J. Cell Sci. 39, I37-H7 (1979) 137Printed in Great Britain © Company of Biologist! Limited

LYMPHOCYTE SURFACE AND CYTOPLASMIC

CHANGES ASSOCIATED WITH TRANSLATIONAL

MOTILITY AND SPONTANEOUS CAPPING OF Ig

D. K. BHALLA, J. BRAUN AND M. J. KARNOVSKYDepartment of Pathology, Harvard Medical School, Boston, Massachusetts 02115,U.S.A.

SUMMARY

Murine B-lymphocytes during translatory motion undergo a series of changes with respectto their morphology and distribution of surface immunoglobulins (Ig). The sequence of eventscomprising these changes was followed by fluorescence microscopy and a correlated detectionof surface features by scanning microscopy on exactly the same cell. A round, presumablynon-motile lymphocyte exhibited a random distribution of Ig and its surface displayed evenlydistributed microvilli. Formation of a ruffled edge at one pole, accompanied by a decreasedfluorescence at this pole marked the initial events of lymphocyte motility. In the subsequentstages, the ruffled edge became progressively prominent and displayed a constriction at its base,while the microvilli were displaced to the opposite pole. Ig in such lymphocytes was localizedat the trailing, microvilli-rich pole. Thin sections of motile lymphocytes revealed Ig, micro-tubules, microfilaments and coated vesicles as the characteristic features of the trailing end.These observations may have bearing on the mechanism of lymphocyte motility and spontaneouscapping of Ig.

INTRODUCTION

Binding of a multivalent ligand causes lymphocyte surface receptors to undergo alateral redistribution in a process that leads to the 'capping' and often endocytosis ofthe ligand-receptor complex (Taylor, Duffus, Raff & DePetris, 1971). Capping, whichis dependent upon temperature and metabolic activity of the cell (Taylor et al. 1971;Unanue, Karnovsky & Engers, 1973) and is sensitive to the intracellular Ca2+ con-centration (Schreiner & Unanue, 1976 a), is often accompanied by cell motility,although the motility and cappingare separable phenomena (Unanue, Ault & Karnovsky,1974). An intracytoplasmic assembly of microtubules and microfilaments has oftenbeen implicated in the control of receptor mobility (Edelman, Yahara & Wang, 1973;DePetris, 1975; Unanue & Karnovsky, 1974; Albertini & Clark, 1975; Schreiner &Unanue, 19766; Nicolson, 1976; Oliver, 1976). Bourguignon & Singer (1977) havesuggested a direct involvement of actin and myosin in the process of capping. Studiesof Schreiner, Fujiwara, Pollard & Unanue (1977), Gabbiani, Chaponnier, Zumbe &Vasalli (1977) and Toh & Hard (1977) have shown that actin, myosin and tubulinco-cap with immunoglobulin or concanavalin A caps.

Lymphocytes transformed by Escherichia coli lipopolysaccharide (LPS), a mitogen-specific for B-lymphocytes, or pretreated with carbamylcholine acquire striking motileforms upon incubation at 37 °C. Such motile cells undergo spontaneous Ig capping in

138 D. K. Bhalla, J. Broun and M. J. Karnovsky

the absence of stimulation by anti-Ig (Schreiner, Braun & Unanue, 1976), thuseliminating the prerequisite of ligand cross-linking of receptors. A non-randomdistribution of 6, ALS-antigen and Con A receptors on uropod-forming prefixedthymocytes has also been reported by DePetris (1978). Recently, Braun, FujiwaraPollard & Unanue (1978) have shown that spontaneous capping of Ig, Fc receptorsand thymus leukaemia antigen is accompanied by the segregation of cytoplasmicmyosin, which can be localized under the cap.

This study was designed to understand further the process of lymphocyte motilitythat leads to spontaneous displacement and polar distribution of Ig. The techniqueemployed was earlier devised by Karnovsky & Unanue (1978) for a correlatedfluorescence and scanning electron-microscopic study of anti-Ig-induced capping ofmouse lymphocytes. This allowed us to carry out a sequential and correlated immuno-fluorescence and scanning electron-microscopic analysis of the cells undergoingspontaneous capping and the results are discussed in terms of surface modificationsand a possible involvement of submembranous contractile filaments.

MATERIALS AND METHODS

Lymphocyte preparation

Spleens from A/St mice (West Senece Laboratories, Buffalo, N.Y.) constituted the sourceof B-lymphocytes. Spleens were excised and single-cell suspensions were prepared usingsterile techniques. The cells (10 x io7) were washed with 40 ml of RPMI 1640 medium supple-mented with 5 % (v/v) heat-inactivated foetal calf serum by centrifugation at 200 g for 8 min.The washing procedure was repeated two more times.

Lymphocyte culture

Cultures were set up in 12 x 75 mm culture tubes (Falcon, Oxnard, Ca.), each tube containing1-5x10' cells in 1 ml of RPMI 1640 medium supplemented with 5% foetal calf serum.Escherickia coli lipopolysaccharide (LPS) (Difco Laboratories, Detroit, Mich.) was added to thelymphocyte suspensions to a concentration of 20 /tg/ml. After 2 days in culture, the lymphocyteswere centrifuged on Ficoll-Hypaque density gradient (Perper, Zee & Michelson, 1968) inorder to eliminate dead cells. The lymphocytes were collected from the interface, washed 3times with Hanks' balanced salt solution (HBSS) containing 10 mM n-2 hydroxyethylpiperazine-N-z ethanesulphonic acid (HEPES) (Microbiological Associates, Bethesda, Md.) by centrifuga-tion at 200 g for 8 min each time and finally suspended to a concentration of 2-5 x io'/ml.Further preparation of cells for fluorescence and scanning electron microscopy was doneessentially in the same manner as described earlier (Karnovsky & Unanue, 1978).

Lymphocyte labelling with anti-Ig and fluorescence microscopy

Coverslips used for collecting the lymphocytes were precleaned by boiling in 10% (v/v) of7X detergent (Flow Laboratories, Hamdon, Ct.) for 30 min, followed by several rinses indouble-distilled water and a final rinse in 100% ethanol. The dried coverslips were lightlyetched in an interrupted series of lines by a diamond scorer and placed in 16-mm-diametermultiwell tissue culture dishes (Costar, Cambridge, Mass.). Cells, 5 x io6, in 0-2 ml of HBSSwere layered on each coverslip, allowed to incubate undisturbed, first at 4 °C for 10 min andthen at 37 CC for 30 min. At this time, fixation was carried out by adding 2 % formaldehyde.After 30 min of fixation, the coverslips containing cells were rinsed at least 3 times with phos-phate-buffered saline (PBS) pH 73. PBS contains 8-0 g NaCl, 02 g KC1, 0-2 g KHaPO«, and

Lymphocyte motility and spontaneous capping 139

0-15 g NaaHPO4 in 1 1. of distilled water. Cells were then treated with 100/tg/ml of fluorescein-conjugated rabbit anti-mouse Ig (FITC-RAMG) for 30 min. The preparation and specificityof the FITC-RAMG has been described elsewhere (Unanue, Perkins & Karnovsky, 1972).The cells were rinsed again with PBS and examined and photographed in a Leitz Orthoplanmicroscope with Ploem epi-illumination.

Scanning electron microscopy

The cells, following examination by fluorescence microscopy, were postfixed in 1 % aqueousosmium tetroxide for 1 h. Dehydration was carried out through a graded series of acetone,followed by critical-point drying in a Samdri pvt-3 critical point drying apparatus using carbondioxide. The coverslips containing cells were then mounted by metal-adhesive tapes onaluminium studs and coated with gold-palladium under vacuum.

Micrographs taken by light and fluorescence microscopy were used to locate the same cellsby scanning electron microscopy, using the etched lines on the coverslips as landmarks. Thecells were photographed in an ETEC scanning electron microscope using an acceleratingvoltage of 20 kV and a tilt angle of 300.

Transmission electron microscopy

Lymphocytes incubated on coverslips at 37 °C for 30 min, as described above, were fixed for1 h by addition of 2-5 % glutaraldehyde (Taab Laboratories, Reading, England) in PBS,pH 73. The cells were thoroughly rinsed with PBS, treated first with FITC-RAMG and thenwith anti-FITC antibody (Abbas, Ault, Kamovsky & Unanue, 197s) conjugated with ferritinby the method of Kishida, Olsen, Berg & Proclop (1975), postfixed with 1 % aqueous osmiumtetroxide, rinsed several times with PBS and dehydrated through a graded series of ethanols.Following absolute ethanol grade, the cells were treated with 0-05 M hafnium chloride (AJfaProducts, Danvers, Mass.) in absolute acetone for 1 h. This treatment is believed to enhance thecontrast of microtubules (Karnovsky, unpublished observation). Cells were rinsed 3 times withabsolute acetone and embedded in Epon by inverting the Epon-filled beam capsules over thecoverslips. Following polymerization, the coverslips were separated by immersing the blocksin liquid nitrogen and ultrathin sections of lymphocyte monolayers were cut by a diamond knifeusing LKB Ultrotome III. Sections were stained with lead citrate and examined in a Philips200 electron microscope.

RESULTS

Murine splenic B-lymphocytes transformed by LPS over a period of 48 h and thenincubated on coverslips at 37 °C for 30 min revealed certain variations with regardsto their morphology and distribution of surface immunoglobulins. A round cellpresumed to be a non-motile B-lymphocyte exhibited a random distribution of Ig onits surface as judged by a diffuse immunofluorescence staining. Scanning electronmicroscopy of such lymphocytes revealed numerous evenly distributed microvilli overthe cell's entire surface (Fig. 1). With the onset of motility, the lymphocytes underwenta series of changes with regard to the immunofluorescence staining pattern and surfacearchitecture. A sequential analysis of the cells in motility revealed that in the initialstages of movement, the advancing end of the cell membrane stretched out, lostmicrovilli and exhibited a ruffled edge. A constriction was often noticed at the baseof the ruffle (Fig. 2). A simultaneous decrease in the immunofluorescence at theruffled end and a corresponding increase at the opposite pole was immediately noticedindicating the beginning of Ig mobility. Most of the microvilli by this time were seen

D. K. Bhalla, J. Broun and M. J. Karnovsky

Fig. i. A round, presumably non-motile cell exhibiting numerous evenly distributedmicrovilli. The cell is diffusely labelled with FITC-RAMG (inset), x 10500; inset,x1300.

Fig. 2. A cell showing ruffles at one end. Microvilli still occupy the major part of thecell surface. A close observation of the fluorescent micrograph reveals a slight decreasein the staining intensity of FITC-RAMG at the ruffled end (inset), x 10000; inset,x 1300.

Fig. 3. A cell exhibiting non-uniform distribution of microvilli, which are denser atthe trailing end (lower left) and reduced at the advancing end (top right). There is anincreased fluorescence intensity at the trailing end and a decreased intensity at theadvancing end (inset), x 10500; inset, x 1300.Fig. 4. A cell showing ruffling activity at 2 opposite poles, while the microvilli occupythe intermediate area. The FITC-RAMG staining seems to be selectively associatedwith the areas occupied by microvilli but depleted from the ruffled edges (inset).x 10000; inset, x 1300.

Lymphocyte motility and spontaneous capping 141

Fig. 5. Cell showing a smooth ruffled edge (top). Microvilli are seen only on the bottomhalf of the cell. Area of the cell surface localized by FITC-RAMG corresponds to themicrovilli-rich bottom half (inset), x 10000; inset, x 1300.Fig. 6. An elongated cell exhibiting a prominent microvilli-free ruffled edge with aconstriction at its base and microvilli at the trailing end. Ig-cap is seen at the trailingend of the cell (inset). The area anterior to the constriction is not visible due to theabsence of fluorescence in this region of the cell, x 9000; inset, x 1300.Fig. 7. A cell exhibiting elongated shape and a distinct contractile ring slightlyposterior to the middle of the cell body. Microvilli are densely packed at the trailingend. A few elongated microspikes seem to anchor the cell to the substratum. A distinctIg-cap is seen at the microvilli-rich trailing end, while the anterior parts of the cellmargin are not stained by FITC-RAMG (inset), x 9000; inset, x 1300.Fig. 8. Major part of the cell surface seen in this micrograph is smooth and exhibitsruffled edges. Microvilli are densely packed on a compact protuberance at the trailingend. FITC-RAMG staining is restricted to the trailing end (inset). The remainderof the cell is not visible, x 9000; inset, x 1300.

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142 D. K. Bhalla, J. Braun and M. J. Karnovsky

to have migrated towards the opposite pole (Fig. 3). Occasionally, ruffles developedat the 2 opposite poles of a cell, with microvilli occupying an intermediate area. Sucha cell exhibited a bipolar cap associated with the microvilli and the Ig-depleted areasrepresented by the 2 ruffled poles (Fig. 4). Under normal circumstances of uni-directional motility, however, the ruffled end in the next stage became more pronouncedwith a distinct constriction at its base. Microvilli and Ig seemed to be selectivelydisplaced from the ruffled edge towards the opposite pole (Fig. 5). Lymphocytes nextexhibited an elongated morphology with a constriction in the middle separating a well

• ' * . •

Fig. 9. Thin section of the trailing end of a motile lymphocyte exhibiting Ig-capdetected by the ferritin-antibody technique (see text for details). Distinct micro-filaments are seen in the microvilli. Note the higher concentration of ferritin particleson the microvilli than on smooth areas of the cell membrane. A number of coatedvesicles (arrows) are seen in the cytoplasm at this pole of the cell, x 37700.

Lymphocyte motility and spontaneous capping 143

developed ruffled end from the opposite pole, which exhibited a dense organizationof microvilli and Ig (Fig. 6). The constriction, still quite prominent and appearing toform a contractile ring slightly posterior to the middle of the cell seemed to restrict themicrovilli and Ig at the trailing end (Fig. 7). Cellular projections, distinctly larger thanmicrovilli were also seen at this end. Such projections, measuring 0-2 fim in diameterand ranging from 2 to 30 /tm in length have been referred to as microspikes, micro-extensions or filopodia by various authors (Albrecht-Buehler, 1976; Albrecht-Buehler & Goldman, 1976; Taylor & Robbins, 1963; Trelstad, Hay & Revel, 1967).In motile lymphocytes, they appeared to anchor the cell to the substratum, while theruffles were stretched at the opposite pole, possibly in an effort to develop new adhesivesites on the substratum. The lymphocyte in the final stage exhibited compact micro-villous accumulation at the trailing end, which was now represented by a shortprotuberance, while the majority of the cell's surface was marked by the presence ofwell defined ruffles but devoid of microvilli (Fig. 8). Similar changes associated withanti-Ig induced capping have earlier been described by Karnovsky & Unanue (1978).

Motile, capped lymphocytes in thin sections showed that Ig was selectively denudedfrom the ruffled areas but associated with microvilli at the opposite pole, as revealedby the ferritin-antibody technique. Furthermore, the ferritin particles in motile cellswere present at higher concentration on microvilli than on the smooth areas of the cellmembrane between them. An accumulation of linearly arranged or a meshwork ofcytoplasmic microfilaments was often noticed at the trailing end. Distinct micro-filaments could also be seen running through the microvilli. Microtubules in mostinstances were observed in the deeper areas of the cytoplasm beneath the cap. Inaddition to microfilaments and microtubules, coated vesicles were constantly seen inthe cytoplasm at the capped pole (Fig. 9). They could be localized, both near thesurface in association with the microfilaments or deeper in the cytoplasm, intermixedwith the microtubules.

DISCUSSION

Lymphocyte surface alterations may be related to the cell's functional state,differentiation stage or the forces in its microenvironment (Van Ewijk, Brons &Rozing, 1975; Roath, Newell, Polliack & Alexander, 1978; Bhalla & Karnovsky,1978). Lymphocytes during locomotion exhibit various changes in their morphologyand they can be distinguished from stationary lymphocytes on the basis of theiramoeboid appearance (Lewis, 1931; Unanue et al. 1974; Schreiner & Unanue, 19766).Previous studies had established that 24-48 h culture of B cells with LPS markedlystimulated a locomotory response of the B cells. The motile cells exhibited Ig in a cap.Non-motile round cells showed Ig in a diffuse pattern. Such spontaneous cappingwas also seen in B cells undergoing locomotion without the previous incubation withany stimuli (Schreiner et al. 1976; Braun et al. 1978). Spontaneous caps did not requiredetection with a bivalent anti-Ig. These observations excluded that LPS was causingIg-capping by cross-linking, and also indicate the spontaneous nature of Ig cappingin motile cells. In the current study, we have relied on lymphocyte shape and surface

144 D- K. Bhalla, J. Braun and M. J. Karnovsky

architecture in determining the motility events. A rounded lymphocyte with itssurface exhibiting evenly distributed microvilli but devoid of any ruffling activitywas characterized as a non-motile cell. Following contact with the substratum,lymphocytes showed certain surface alterations suggestive of motility. The progressivestages of movement were assessed by the relative amount of surface ruffling, displace-ment of microvilli towards one pole and deviation of the cell form from the roundedappearance.

Abercrombie, Heaysman & Pegrum (1970) have suggested that the lamellipodia atthe edge of a moving fibroblast result from the assembly of new surface, which may befavoured by the longitudinal pull applied to the plasma membrane by the cytoplasmicmicrofilaments (Harris, 1976). Function of microfilaments in cell locomotion andsurface ruffling has also been suggested by others (reviewed by Goldman et al. 1973;Korn, 1978). Our scanning electron-microscopic observations indicating a ruffledadvancing end and a microvilli-rich trailing end of the motile lymphocytes areconsistent with the suggestion of a more stretched anterior region of the plasmamembrane and the least stretched region at the posterior end (Harris, 1976). In lightof such studies, it seems reasonable to speculate that during the initial stages of motilityand in the course of local stretching of plasma membrane in the ruffle, a link betweenmicrofilaments and certain membrane proteins is established. In this type of linkage,certain membrane receptors are selectively recognized by the underlying micro-filaments without prior crosslinking of the receptors into aggregates, which has beenpreviously proposed to be a requirement for actin or myosin mediated capping(Bourguignon & Singer, 1977). Subsequently, the displacement of microfilaments andthe linked proteins may occur in a coordinated manner. According to Borisy &White's (1978) model for cytokinesis in animal cells, a signal emitted from the poles ofa mitotic spindle perturbs the filamentous net underlying the plasma membrane suchthat the tension at the cell surface becomes greater in the equatorial than in the polarregions. As a result of this tension imbalance, the elements of the net become re-distributed and concentrated at the equator, followed by a constriction leading tocleavage of the cell. Such a model may also help explain the formation of a constrictionseen behind the ruffled edge in motile lymphocytes. However, the nature of the signalresponsible for the formation of the constriction is not clear. Since the local membraneconstrictions in a motile lymphocyte appear to proceed from the advancing to thetrailing end, they may be envisaged as the cellular specializations favouring thedisplacement of the complexes of ligand-receptor and locally contracted microfilamentsto the trailing end. Microspikes seen at the trailing end of motile lymphocytes mightbe vital for anchorage and cell motility. Their role in particle transport, cell locomotionand as sensory organs have been discussed by Albrecht-Buehler (1976) and Albrecht-Buehler & Goldman (1976). The selective capping of Ig but not 6 and H2 may reflecta difference in the nature of insertion of these receptors in the membrane and theirinteraction with integral membrane proteins and/or with actin and myosin. Presenceof a meshwork or linearly arranged microfilaments below the cap probably serves tomaintain the cell's motile form and restricts Ig in the cap. Accumulation of micro-filaments at the trailing end of the motile lymphocytes and the fact that the density of

Lymphocyte motility and spontaneous capping 145

ferritin particles is more on the microvilli, which are clearly rich in microfilaments,further supports the suggestion of an association between microfilaments and receptorsduring cell motility. Although a physical linkage between Ig and microfilaments duringspontaneous capping seems quite likely, the function of microfilaments in balancingthe forces created by regional alterations in the physical properties of the cappedmembrane (Albertini, Berlin & Oliver, 1977) may be equally important.

A directional flow of lipids alone (Bretscher, 1976) or of lipids and proteins (Harris,1976) of plasma membrane has been hypothesized to explain the capping of surfaceantigens. Bretscher (1976) has suggested coated vesicles as a means for lipid resorptioninto the cell. General occurrence of the coated vesicles in the cytoplasm at the trailingend of motile lymphocytes supports the recycling phenomenon; however, it is hard tosay if the recycling accompanying motility involves only lipids (Bretscher, 1976) ormembrane integral proteins and carbohydrate components also (Harris, 1976).Studies showing an association of coated pits with stress fibres in human fibroblasts(Anderson et al. 1978) and the occurrence of coated vesicles in association withmicrofilaments and microtubules at the trailing end of motile lymphocytes may suggesta possible role of contractile elements in membrane-recycling.

In conclusion, then, using the correlated immunofluorescence and scanning electronmicroscopy, this study presents a systematic evaluation of the surface features of thelymphocytes undergoing motility and spontaneous capping of Ig. The observationsreported may be relevant to the mechanism underlying motility and capping. Thesuggestions are supported by the thin-section observations. The study is also con-sistent with the hypothesis suggested earlier that the ruffles at the anterior edgereflect a degree of membrane flow from that pole posteriorly (Karnovsky & Unanue,1978).

We thank Dr E. R. Unanue for his advice, Mr Robert Rubin for photographic help and MsKay Cosgrove for assistance in manuscript preparation. This work was supported by GrantsAI 10677 and GM 01235 from the N.I.H., U.S.P.H.S.

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(Received 2 February 1979)