the strongestfluorescence is thought to arisefrom excited evanescentradiation arisingfromtwo intersecting molecules that are closest to the monolayer, probably FITClaser beams undergoing total internal reflection in the quartz IgE moleculesbound both to Fc receptors in the plasma microscope slide. The sharp contrast between bright and dark -" regions provides compelling evidence that the fluorescence does not arise from scattered radiation and that the objective

FIG. 4. Bright field optical photomicrograph of RBL cells bound to the hapten-containing lipid monolayer in the presence of specific IgE. The temperature is 25 "C. Three cells in the lower right-hand corner of the photograph are out of focus and are not bound to the monolayer. The field of view is 140 X 180 pm. FIG. 2. Fluorescence of FITC-IgE bound to l i p i d h a p t e n I i n a solid monolayerof DPPC. The fluorescence is excited by 488-nm

evanescent radiation undergoing internal reflection in a quartz. slide (see Fig. 1. lower). The interference pattern is due to the interference of the two focused intersecting coherent laser beams in the quartz slide (Fig. 1, upper).The pattern period is 5.24 prn. The field of view is 165 X 215 pm.

FIG. 5. Fluorescence photomicrograph of monolayer-bound

RBL cells. Fluorescence of FITC-IgE associated with the specific antibody-dependent binding of HBL cells to the lipid hapten I-containing monolayer on the lower half of the quartz microscope slide.

As discussed in the text, fluorescence is only excited for those FITCIgEmolecules that are within -111.6 nm of the lipid membrane surface, and the strongest fluorescence is thought to be due to IgE FIG. 3 . Fluorescence of FITC-IgE excited by evanescent ramolecules bound to both the lipid hapten and to Fc receptors on the d i a t i o n a r i s i n g f r o mtwo intersecting laser beams undergoing RBL cell membrane. The highly punctate distributionof fluorescence total internal reflection. The cells are bound to lipid hapten I is believed to be due to filopodia-like attachment structures, rather containing monolayers on surface of the quartzmicroscope slide. The than two-dimensional clustering or patching of IgE molecules shared stripped pattern arises from the interference of the two laser beams. by two coplanar membranes. The field of view, the same as Fig. 4, is The field of view is 140 X 180 pm. also 140 x 180 pm.

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membrane of the RBL cell and to lipid haptens in the monolayer membrane. An effect of Mg2+ and Ca2’ on the shape of monolayerbound RBL cellscouldbeobserved by (i) measuring the apparent diameter of the cells with evanescent wave stimulation of fluorescence and then with the usual bright field illumination, and (ii) measuring the distance between the focal planes for the two types of illumination. With evanescent wave illumination the apparent diameter of the cell is a measure of the area of the cell in close contact (-100 nm) with the bilayer membrane, and the focal plane is within 0.5 pm of the lipid monolayer (determined by the objective lens of the microscope). Under bright field illumination the objective lens is adjusted so as tobe focusedin the plane of the cell with the largest diameter. RBL cells in the presence of Ca2+and/or Mg2’ in general had larger diameters than cells without M C and Ca2+in the buffer. This is true for both evanescent wave excitation as well as bright field illumination. In the presence of Ca2+or Mg2+ the two diameters were nearly the same, whereas in the absence of both of these ions the two diameters were markedly different, the bright field diameter being -50% larger than the diameter seen with evanescent wave excitation. The depth of the cell, another indication of how much it had spread on the monolayer membrane, could be gauged by the difference in the two focal planes. In the absence of these divalent ions in the buffer, cells spread out less on the monolayer, and they remained more rounded and hung further down from the membrane than in the presence of either of these ions. In one experiment, not described here in detail, RBL cells coated with FITC-IgE were mixed with lipid hapten I-containing liposomes and placed on the stage of an epifluorescence microscope at 37 “C. As expected, the RBL cells were uniformly fluorescent, with an enhanced but uniform fluorescence in the regions of RBL cell membrane-liposome membrane contact. Unexpectedly, we also observedsignificant fluorescence over the entire surface of liposomes in contact with the RBL cells, but not on the surfaces of liposomes at greater distances from the RBL cells, and not in contact with the RBLcells. It is possible that the liposome-associated fluorescence arises from the fluorescent proteolytic cleavage products of FITC-IgE that diffuse away from the RBL cells and stick nonspecifically or specifically to the liposomes. In control experiments we incorporated 1 mol % of the fluorescent lipid probe NBD-PE as well as 2 mol % lipid haptenI in aDPPC monolayer to which basophils were bound. By using evanescent wave excitation we were able to demonstrate that the lipid monolayer under the basophils retained its integrity; they remained diffusely fluorescent and had the same fluorescence as the monolayer membrane not under the basophils. It was noted, however, that on prolonged illumination the fluorescent lipids under the basophils bleached morerapidly than thesurrounding fluorescent lipids. This enhanced photobleaching has been shown to depend on the release of OZ-.’

molecule, originates from molecules closestto thelipid monolayer, because this is the region where the exciting radiation is most intense. The “patchy” regions of most intense fluorescence observed between the cells and the monolayers (Fig. 5) could originate in one of three ways. (i) All FITC-IgE molecules might be equidistant from the monolayer but have a patchy nonuniform lateral distribution. Alternatively, the plasma membrane of the RBL cell in the interface region might not be coplanar with the lipid monolayer, and the strongest fluorescence could arise from (ii) larger numbers of FITC-IgE molecules not close to the lipid monolayer, or (iii) FITC-IgE molecules close to the lipid monolayer, and in the most intense radiation field. Our visual observation of this fluorescence from these cells withrelatively intense bleaching radiation clearly demonstrates that themost strongly fluorescent regions bleach most rapidly. This immediately indicates that the most strongly fluorescent regions arise from FITCIgE molecules that are closest to the monolayer, option (iii) above. This conclusion is further reinforced by numerous observations in this laboratory that, under conditions of epifluorescence microscopy where all regions of a fluorescently labeled cell are exposed to bleaching radiation, the contrast in the nonuniformity in fluorescence due to patches of fluorescence is enhanced rather than suppressed. Thus we have confidence in our conclusion that the strongly fluorescent patches seen in Fig. 4 arise from molecules of FITC-IgE that are close to the monolayer, presumably bound to both lipid haptens in the monolayer and to Fc receptors on the basophil. The patchy distribution of fluorescence seen here at the monolayer membrane-basophil membrane interface isnot seen using conventional epifluorescence microscopy (see also Reference 1).We believe that this patchy distribution of IgE fluorescence is characteristic of the specific membrane-membrane interaction that leads to membrane-membrane adhesion and basophil degranulation. Our results are consistent with the concept that enhanced receptor concentration in the contact region givesrise to the triggering that leads to basophil degranulation. For example, if the bright fluorescent points seen in Figs. 3 and 5 arise from the fluorescent IgE-Fc receptors that have migrated alongfrlopodia-like structuresto points of basophil membrane-monolayer membrane contact (where lipid hapten-IgE bonds are formed), then anenhanced concentration of Fc receptors in these contact regions could lead to Fc-Fc receptor complexes and cell triggering. This corresponds to one aspect of the “equilibrium receptor complex model” described in a companion paper (2). We do not believe that these “patches” of fluorescence can be thought of in terms of immobilized clusters. Most, if not all, of the fluorescent IgE at the RBL-monolayer target interface appears to be in rapid reversible equilibrium with fluorescent IgE at distances 5100 nm removed from the membrane-membrane contact region. That is, in preliminary experiments we have observed that the fluorescence at the interface stimulated by evanescent radiation recovers in times of the order of 100 ms following an intense burst of laser radiation that bleaches the fluorescence in the region of membrane-membrane contact. This time corresponds to a lateral DISCUSSION diffusion of IgE-Fc receptors for distances of the order of -60 The present work describes the spatial distribution of flu- nm, i.e. of the order of penetration depth do of the evanescent orescent FITC-IgE molecules within distances d do = 112 radiation. This calculation assumes an IgE-Fc receptor lateral nm betweena planar lipid monolayer and specifically adherent diffusion coefficient that is equal to 2 X 10”’ cm2/s (23). RBL cells. As discussed under “Materials and Methods,” the (Details of these calculations will be given elsewhere.) distance do is the distance from the monolayer surface a+ Some of the smallest fluorescent spots seen under the RBL which the intensity of the exciting radiation falls to l / e of its cells inFigs. 3 and 5 are estimated to have widths of the order value at the surface. The strongest fluorescence, per IgE ofO.1pm or less. Rough estimates of the area of closely packed * D. Hafeman, R. Weis, K. Balakrishnan, and H. McConneU, un- antibody molecules suggestthat thefluorescent spots contain on the order of 100 antibodies, or less. Presumably each spot published data.

Contact Regions between Plan:arMembranes Basophils and would besufficient to provide a signal for partial degranulation if the fluorescence of each spot arises from IgE antibodies bound to both the lipid haptens and Fc receptors (3-7, 24). Although the monolayer targets do trigger a specific antibody- and hapten-dependent release of serotonin, this release is somewhat lower than thereleases observed with liposomes (see “Results” and Ref. 2). Lawson et al. (25) have noted that mast cell degranulation can be localized to discrete areas of the cell. The lower release seen with monolayers could bedue to the triggering of a lower number of local units in the RBL cells, since the monolayer stimulus is topologically limited in its contact with the RBL cells. In previous work Schlessinger et al. (23) have used epfluorescence microscopy to studythe distribution and motion of fluoresceinated IgE molecules bound to rat peritoneal mast cells. They have reported that theaddition of antfluorescein antibodies to thesecells with FITC-IgE leads to degranulation and the formation of small ( ~ pm) 1 “microaggregates.” It is doubtful that these microaggregates are physically equivalent to the patchy distribution we observe with evanescent wave excitation although they may be functionally equivalent. Acknowledgments-We thank Dr.V. von Tscharner forhis help in the design of the prisms used in this work. REFERENCES 1. Cooper, A. D., Balakrishnan, K., and McConnell, H. M. (1981) J. Biol. Chem. 256,9379-9381 2. Balakrishnan, K., Hsu, F. J., Cooper, A. D., and McConnell, H. M. (1982) J. Biol. Chem. 257,6427-6433 3. Siraganian, R. P., Hook, W.A., and Levine, B. B. (1975) Immunochemistry 12, 149-157 4. Ishizaka, K., and Ishizaka, T. (1969) J. Immunol. 103. 588-595

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