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J. ellSci.39, I37-H7 (1979) 137

Printed in Great Britain Company ofBiologist Limited

LYMPHOCYTE SURFACE AND CYTOPLASMIC

CHANGES ASSOCIATED WITH TRANSLATIONAL

MOTILITY AND SPONTANEOUS CAPPING OF Ig

D.

K. BHALLA,

J.

BRAUN

AND

M. J. K ARN OVSK Y

Departmentof Pathology, HarvardMedicalSchool, Boston,Massachusetts02115,

U.S.A.

SUMMARY

Murine B-lymphocytes during translatory motion undergoaseries of changes with respect

to their morphology and distribution of surface immunoglobulins (Ig). The sequence of events

comprising these changes was followed by fluorescence microscopy anda correlated detection

of surface features

by

scanning microscopy

on

exactly

the

same cell.

A

round, presumably

non-motile lymphocyte exhibited a random distributionofIg and its surface displayed evenly

distributed microvilli. Formation

of a

ruffled edge

at

one pole, accompanied

by a

decreased

fluorescence atthis pole marked the initial eventsoflymphocyte motility. Inthe subsequent

stages, 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 localized

at the trailing, microvilli-rich pole. Thin sections

of

motile lymphocytes revealed Ig, micro-

tubules, microfilaments and coated vesiclesas the characteristic features of the trailing end.

These observations may have bearing on the mechanism of lymphocyte motility and spontaneous

cappingofIg.

INTRODUCTION

Binding

of

a multivalent ligand causes lymphocyte surface receptors to u ndergo

a

lateral redistribution in a process that leads to the 'capping' and often endocytosis of

the ligand-receptor complex (Taylo r, Duffus, Raff

DePetris, 1971). Capping, which

is dependent upon temperature and metabolic activity of the cell (Taylor et al.1971;

Unanue, Karnovsky & Engers, 1973) and

is

sensitive

to

the intracellular Ca

2+

con-

centration (Schreiner

&

Unanue, 1976

a), is

often accompanied

by

cell motility,

althoughthemotility and capping are separable phenomena (Una nue, Ault Karnovsky,

1974). An intracytoplasmic assembly

of

microtub ules and microfilaments has often

been 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) have

suggested a direct involvement of actin and myosin in the process of capping. Studies

of Schreiner, Fujiwara, Pollard & Unanue (1977), Gabbiani, Chaponnier, Zumbe &

Vasalli (1977) and Toh & Hard (1977) have shown that actin, myosin and tubulin

co-cap with immunoglobulin

or

concanavalin A caps.

Lymphocytes transformed byEscherichia colilipopolysaccharide (LPS), a mitogen-

specific for B -lymphocytes, or pretrea ted w ith carbamylcholine acquire striking motile

forms upon incubation at 37 C. Such motile cells undergo spontaneous Ig capping in

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

the absence of stimulation by anti-Ig (Schreiner, Braun & Unanue, 1976), thus

eliminating the prerequisite of ligand cross-linking of receptors. A non-random

distribution of 6, ALS-antigen and Con A receptors on uropod-forming prefixed

thymocytes has also been reported by DePetris (1978). Recently, Braun, Fujiwara

Pollard & Unanue (1978) have shown that spontaneous capping of Ig, Fc receptors

and thymus leukaemia antigen is accompanied by the segregation of cytoplasmic

myosin, which can be localized under the cap.

This study was designed to understand further the process of lymphocyte motility

that leads to spontaneous displacement and polar distribution of Ig. The technique

employed was earlier devised by Karnovsky & Unanue (1978) for a correlated

fluorescence and scanning electron-microscopic study of anti-Ig-induced capping of

mouse lymphocytes. This allowed us to carry out a sequential and correlated immuno-

fluorescence and scanning electron-microscopic analysis of the cells undergoing

spontaneous capping and the results are discussed in terms of surface modifications

and a possible involvement of submembranous contractile filaments.

MATERIALS ANDMETHODS

Lymphocyte preparation

Spleens from A/Stmice (West Senece Laboratories, Buffalo, N.Y.) constitutedthesource

of B-lymphocytes. Spleens were excised

and

single-cell suspensions were prepared using

sterile techniques. T he cells (10 x io

7

) were washed with 40 mlofRPMI 1640 medium supple-

mented with5 (v/v) heat-inactivated foetal calf serum bycentrifugation at200g for 8 min.

The washing procedure was repeated two more times.

Lymphocyte

culture

Cultures w ere set up in 12 x75mm cultu re tubes (Falcon, Oxnard, Ca.), each tube containing

1-5x10'

cells in 1ml of RPMI 1640medium supplemented with 5 foetal calf serum.

Escherickia coli

lipopolysaccharide (LPS) (Difco Laboratories, Detroit, Mich.) was added

to the

lymphocyte suspensions to a concentrationof20/tg/m l. After2days in culture, the lymphocytes

were centrifuged on Ficoll-Hypaque density gradient (Perper, Zee &Michelson, 1968) in

order to eliminate dead cells.The lymphocytes were collected from the interface, washed3

times with Hank s' balanced salt solution (HBS S) containing10 mMn-2hydroxyethylpiperazine-

N-z ethanesulphonic acid (HEPES) (Microbiological Associates, Bethesda, Md .) by centrifuga-

tion at200g for 8mineach time and finally suspended to aconcentration of 2-5xio' /ml.

Further preparation of cells forfluorescenceand scanning electron microscopy wasdone

essentiallyin thesame man nerasdescribed earlier (Karnovsky & Unanue, 1978).

Lymphocytelabellingwith anti-Ig andfluorescence microscopy

Coverslips usedforcollectingthelymphocytes were precleanedbyboilingin10 (v/v) of

7X detergent (Flow Laboratories, Hamdon, Ct.) for 30 min , followed by several rinsesin

double-distilled waterand a final rinse in 100 ethanol. The dried coverslips were lightly

etchedin an interrupted seriesoflinesby a diamond scorer andplacedin 16-mm-diameter

multiwell tissue culture dishes (Costar, Cambridge, Mass.). Cells, 5xio

6

,in0-2ml ofHBSS

were layeredoneach coverslip, allowed to incubate undisturbed, first at 4Cfor10 minand

thenat 37

C

C for30 min.At this time, fixation was carriedout byadding2 formaldehyde.

After 30 minoffixation,thecoverslips containing cells were rinsedatleast3times with phos-

phate-buffered saline (PBS)pH73 . PBS contains 8-0gNaCl, 02 gKC1, 0-2g KH

a

PO,and

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Lymphocyte motility andspontaneous capping 139

0-15 g N a

a

HPO

4

in

11.

of

distilled water. Cells were then treated with 100 /tg/m l

of

fluorescein-

conjugated rabbit anti-mouseIg (FITC-RAMG) for30 min.Thep reparationandspecificity

of the FITC-RAMG has been described elsewhere (Unanue, Perkins &Karnovsky, 1972).

The cells were rinsed again with PBSandexamined andphotographed in a Leitz Orthoplan

microscope with Ploem epi-illumination.

Scanning electron microscopy

The cells, following examinationbyfluorescencemicroscopy, were postfixed in1 aqueous

osmium tetroxide for 1h. Dehydration wascarried outthrough a graded series ofacetone,

followedbycritical-point dryingin aSamdri pvt-3 critical point drying apparatus using carbon

dioxide. The coverslips containing cells were then mounted by metal-adhesive tapes on

aluminium studsandcoated with gold-palladium under vacuum.

Micrographs takenbylightandfluorescencemicroscopy were usedtolocatethesame cells

by scanning electron microscopy, usingtheetched lineson thecoverslipsaslandmarks.The

cells were photographed

in an

ETEC scanning electron microscope using

an

accelerating

voltageof20 kVand atilt angleof30

0

.

Transmission electron microscopy

Lymphocytes incubatedoncoverslipsat37 Cfor30 min, as described above, were

fixed

for

1h by addition of 2-5 glutaraldehyde (Taab Laboratories, Reading, England) in PBS,

p H 7 3 .Thecells were thoroughly rinsed with PBS, treated first with FIT C-R AM Gandthen

with ant i-F IT C antibody (Abbas, Ault, Kamovsky & Una nue, 197s) conjugated with ferritin

bythemethodofKishida, Olsen, Berg & Proclop (1975), postfixed with 1 aqueous osmium

tetroxide, rinsed several times with PBSanddehydrated throughagraded seriesofethanols.

Following absolute ethanol grade, the cells were treated with 0-05M hafnium chloride (AJfa

Produ cts, D anvers, Mass.) in absolute acetone for

1

h. This treatment is believed to enhancethe

contrastofmicrotubules (Karnovsky, unpublish ed observation). Cells were rinsed 3 times with

absolute acetone

and

embedded

in

Epon

by

inverting

the

Epon-filled beam capsules over

the

coverslips. Following polymerization,

the

coverslips were separated

by

immersing

the

blocks

in liquid nitrogen and ultra thin sections of lymphocyte monolayers were cut by a diamond knife

using LKB Ultrotome

III.

Sections were stained with lead citrate

and

examined

in a

Philips

200 electron microscope.

RESULTS

Murine splenic B-lymphocytes transformed by LPS over a period of

48

h and then

incubated on coverslips at 37 C for 30 min revealed certain variations with regards

to their morphology and distribution of surface immunoglobulins. A round cell

presumed to be a non-motile B-lymphocyte exhibited a random distribution of Ig on

its surface as judged by a diffuse immunofluorescence staining. Scanning electron

microscopy of such lymphocytes revealed numerous evenly distributed microvilli over

the cell's entire surface (Fig.

1).

With the onset of motility, the lymphocytes underwent

a series of changes with regard to the immunofluorescence staining pattern and surface

architecture. A sequential analysis of the cells in motility revealed that in the initial

stages of movement, the advancing end of the cell membrane stretched out, lost

microvilli and exhibited a ruffled edge. A constriction was often noticed at the base

of the ruffle (Fig. 2). A simultaneous decrease in the immunofluorescence at the

ruffled end and a corresponding increase at the opposite pole was immediately noticed

indicating the beginning of Ig mobility. Most of the microvilli by this time were seen

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D.

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

Fig. i. A round, presumably non-motile cell exhibiting numerous evenly distributed

microvilli. Th e cell is diffusely labelled with FIT C- R A M G (inset), x 10500; inset,

x1300 .

Fig. 2. A cell showing ruffles at one end . M icrovilli still occupy the major part of the

cell surface. A close observation of the fluorescen t micrograph reveals a slight decrease

in the staining intensity of FIT C -R AM G at the ruffled end (inset), x 10000; inset,

x 1300.

Fig. 3. A cell exhibiting non-uniform distribution of microvilli, which are denser at

the trailing end (lower left) and reduced at the advancing end (top right). There is an

increased fluorescence intensity atthe trailing end andadecreased intensity atthe

advancing end (inset), x 10500; inset, x 1300.

Fig. 4. A cell showing ruffling activity at 2 opposite poles, while the microvilli occupy

the intermediate area. The FITC-RAMG staining seems to be selectively associated

with the areas occupied

by

microvilli but depleted from the ruffled edges (inset).

x 10000 ; inset x 1300.

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Lymphocyte motility and spontaneous capping

1 4 1

Fig. 5. Cell showing a smooth ruffled edge (top). Microvilli are seen only on the bottom

half of the cell. Area of the cell surface localized by FIT C -R A M G correspo nds to the

micro villi-rich botto m half (inset), x 100 00; inset, x 1300.

Fig. 6. An elongated cell exhibiting a prominent microvilli-free ruffled edge with a

constriction at its base and microvilli at the trailing end. Ig-cap is seen at the trailing

end of the cell (inset). The area anterior to the constriction is not visible due to the

absen ce of fluorescence in this region of the cell, x 900 0; inset, x 1300.

Fig. 7. A cell exhibit ing elongated shape and a dist inct contracti le ring sl ightly

posterior to the middle of the cell body. Microvilli are densely packed at the trailing

end. A few elongated microspikes seem to anc hor the cell to the substratu m. A d istinct

Ig-cap is seen at the microvilli-rich trailing end, while the anterior parts of the cell

margin are not stained by F IT C -R A M G (inset), x 9000; inset , x 1300.

Fig. 8. Major part of the cell surface seen in this micrograph is smooth and exhibits

ruffled ed ges. Microvilli are densely packed on a compact p rotube rance at the trail ing

end. FI TC -R A M G sta ining is rest r ic ted to the tra i ling end ( inse t ). The remainder

of the cell is not visible, x 900 0; inset, x 1300.

CEL 9

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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 developed

at the 2 opposite poles of a cell, with microvilli occupying an intermediate area. Such

a cell exhibited a bipolar cap associated with the microvilli and the Ig-depleted areas

represented by the 2 ruffled poles (Fig. 4). Under normal circumstances of uni-

directional m otility, however, the ruffled endinthe nextstagebecame more pronounced

with a distinct constriction at its base. Microvilli and Ig seemed to be selectively

displaced from the ruffled edge towards the opposite pole (Fig. 5). Lymphocytes next

exhibited 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-cap

detected by the ferritin-antibody technique (see text for details). Distinct micro-

filaments are seen in the microvilli. No te the higher concentration of ferritin particles

on the microvilli than on smooth areas of the cell membrane. A number of coated

vesicles (arrows) are seen in the cytoplasm at this pole of the cell, x 37700.

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Lymphocyte motility andspontaneous capping 143

developed ruffled end from the opposite pole, which exhibited a dense organization

of microvilli and Ig (Fig. 6). The constriction, still quite prominent and appearing to

form a contractile ring slightly posterior to the middle of the cell seemed to restrict the

microvilli and Ig at the trailing end (Fig. 7). Cellular projections, distinctly larger than

microvilli were also seen at this end. Such projections, measuring 0-2 fim in diameter

and ranging from 2 to 30 /tm in length have been referred to as microspikes, micro-

extensions or filopod ia by various authors (A lbrecht-Bueh ler, 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 the

ruffles were stretched at the opposite pole, possibly in an effort to develop new adhesive

sites on the substratum. The lymphocyte in the final stage exhibited compact micro-

villous accumulation at the trailing end, which was now represented by a short

protuberance, while the majority of the cell's surface was marked by the presence of

well defined ruffles but devoid of microvilli (Fig. 8). Similar changes associated with

anti-Ig induced capping have earlier been described by Karnovsky & Unanue (1978).

Mo tile, capped lymphocytes in thin sections showed that Ig was selectively denuded

from th e ruffled areas but associated wi th microv illi at the opposite pole , as revealed

by the ferritin-antibody technique. Furthermore, the ferritin particles in motile cells

were present at higher concentration on microvilli than on the smooth areas of the cell

membrane between them. An accumulation of linearly arranged or a meshwork of

cytoplasmic microfilaments was often noticed at the trailing end. Distinct micro-

filaments could also be seen running through the microvilli. Microtubules in most

instances were observed in the deeper areas of the cytoplasm beneath the cap. In

addition to microfilaments and microtubules, coated vesicles were constantly seen in

the cytoplasm at the capped pole (Fig. 9). They could be localized, both near the

surface in association with the microfilaments or deeper in the cytoplasm, intermixed

with the microtubules.

DISCUSSION

Lymphocyte surface alterations may be related to the cell's functional state,

differentiation stage or the forces in its m icroenvironment (Van Ewijk, Brons &

Rozing, 1975; Roath, Newell, Polliack & Alexander, 1978; Bhalla & K arnovsky,

1978). Lymphocytes during locomotion exhibit various changes in their morphology

and they can be distinguished from stationary lymphocytes on the basis of their

amoeboid appearance (Lewis,

1931;

Unanuee t

al.

1974; Schreiner

Unan ue, 19766).

Previous studies had established that 24-48 h culture of B cells with LPS markedly

stimulated a locomotory response of the B cells. Th e m otile cells exhibited Ig in a cap.

Non-motile round cells showed Ig in a diffuse pattern. Such spontaneous capping

was also seen in B cells undergoing locomotion without the previous incubation with

any stimuli (Schreineret

al.

1976; Braunet

al.

1978). Spontaneo us caps did not require

detection with a bivalent anti-Ig. These observations excluded that LPS was causing

Ig-capping by cross-linking, and also indicate the spontaneous nature of Ig capping

in motile cells. In the current study, we have relied on lymphocyte shape and surface

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

architecture in determining the motility events. A rounded lymphocyte with its

surface exhibiting evenly distributed microvilli but devoid of any ruffling activity

was characterized as a non-motile cell. Following contact with the substratum,

lymphocytes showed certain surface alterations suggestive of motility. Th e progressive

stages of movement were assessed by the relative amou nt of surface ruffling, displace-

ment of microvilli towards one pole and deviation of the cell form from the rounded

appearance.

Abercro mbie, Heaysman & Pegru m (1970) have suggested that th e lamellipodia at

the edge of a moving fibroblast resu lt from the assembly of new surface, which may be

favoured by the longitudinal pull applied to the plasma membrane by the cytoplasmic

microfilaments (Harris, 1976). Function of microfilaments in cell locomotion and

surface ruffling has also been suggested by others (reviewed by Goldman et al.1973;

Korn, 1978). Our scanning electron-microscopic observations indicating a ruffled

advancing end and a microvilli-rich trailing end of the motile lymphocytes are

consistent with the suggestion of a more stretched anterior region of the plasma

membrane and the least stretched region at the posterior end (Harris, 1976). In light

of such studie s, it seems reasonable to speculate that during the initial stages of motility

and in the course of local stretching of plasma membrane in the ruffle, a link between

microfilaments 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 been

previously proposed to be a requirement for actin or myosin mediated capping

(Bourguignon Singer, 1977). Subsequently, the displacement of microfilaments and

the linked proteins may occur in a coordinated manner. According to Borisy &

W hite's (1978) model for cytokinesis in animal cells, a signal emitted from the poles of

a mitotic spindle perturb s the filamentous net underlying the plasma mem brane such

that the tension at the cell surface becomes greater in the equatorial than in the polar

regions. 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 to

cleavage of the cell. Such a model may also help explain the formation ofaconstriction

seen behind the ruffled edge in motile lymphocytes. How ever, the na ture of the signal

responsible for the formation of the constriction is not clear. Since the local me mbrane

constrictions in a motile lymphocyte appear to proceed from the advancing to the

trailing end, they may be envisaged as the cellular specializations favouring the

displacement of the complexes of ligand-recep tor and locally contracted microfilaments

to the trailing end. Microspikes seen at the trailing end of motile lymphocytes might

be vital for anchorage and cell motility. The ir role in particle trans port, cell locomotion

and as sensory organs have been discussed by Albrecht-Buehler (1976) and Albrecht-

Buehler & Goldman (1976). The selective capping of Ig but not 6and H2 may reflect

a difference in the nature of insertion of these receptors in the membrane and their

interaction with integral membrane proteins and/or with actin and myosin. Presence

of a meshwork or linearly arranged microfilaments below the cap probably serves to

maintain 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

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Lymphocyte motility andspontaneous capping

145

ferritin particles is more on the microvilli, which are clearly rich in microfilaments,

further suppo rts the suggestion ofanassociation between microfilaments and receptors

during cell motility. Although a physical linkage between Ig and microfilaments during

spontaneous capping seems quite likely, the function of microfilaments in balancing

the forces created by regional alterations in the physical properties of the capped

membrane (Albertini, Berlin & Oliver, 1977) may be equally important.

A directional flow of lipids alone (Bretscher, 1976) or of lipids and pro teins (H arris,

1976) of plasma membrane has been hypothesized to explain the capping of surface

antigens. B retscher (1976) has suggested coated vesicles as a means for lipid reso rption

into the cell. General occurrence of the coated vesicles in the cytoplasm at the trailing

end of motile lymphocytes supports the recycling phenomenon; however, it is hard to

say if the recycling accompanying motility involves only lipids (Bretscher, 1976) or

membrane 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 with

microfilaments and mic rotubule s at the trailing end of motile lymphocytes may suggest

a possible role of contractile elements in membrane-recycling.

In conclusion, then, using the correlated immunofluorescence and scanning electron

microscopy, this study presents a systematic evaluation of the surface features of the

lymphocytes undergoing motility and spontaneous capping of Ig. The observations

reported may be relevant to the mechanism underlying motility and capping. The

suggestions are supported by the thin-section observations. The study is also con-

sistent with the hypothesis suggested earlier that the ruffles at the anterior edge

reflect 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 Ms

Kay Cosgrove for assistance in manuscript preparation. This work was supported by Grants

AI 10677 and GM 01235 from the N.I.H., U.S.P.H.S.

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Received

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