Proc. Natl. Acad. Sci. USAVol. 92, pp. 6976-6980, July 1995Cell Biology

Large secretory structures at the cell surface imaged withscanning force microscopy

(secretory granules/rat basophilic leukemia cells/exocytosis/IgE receptor activation)

ANNAMMA SPUDICH*tt AND DAVID BRAUNSTEIN§ODepartments of *Molecular and Cellular Physiology and §Biochemistry, Stanford University, Stanford, CA 94305

Communicated by James A. Spudich, Stanford University School of Medicine, Stanford, CA, April 6, 1995

ABSTRACT Scanning force microscopy was used to im-age rat basophilic leukemia (RBL-2H3) cell surfaces underdifferent stimulation conditions that either permit or inhibitsecretion. Cross-linking the surface IgE receptors with dini-trophenol-conjugated bovine serum albumin initiates secre-tion in RBL cells with concomitant spreading of the cell body.Structures at the cell surface 1.5 ,lm in diameter relate tosecretion both spatially and temporally. The position of thesesurface pits and their sizes suggest that they may be related tothe dense-core granules positioned along the cytoskeletalfilaments in detergent-extracted, unactivated RBL cell pro-cesses. Topographic scanning force microscopy images ofRBLcell surfaces at 2, 5, and 35 min after activation show that thesestructures persist and change in cross-sectional profile withtime after activation. These structures may be related to themembrane retrieval mechanism of cells after intense stimu-lation.

Secretion is one of the critical events of the signal-transductioncascade in many eukaryotic cells. Studies of events connectedwith secretion have mainly used biochemical and electrophysi-ological techniques (1-8). Many biochemical events of thesignal-transduction pathway that leads to the secretion ofhistamine and serotonin have been reported in rat basophilicleukemia (RBL-2H3) cells (9-11). RBL-2H3 cells stimulatedby cross-linking the IgE receptors undergo significant changesin cell morphology within 3-5 min after activation (1, 12, 13).These activation-induced shape changes are coincident withstimulated secretion of serotonin and histamine (2, 13). Themaximum rate of secretion occurs at -3 min after antigenstimulation. Receptor cross-linking in the absence of externalCa2+ inhibits secretion but does not inhibit the activation-induced cell spreading (1). Phorbol ester (phorbol 12-myristate 13-acetate; PMA) stimulation induces cell spreadingwithout secretion, and treatment with the calcium ionophoreA23187 causes a low level of secretion to occur withoutsignificant cell spreading.

Visualization of secretion-related events at the cell surfacehas been difficult due to the transient nature of such events andthe difficulty of localizing such secretory structures. Imagingsecretion events has mainly relied on freeze-fracture tech-niques and electron microscopy (14-17). Using scanning forcemicroscopy (SFM) and different stimulation conditions thateither permit or inhibit secretion, we demonstrate structures atthe cell surface of RBL cells that are - 1.5 ,tm in diameter andare coupled with secretion temporally and spatially. AlthoughSFM was originally conceived as an instrument to provideimages of the surface topography of nonconductive materialsat near-atomic resolution (18), many of the current applica-tions of SFM are on the scale of 10 nm-10 ,tm.Many samples of biological interest, such as protein-nucleic

acid complexes and protein assemblies, have been successfully

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

imaged at high resolution with SFM (19-22). The SFM is alsoproving useful for studying cell-surface structures, subcellularorganelles, and surface dynamics of living cells at high reso-lution (12, 23-25). For fixed preparations, SFM has importantadvantages over other microscopy techniques that providehigh resolution, such as scanning electron microscopy. Oneadvantage, in particular, is the ability to image samples closerto the native structure by avoiding metal coating. Anotheradvantage is the ability to derive the cross-sectional profile ofsurface structures from an SFM topographic image withoutthe need for thin sectioning. In addition, the ability to work atambient pressures and the minimum sample preparation re-quired make this technique suitable for imaging surface struc-tures that are difficult to preserve and visualize with opticalmicroscopy. Cells were imaged by SFM before and afterstimulation by cross-linking the IgE receptors under conditionsthat permit secretion and cell spreading.

MATERIALS AND METHODSCell Culture. Cells were cultured and activated with antigen

as described (12). Low density cultures (2 x 105 cells per ml)primed with anti-DNP IgE at 0.5 ,ug/ml (Sigma) were culturedovernight on glass coverslips at 37°C. Antigen-induced secre-tion was initiated by treatment with 2,4-dinitrophenol (DNP)-bovine serum albumin (BSA) (100 ng/ml; Sigma) in Ringer'ssolution [135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.8 mMCaCl2, 20 mM Hepes (pH 7.4), 5 mM glucose]/BSA (0.1mg/ml). For other activation protocols cells were treated withPMA (TPA) at 20 ng/ml (Sigma) in Ringer's solution for 5 minat room temperature or with calcium ionophore A23187 at 2.6ng/ml (Sigma). Stimulation was terminated by fixation in 2%formaldehyde/Ringer's solution for 10 min. After fixation,cells were washed twice with Ringer's solution and dehydratedthrough an ethanol series (30, 50, 70, 85, 90, 95, and 100%; 10min per dilution). The 100% ethanol was completely ex-changed with hexamethyldisilizane (Electron Microscopy Sci-ences, Fort Washington, PA) followed by a 10-min treatmentin 100% hexamethyldisilizane. The samples were then airdried.SFM. Scanning force images were taken by using an Auto-

probe LS SFM (Park Scientific Instruments, Sunnyvale, CA)with a 100-,tm piezoscanner that has Scanmaster real-timeoptical-scan correction. Two hundred-micrometer-long SiN3Microlevers (Park Scientific Instruments) with oxide-sharpened pyramidal tips of force constants 0.03 N/m wereused for the imaging. Samples were imaged with 10-9-10-8

Abbreviations: SFM, scanning force microscopy or microscope; RBL,rat basophilic leukemia; DIC, differential interference contrast; DNP,2,4-dinitrophenol; PMA, phorbol 12-myristate 13-acetate; BSA, bo-vine serum albumin.tPresent address: Department of Biochemistry, Stanford UniversitySchool of Medicine, Stanford, CA 94305.tTo whom reprint requests should be addressed.lPresent address: Park Scientific Instruments, 1171 Borregas Avenue,Sunnyvale, CA 94089.

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N, at a rate of 2 Hz per scan line, 256 x 256 pixels per image.Images were tilt-corrected by using Autoprobe image-processing software. Final processing of all images was done byusing Adobe PHOTOSHOP and Adobe ILLUSTRATOR.Transmission Electron Microscopy. For transmission elec-

tron microscopy, cells were grown on Formvar-coated nickelgrids overnight at 37°C. Unactivated cells were extracted with0.5% Triton X-100/0.25% glutaraldehyde in a modified Ring-er's solution (13). After extraction, cells were washed threetimes in Ringer's solution/5 mM EGTA and fixed in 1%glutaraldehyde/Ringer's solution/5 mM EGTA for 20 min at22°C. After a 5-min treatment with 0.2% tannic acid in water,cells were postfixed with 0.5% OS04 in water for 15 min.Dehydration and subsequent steps were as described (13).

Fluorescent Labeling. Fluorescent staining of the activatedRBL cell surface used a monoclonal antibody shown previ-ously by Bonifacino et al. (26) to detect secretory granulemembranes of RBL cells. A modification of their procedurewas used to label the cells. Cells were cultured overnight onglass coverslips in 6-well plates and stimulated by receptorcross-linking as described above. After stimulation for 5 min,the coverslips were immediately cooled to 4°C and incubatedin blocking buffer (1% BSA/2% normal goat serum/Ringer'ssolution) at 4°C for 30 min. Cells were then incubated with theprimary antibody diluted 1:10 in blocking buffer for 2 hr at 4°C.After incubation with the primary antibody (5G10), the cellswere rinsed three times with ice-cold blocking buffer and fixedfor 10 min in 2% formaldehyde/Ringer's solution. Fixed cellswere rinsed twice with 0.1 M glycine/phosphate-bufferedsaline for 5 min each and incubated for 1 hr at room temper-ature with the fluorescein isothiocyanate-labeled goat anti-mouse antibody (Organon Teknika-Cappel) diluted 1:25 inblocking buffer. Cells were then washed and mounted inMowiol as described (13).

Fluorescence and Differential Interference Contrast (DIC)Microscopy. One-micrometer sections of the labeled cells wereimaged (27) both in the fluorescence mode and by DIC optics.Images were processed by using Adobe PHOTOSHOP and AdobeILLUSTRATOR.

RESULTSTopographic SFM images of RBL cells before activation andat 5 min after activation under different conditions are shownin Fig. 1. Unstimulated cell surfaces display fine filopodia anduneven surface topography (Fig. la). These images are con-sistent with electron microscopy and DIC images of cellsbefore stimulation (12). Stimulation with DNP-BSA at 100ng/ml for 5 min results in the cell spreading associated withactivation (Fig. lb). Prominent on the cell surface, primarilyalong the center line of processes that extend outward from thenucleus are numerous pits that are 1.7 ± 0.5 ,um (n = 50) indiameter. Each pit is surrounded by an elevated collar. Suchstructures were observed in stimulated cells in three separateexperiments. The size and distribution of these collared pitsare consistent with their being derived from the electron-densegranules present in the cell cortex of unactivated cells andthose found associated with the filaments present in transmis-sion electron microscopy images of unactivated, detergent-extracted cells (Fig. 2).To correlate these structures with secretion, cells were

stimulated with DNP-BSA as above but in the presence of 5mM EGTA to chelate external Ca2l. Activation in EGTA waspreviously shown to inhibit secretion but permit activation-induced cell spreading (1). Fig. lc shows the process of a cellat 5 min after activation in EGTA. Cell spreading is evident,but the collared pits in secreting cells are absent from thesesamples. In other experiments cells treated with calciumionophore A23187, previously reported to cause a low level ofsecretion without significant cell spreading (1), show collared

FIG. 1. SFM images of RBL cell processes acquired under condi-tions that induce or inhibit secretion. Images are oriented so the cellnucleus (not shown) would appear below the bottom edge (a).Unactivated cell process showing smooth surface topography andnumerous microspikes. (b) Cells stimulated for 5 min with DNP-BSAat 100 ng/ml show numerous 1.7 + 0.5-,um-sized surface pits (n = 50),each collared by an elevated annulus. (c) Cells stimulated for 5 minwith antigen in the presence of 5 mM EGTA. (d) Cells treated with 0.5mM calcium ionophore A23187 show surface pits but retain micro-spike morphology. (e) PMA (TPA) at 20 ng/ml causes cell spreadingand changes in surface topography without forming surface pits. (f)Histogram of number of surface pits per jum2 of cell-surface areaimaged in a-e. Thirty images were analyzed for each point. Surface pits>0.5 ,um were counted for the quantitation. (Bars = 5 ,um.)

pits similar in size and morphology to those induced by antigenactivation (Fig. ld). Cells stimulated with PMA alone (Fig. le)show cell spreading and changes in surface topography, but thecollared pits were not present in these cells. The changes insurface topography detected here with PMA treatment areconsistent with effects of PMA on the actin cytoskeleton (28).The number of these collared pits per 100-,m2 surface areaunder these various conditions is quantitated in Fig. lf. Thenumber of these structures observed for cells activated underconditions that permit secretion compared with that observedfor nonsecreting cells suggests that the collared pits areconnected with the secretion process and possibly are exocy-totic in nature.SFM images of the surfaces of secreting cells at 2 min, at 5

min, and at 35 min after stimulation show changes in topog-raphy of the secretion-induced structures with time afterstimulation (Fig. 3). The profiles of the surface structure at 2min after activation suggest the beginning of an annularstructure. At 5 min after activation, when maximum secretion

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FIG. 2. Transmission electron micrograph of an RBL cell process shows numerous large, electron-dense granules positioned along the centerof the cell process. (X1O,OO0.)

of serotonin can be measured biochemically (2), the surfacesof the cells show numerous collared pits of -1.5 ,um indiameter. Cross-sectional profiles of these pits at 5 min afteractivation show that the collars that surround them projectabove the cell surface (Fig. 3 Insets). At 35 min after activationthe pits appear collarless. The lack of a discernible collar at 35min implies that the underlying membrane architecture thatforms the collar at 5 min changes over time. Because of thegeometry of the tip used in SFM, only those parts of the cellsurface that contact the probe tip are imaged. So, although therim of the pit can be accurately imaged, any part of the pitrecessed beneath the rim is inaccessable to the probe tip. Also,due to the finite aspect ratio of the tip, the true depth profileof the pits cannot be gauged accurately. In spite of theselimitations, SFM has demonstrated that it can accuratelydepict the surface structure of macromolecular samples (12,21).The collared pits that appear upon activation probably relate

to dense-core granules of 0.5-,um diameter that are observedby both detergent-extracted (Fig. 2) and thin-sectioned elec-tron micrographs (1) of activated RBL cells. The position ofthese dense-core granules positioned along the cytoskeletalfilaments overlaps with the collared pits seen at the cell surfaceby SFM after secretion. The location and the size of thesegranules are consistent with the possibility that the surface pitsarise by fusion of the granules with the cell surface. Fusion ofthe granule with the plasma membrane may stretch out the fullsurface area of the granule into a collared pit structure with agreater apparent diameter. If the total surface area of a sphereof diameter d were spread out into a circle of diameter D, therelationship of the two diameters would be 2d = D. A granuleof 0.5 ,um in diameter could therefore appear as a patch of-1.0-,Am diameter.The secretory nature of these collared pits was further

suggested by staining activated RBL cells with an antibody thathas been shown by Bonifacino et al. (26) to stain secretorygranule membranes at the cell surface. Fig. 4a shows a singleoptical section taken with a laser scanning confocal micro-scope imaged in the DIC and fluorescence modes. In thisnarrow plane of focus only the top surface of the stimulatedcell is in focus in the DIC image. Fig. 4b shows patches ofpunctate staining primarily along the process. The coplanarityof the two images demonstrates that the patches are localizedat the cell surface; such patches were not seen in the focal planeof the cytoplasm or on the surface of unstimulated cells (data

not shown). The staining pattern is similar in location anddistribution to the collared pits observed in the SFM topo-graphic images described above. On the basis of these obser-vations, it is reasonable to speculate that the fluorescentpatches seen from the confocal image and the annular pits seenin the SFM images represent the same structures. Bonifacinoet al. (26) reported the persistence of the membrane antigenstaining of secretory granules at 45 min after activation. Thisresult is consistent with our SFM images showing surface pitsat 35 min after activation (Fig. 3).

DISCUSSIONAlthough the collared pits seen in Figs. 1 and 3 are probablylargely products of exocytotic events, many of them couldrepresent endocytic events. The presence of large endocyticstructures at the cell surface was proposed by a number ofprevious studies (28). The diameter of the collared pits issignificantly larger than that of coated pits (- 100 nm) (7).While coated vesicles have been proposed to account for thebulk of endocytosis in mammalian cells, the movement ofplasma membrane proteins into cells has also been proposedto occur by other mechanisms (7). In chromaffin cells, afterintense stimulation, endocytic uptake of membrane subse-quent to exocytosis may occur via large vacuoles throughclathrin-independent mechanisms (28).

Furthermore, Thomas et al. (7) showed that in single pitu-itary cells, membrane retrieval (as measured by downwardcapacitance changes) occurs by structures larger (-1.5 ,um indiameter) than coated vesicles and that these structures canremain at the cell surface for several minutes. Rosenboom andLindau (8) also demonstrated endocytosis via large vacuoles ofsimilar size (2 ,um) detected as downward capacitance changesin single pituitary nerve terminals. In cells that show pro-nounced ruffling activity upon stimulation by growth factorsand phorbol esters, large vacuoles described as macropinocyticvesicles have been described (29). Ruffling activity induced bySalmonella binding to the surface of HEp-2 cells or antigenactivation ofRBL cells induces uptake of noninvasive bacteria,possibly through a macropinocytosis mechanism (27).The SFM images presented here, as well as the data reported

by Bonifacino et al. (26), suggest that the receptor activation-induced structures persist >35 min after secretion is initiated.If these structures represent a mechanism of non-clathrin-mediated membrane retrieval, it is unclear what fraction of the

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FIG. 3. Changes in the secretory pit at various times afteractivation of three different cells. (a) Two minutes after stimulationwith DNP-BSA the cell surfaces show numerous projections thatcould be early stages of secretion events. Many collared pits arealready visible at this time. (b) At 5 min after activation, at the peakof secretion, deep pits of -1.5 ,um in diameter are more prevalentthan at 2 min. These structures show collars that in the topographicimage appear to be projecting from the surface, and occasionally pitswith spherical inclusions are also detected in these samples (seearrowhead). (c) At 35 min after the initiation of secretion, pit-likestructures still persist at the cell surface. These lack the surroundingcollar-like projection seen at earlier time points. Insets (a-c) Cross-sectional profiles derived from the SFM topographs of specific pits(indicated by connected arrows). Profiles show that the collarsurrounding a pit in the 2-min and 5-min time points after activation isabsent at 35 min. Vertical scale in Insets is arbitrary. (Bars = 4 ,um.)

FIG. 4. Localization of secretory granule membranes at the cellsurface 5 min after stimulation imaged by scanning confocal DIC andfluorescence microscopy. Images of an activated cell at the same planeof focus in the DIC mode (a) and in the fluorescence mode (b),showing the position of secretory granule membrane at the cellsurface. (Bars = 5 ,tm.)

exocytotic granule membrane is recaptured in these slow-uptake structures. Perhaps retention of the pits at the cellsurface at 35 min reflects a saturation of the endocyticpathway, as described by Thomas et al. (7), after intensestimulation by the high antigen concentration used in theseexperiments.Although there have been earlier SFM studies of RBL

cell-surface morphology (1, 11), the collared pits reportedhere were not seen in those studies. This difference may bedue, in part, to our use of low-density cell cultures, whichallows single cells to assume a spread morphology. More ofthe cell surface is exposed under these conditions. Further-more, the use of high-aspect-ratio oxide-sharpened SiN3cantilevers provided the necessary resolution and definitionto cleanly resolve structures on the cell surface. The inertnature of the SiN3 surface also results in little tip-sampleinteraction in the form of tip sticking during imaging. Thesefactors in combination with the mild fixation and samplepreparation conditions used here allowed us to visualizethese secretion-induced structures.

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We thank Dr. J. A. Spudich and Dr. Hans Warrick for criticallyreading the manuscript and Dr. Suzanne Pfeffer for use of the cellculture facility. We are grateful to Dr. J. Bonifacino for providing uswith the antibody (SG10) to secretory granule membranes. Finally, wethank Park Scientific for the loan of the Autoprobe LS SFM and, inparticular, Marco Tortonese for the gift of oxide-sharpened SiN3cantilevers. This work was supported by National Institutes of HealthGrant GM30387 to Prof. J. A. Spudich of the Department of Bio-chemistry (Stanford University) and National Science FoundationGrant ECS 8917552 to Prof. C. F. Quate of the Department ofAppliedPhysics (Stanford University). A.S. was supported by a Faculty Grantfrom the Program in Molecular and Genetic Medicine at StanfordUniversity, and D.B. was supported by National Institutes of HealthPostdoctoral Fellowship PHS AR08282-02.

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