Organization of lymphocyte plasma membrane. Surface protein-membrane matrix interactions in B-cell lines of different stages of differentiation

Ce~ Differentiation, 22 (1988) 233-244 Elsefier Sc~ntificPub~shers Irdand, Ltd.

233

CDF 00468

Organization of mphocyte phsma membrane. Surface protein-membrane m fix infractions in B-cell lines of different stages of differentiation Guido Trenn

1, H~ime Takayama

~, Wendy F. Davidson 3, Herbert C. Morse III 2 and M~hail V. Sitkovsky 1

Laboratories of t Immunology and 2 Immunopathology, Na~onal Institute of Allergy and Infectious Diseases and ~ Laboratory of Genetics, National Cancer lnstitut~ National Institutes of Health, Bethesda, MD 20892, U.S.A.

(Accord ~ August 1 ~

Composition of surface proteins and t h o r interactions with cytoskeleton or membrane ma~ix were compared in tumor B-cell fines of different stages of B-lymphocyte maturatio~ All studied B-cell lines were found to share a dmilar set of cell surface prot~n~ which are fight~ associated with the cytoske~to~ The increase in amount of detergent-unex~actable cell surface proteins with B-cell maturation suggested that differentiation of B ~mphocytes was accompanied by dev~opment of spe~fic interactions between sudace proteins and ~emen~ of the cytoskeleton or membrane ma~i~ U~ng a recently dev~oped procedure for ~mphocyte plasma membrane ~actionation we demons~ate changes in dis~ibution of cell sudace protons in membrane ma~ix-dch and membrane ma~ix-poor plasma membrane fractions during B-~mphocyte maturatio~ Thu~ cell sudace proteins of the mature B-cell line MOPC-315 were predominant~ found in the plasma membrane veeries of a high buoyant den~ty. These vesicles most~ cont~ned plasma membrane proteins fight~ assoc~ted with ~ements of the membrane ma~ix. In immature B cells 0ine 70Z3) ~rtu~ly all surface proteins were detected in both low and high buoyant den~ty membrane v e ~ c ~ The tendency to increased associations between surface proteins and cytoske~ton/membrane ma~ix with maturation of B cells could not be expl~ned by increased amoun~ of fi~mentous actin, ~nce no correction was found between the amount of ~obular or filamentous actin and the degree of sudace protein-cytoskeleton (membrane matrix) interactions. B-Lymphocyte; D ~ n f i a f i o n ;

~ma

memb~n~ Membrane ma~ix; C y ~ s k ~ e ~ n

Correspondence address: G. Trenn, Laboratoryof Immunology,

Introduction

Bld~ 1~ Room 11N-311, NIH, Bethesd~ MD 20892, U.~A. Abbreoia~ons: Ab, antibody; sA~ surface antigen; PMSF, phenylmethylsulfonylfluofid~ PBS,phosphate-bufferedsaline; A~ antigen; EGTA, ethyleneglycol-bi~aminoethylether) tetraacetic a~d; ATP, adenofine 5Ltfiphosphate; DNaseL deoxyfibonudease I; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel electrophoresis;sIgM, cell surface IgM.

The c y ~ a s m of most mammalian cells cont~ns a ~ g ~ y c o m p ~ stru~ured meshwo~ of fi~men~us and irregular connecting demems that ~ r m the so called c y ~ s k d ~ ~amework (W~osewick and Po~e~ 1976; Schliva, 1986). T ~s

0045~039/88/$0330 © 1988 Else~er SoenfificPublishers ~dan& Ltd.

234 framework of cytoplasmic fiber~ which maintains its ultrastructural integrity after ex~action with non-ionic detergen~ in appropriate buffe~s g operationally refe~ed to as the cytoskdeton (Brown et M., 1976; Gilbert and Fulton, 1985). The inter actions of the cytoskdeton with surface protons are befieved to regulate the functions of cell surface receptors (e.g, affinity of the nerve growth factor receptor - Schechter and BothwdL 1981L Whim ~milar influence of cytoskdeton on the properties of Ag receptors in lymphocytes has not been documenmd, it has been shown that infractions of antigen with receptors or monodonal antibod~s with surface antigens induce changes in cytoskdetal organization and assodation of sAgs with the cytoskdeton (Braun et M., 1982; Woda and McFadden, 1983). Reorganizations of the microtubule organifing ~enter (MTOC) and the Golgi apparatus were found after interaction of cytotoxic T lymphocytes (Goger et M., 1982) and T-hOper lymphocytes (Kuppfer et M, 1986) with Ag-bearing target cells. The plasma membrane itsdf has been shown to be assodated with a detergent unex~actabM continuous layer of protons which is referred to by authors and throughout this Iep¢rt as the membrane matrix which ~ distinct from the cytoskeleton (Mesher et M., 1981). The structure of the dements of the cytoskdeton and membrane matrix in lymphocytes ~ now the subject of inten~ve research, and it was recently suggested that an actin-containing dete> gent insolubM matrix in lymphocytes is ~milar to the actin-ffee membrane skdeton of red blood cells (Mesher et al., 1981; Apgar et M., 1985; Apgar and Mesheg 1986). It has also been shown that cros>finked surface immunoglobufins are assooated with such insoluble matrix (Braun et M., 1982). A~uming that a~odations of surface receptors with underlying proteins of the membrane matfix/cytoskdeton are important for thor Mgnal-~ansdudng functions and biomechanicM properties of lymphocytes, it seemed necessary to investigate how these assodations change during the functional response or differentiation of lymphocytes. It was Mready demon~rated in other cellular sy~ems (e.g, cuRured muscle cell~ that the number of cytoskOeton-assodated surface re-

ceptors (acetylchofine receptors) increased with maturation (Prives et ~., 1982). Despite extensive ~udies of the antigen~ changes on the surface of differentiating B lymphocytes (Hammerling et al., 1976; Davidson et ~., 1984), fitfle is known about interactions of these antigens with the membrane/cytoskeleton. Howeve~ breakthroughs in the understanding of the s~ucture of B- and T-cell receptors and availability cf n u d o c a~d probes and monoclonal antibodies to the surface markers allowed estabfishment of B-cell fines which could be arranged in ascending order of maturation (Davidson et al., 1984), representing different stages of B-cell dib ferentiation. Ufing such cell fines it is posfible to have enough material for biochemic~ ~udies of plasma membrane organization at the d o n ~ ~vd. In this repo~ we describe the organ~ation of plasma membranes in B-cell fines, which represent defined ~ages of B-cell differentiation. These ~udies were greatly aided by the use of a merebran_e ~actionation procedure devdoped to ~olate lymphocyte plasma membrane domains which di~ fer in amount of membrane matrix and in degree of membrane matrix-surface protein interactions. We ~so document differences in the amount of globular and filamentous actin in different B-cell fines, fince actin is impficated in many cellsurface-mediated activities of lymphocytes (Braun, 1983).

Materials and Methods B-cell 6nes

Characteristics of the B-cell fines we used are described in Table I. The B-cell fines in Table I are presented in order of their maturation. (For more detailed characterization of B-cell fines under investigation see Kim et al., 1979; Lanier et al., 1981; Paige et al., 1981; Mushinski et al., 1987.) D N a s d inhibition assay

For determination of cell~ar actin concentration the D N ~ inhihition assay was used as described dsewhere (Blikstad ~ ~., 1978; Fox et ~., 1~1) w i ~ minor modification. Briefly, 8 × l0 s

235 TABLE I Call surface marke~ of ~ f ~ n t

~c~

f i ~ s reed ~ r ~ e p ~ s e n t ~ u ~

Surface antigens

NFS-112 NFS-1135

70Z3

WEHI 231

L10A

BALTELM SJL-4/2

MOPC 315

L~17 f f c ~ B2~ ~B Ia ~g

+ +++ +++

+ +++

+ +++ +++

+ +++ ++

+ +

-

+ +++ +++

_

+

+

+

+ +

+ +

+ +

~g~/~ slgM sIgD Ig secretion PC-1

_

+

-

-

+

_

_

+

+ + +

-

+

+ +

+ + +

N F ~ l l 2 and NF~1135: p~-pr~B-cell; 70Z3: pr~B-ce~; WEHI 231 and L10A: ~mphocytic stage of development; BALTELM and SJL-4/2: immunoblast~ cell; MOPC~15: ~ a s m a cell.

cells per samp~ were suspended in 140 #1 phosphate-buffered saline (PBS). Ex~action of cells with 1% Triton X-100 (100 mM Tri~HCI, pH 7.4) was performed for 30 min at 4°C in the presence of 2 mM ~h~ene glycol-hi~aminoeth~ ~heO tetraacetic a~d (EGTAL 1 mM phen~m~h~sulfon~ fluoride (PMSF), 10 # g / m l ~upepfin and 5 # g / m l antipaim The incubation was fo~owed by centrifug~ion in an Eppendorf centrifuge for 15 min at 4°C and amount of glob~ar actin was measured in the supern~ant. For determination of total actin 150 #1 of depdymerization buffer (1.5 M guanidin~HCl, 1.0 M N ~ a c ~ 1.0 mM Na ATE 6 mM CaC12 d i ~ v e d ~ 20 mM TrisHCI, pH. 8.4) was added to samples after extraction with 1% Triton X-100. Samples were incubated on ~e for 10 rfii~ spun for 20 min in an Eppendorf centrifuge and amount of total acfin was estimate& In a control assa~ 10 #1 DNaseI solution (0.1 mg/ml DNasd (~gm~ No. D-4527)), 0.1 mM CaC12, 0~1 mM PMSF in 50 mM Tfis-HCI (pH 7.5) and 1.5 ml of a DNA solution (40 # g / m l DNA ~om calf thymus, ~gma N ~ D-1501L 1.8 mM C9C1~, 4 mM MgSO4 in 100 mM Tfis-HC1 (pH 7.5) were directly mixed in a cuvette with 20 #1 of a ~ - ~ e e sample buffe~ The enzymatic reaction was monitored over a time period of 4 min at room ~mperature by the ~crement of absorbance at 260 nm (Beckman spec~ophotome~O. The flope of the hnear pa~ of the ob~ined curve was

determined. Consequently, various amounts of actin were added to the DNA/DNase mixtur~ The degree of inhibition of this enzymatic reaction was plotted again~ the different actin concentrations and thus a ~andard curve was obtained. The globular acfin concen~ation of a given sample was determined by adding 20 gl Triton X-100 soluble material (equivalent to 1 × 105 cell~ to the DNA solution. For determination of total actin a new negative control to correct the effect of guanidine-HCl was introduced. Activity of DNaseI in the presence of guanidine-HC1 and in the absence of actin was measured since the guanidine-HC1 containing depolymerization buffer changes both the magnitude and the time course of ~ght absorbance. The concenUafion of filamentous actin was calculated as the difference between amounts of globular and total actin for an equivalent of 1 × 105 cells. The mean value ± SEM was determined from several expefimenU.

Iodination of cell surface pro~ins For l~Idabeling of cell surface protein the glucos~oxidas~lactoperoxidase catalyzed method was used (Coope~ 1977). Briefl~ 50 × 106 cells were extensively washed and resuspended in a 20 mM D-~ucose/PBS solution (1 ml). Lactope~ oxidase (Cooper Biomedical, lot No. 30825131) was added to a final concentration of 180 U/ml. One hundred #Ci Na 1~I 0MS-3~ Amersham) were added to the cell suspenfion. The ~beling

236

reaction was staaed by ad~tion of ~ucose o ~ d a ~ s~ufion (~gm~ No. G-6500) to a fin~ concen~ation of 4 U/ml. A f ~ ~cubation at room ~ m p ~ ature for 15 min cells were washed s~ times ~ a 10 mM KI/PBS s~ution and resuspended ~ P B S - b u f ~ The amount of de~rgent extractable radioactive h b d e d m ~ e f i ~ was de~rmined by s o ~ f i f i n g cells wi~ the n o ~ o ~ c d ~ g e n t Non~ P-40 (NP-4~ (~gm~ No. N~50~. Cells w~e ex~ac~d wilh 0.5% NP-40, 0.01 M Tri~HC1 (pH 7.~, 0.15 M NaC1, 0.02% NaN 3. P r o ~ y f i s w ~ m~d by ~ e ad~fion of ~upeptin (10 ~g/mlL antip~n (10 ~g/ml) and PMSF (1 mM). The m i x ~ was ~ c u b ~ e d at 4°C for 30 min and • en centrifuged at 10000 × g for 15 min. TCApre~pitab~ ~ d ~ a c t i ~ f f ~ supernatants and ra~oa~iviff of NP-40 ~soluble materi~s ~ e ~ meas u e d by a 7~oun~r (Beckman). The NP-4~s~ub ~ / i n s ~ u ~ e protons were further an~yzed by s o , u r n dodec~ s ~ p ~ y a ~ a m i d e gd dectrophoresis (SD~PAGE) and autoradiography. SD~PAGE was performed according to Laemmli (197~.

Immunoblot procedures After SD~PAGE the protein bands w~e b ~ e d onto n~roce~ulose paper accor~ng to ~ e procedure described by Tow~n et ~. (197~. Blotting was conduced at 100 mA for 3 h (4°C). The ~ o c d l ~ o ~ paper ~.45 ~m pore s ~ Lot No. 4023/5, Sc~cher&Schuell) was then washed in blotting buffer (100 ml cont~n: 10 ~1 antifoam emdficn (No. A-5758, ~ g m ~ , 0.5 ml 2% NaN 3, 49.5 ml ~stil~d water and 50 ml 0.1 M Tris buffer) to w~ch 5 g/100 ml non-fat dry milk was added to prevent non-spedfic ~ n d ~ & The same buf~r was used for incubation of ~ o c e l l ~ o s e pap~ wi~ rab~t anti-mou~ ~ c h ~ n spe~fic antibody (5 ~g/ml) (Zyme& Lot No. 50201) for 1 h at ~oom ~ m p ~ a m ~ . A f ~ w ~ n g with PBS and PBS c o n t ~ n g 0.05% Tween 20 (No. P-1379, ~gm~ ~e ~ocellu~ paper w ~ ~ c u b ~ e d wi~ ~ I - h b d e d protein A (Amersham) at appro~m ~ d y 100 000 cpm/ml. Finally, ~ e ~ o c d ~ paper was washed ag~n and ~r dried before autora~ography w ~ performed.

Purification and fractionation of p~sma membran~ To study ~ e compofition of proteins in phsma

membranes we labded cell surface protons according to the procedure described above. Calls were then incubated in 40 mM Tris buffer (pH 7.4, call concentration 108/mt) on ice for 10 min and spun at 1500 rpm for 5 min. Calls were resuspended in 40 mM Tfis buffer contNning 1 mM ZnC12, leupeptin (10 ~g/ml), antipNn (5 ~g/ml) and 1 mM PMSF. After incubation on ~e for 5 min, cells were disrupWd Nther by a Dounce homogenizer (7 ml capadty; Wheaton, N J) or by nitrogen ca~tation. The degree of call di~upfion was monitored under the microscope. The homogenate was centrifuged at 500 rpm for 1 min to separate the nudN and call debris. The supe~ natant was col~cte& transferred to high speed centrifuge tubes and centrifuged at 10000 rpm for 10 min at 4°C (SorvM1 SS-34 rotoO. The pallet was resuspended in 1 ml of 20% sucrose/40 mM Tri~HCI solution. The discontinuous sucrose gradient (1.7 ml of 65%, 3.4 ml of 55%, 45%, and 35% sucrose solution) was ovedNd with resuspended homogenized matefiN and centrifugation was performed (4°C, 10000 rpm) for 12 h ufing SW40 Ti rotor (Beckman) and L-8 ul~acentrifuge (Beckman). The gradient was ~acfionated by inserting a capillary into the centrifuge tube. Fractions of 0.5 ml were collec~d by confinuou~y aspirating suspenfion ~om the bottom of the tube ufing a peristaltic pump. The pellets were resuspended in 40 mM Tri~HC1 solution. RadioactNity of each ~acfion was measured and fractions with the highest counts were pooled. Thus four mNor ~actions and the resuspended pellet ~action were obtained. These m~or fractions were spun for 3 h at 49000 rpm (50 Ti roto0. The prints were resuspended in 20 mM Tfis-HCI buffer contNning 0.15 M NaC1 (pH 7.4) and protons were solubilized with NP-40 buffer as described above.

Res~

Associaaon of cell surface proteins with cytoske&ton in dtfferent B-cell #nes To evaluate the amount of cell surface antigens associated with the cdlular matrix at different stages of B-cell maturation we iodinated the cell

A

B

kDa

kDa

A

B

C

- 200

-

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97

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97

-

68

-

68

-

30

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30

D

A

B

C

200

D

C

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Fi~ 1. SDS-PAGE analysis of cell surface protons of different B-ce~ fne~ 50 × 106 cells were radiolabded with z~ I, solubifzed and protons were an~yzed by SDS-PAGE and autoradiography as described in Materials and Methods (Pand A) NP-40-insolub~ (cytoskeleton-associated) B-cell su~ace prot~n~ Lane A, pre-B-cell fne 70Z3. Lane B, lymphocytic cell fne WEHb231. Lane C, lymphocytic cell fine L10A. Lane D, plasma ce~ ~ne MOPC-315. Arrows indicate protein bands shared by all ~udied cell fnes. The molecular weights of these proteins are (from top to bottom): 61000, 5100~ 43 500, 33000 and 28 500. (Pand B) NP-40~oluble B-cell surface proteins. Lane A, pr~B-ce~ line 70Z3. Lane B, lymphocytic cell fine WEHI-231. Lane C, lymphocytic cell fne L10A, Lane D, plasma cell ~ne MOPC-315. (Pand C) Same lanes as shown in pand A a~er a shorter exposure fim~

238

surface protdns of different B-cell fines. A~er extraction with the non-~nic de~rgent NP-40, the NP-40-s~u~e and - ~ s o h b ~ ~actions were an~yzed by SD~PAGE followed by autora&ography (Fig. 1). Pr~B-cdl fine 70Z3 had the ~ast amount of NP-40-une~racmb~ cell surface pr~ ~ins (Fig. 1A/C). An increase in the number and amount of NP-40qns~u~e surface protons was detected when more m~ure cell fines WEHP231 and L10A were an~yzed. The plasma call fine MOPC-315 showed the ~ g h ~ t amount of NP-4~ ~ s ~ u ~ e surface proteins. It shou~ be p ~ n ~ d out that ~ f ~ n c e s in amount of ~ n ~ a b ~ cell surface proteins (both N P - 4 0 ~ u b ~ and qnsoluble) may account for the ~ f ~ r e n t in~nfit~s of proton bands among ~sted call fines. Howeve~ this a m b ~ was ~s~ved when rat~ of NP-40s~ub~ to NP-4~ins~u~e t25I-surface h b d ~ d protons was compared; this ratio ~creased in more m ~ u ~ B-cell fines and was higher in MOPC 315 call fine. Comparison of the compofition of 70Z 3

40

~°~ ,._E -~.

NP-40-unextractable materi~ in four B-cell fines reve~ed fimilar protein pat~rns in the subset of cell surface protdns with molecular w~ght brow 68000 (identified by arrows on Fig. 1A). This subset of protons with molecular wdgh~ of 28 500, 33000, 43 50~ 51 000 and 61000, were found in ~1 four cell fines. Bands with higher mo~cular wdgh~ (espeo~ly proton bands with molecular wogh~ of 100000, 113000 and 145000) appeared in the more mature B-call fines. These resul~ may indicate that some of the cytoskd~on-a~odated call surface proteins (marked with a~ows in Fig. 1A) are necessary for bafic cellular functions during ~1 stages of B-cell differentiation and are therefore conserved during maturation. In the B-cell fines we ~s~& the pat~rns of surface proteins not a ~ o d a ~ d with cytoskd~on (NP-40-solub~ ~acfion) were quire dif~rent (F~. 1B). While the less m~ure B cells showed a high amount of NP-40-solub~ surface anfigen~ MOPCOl5 had d r a m a t i c ~ fewer extractable

i 30'

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NP 40 unsNuNe

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1

2 3 membranefraction

4

Fi R 2. Di~fibution of cell surace h b d e d protons among B~dl ~asma membrane ~acfions of different buoyant denfitie~ B cells were call surface h b d e d with 1~I, ~srup~d by homoge~z~ion in Dounce homoge~zer and ~asma membrane ~ a ~ n s were obt~ned a~er sucrose denfi~ gra~ent centri~gation as described m Ma~fi~s and M~hod~ Hasma membrane fractions were e x h a l e d wi~ NP~0 and ra&oactivi~ in N ~ 4 0 ~ u b ~ and N ~ 4 0 q n s ~ u ~ e fractions was counted. Fraction 1 contained the dense vefide~ w ~ fraction 4 ~ e f i g h ~ vesicles. Bars m ~ c a ~ percent of radioactivity in the ~ven ~asma membrane fraction. Tot~ ra&oactififf ~ ~act~ns 1, 2, 3, and 4 ~us p d ~ t was con~dered ~ be 100%. R a d ~ a c t i ~ m pell~s of 70Z3 was 40% of ~tal, w ~ ~ r MOPC-315 it was 52% of ~ t ~ . The h~ched bars repre~m ~ e proportion of NP-4~unex~a~a~e ra~oacfifi~ in each ~asma membrane fraction.

239

surface antigens. Thus, the m~ofity of cell surface protons in the MOPC-315 cell fine are attached to the cdlular matri~ The pat~rn of NP-40-soluble proteins presen~d in Fig. 1B is much more comp~x and different among the four cell fines than the more uniform pat~rn of NP-40-insoluble prot~ns displayed in Fib 1A/C. Maturation of B cells seems to be asso~ated with both the appearance of new cell surface antigens (NP-40-soluble and -insolub~) and the disappearance of NP40-soluble surface protons ori~nally present at earlier stages of devdopment. These resul~ demonstrate that changes in compofifion and organization of cell surface protons are extenfive and are accompanied by changes in thor interaction with the cytoskdeton and/or membrane matfi~ The data presented in Fib 1A-C do not ~low us to distinguish between surface proton in~ra~ tions with the cytoskdeton or the membrane matrix. To address this question we took advantage of a recently devdoped procedure for ~olation of lymphocyte plasma membrane ~actions of different buoyant denfities (Takayama et ~., in preparation).

A

200

97 68

43 26 3

P

4

D~t~but~n of BmeH surface protons in dtfferent plasma membrane fractions After ,fracfionafion of plasma membranes by isopycnic centfifugatio~ the amount of tot~ NP40-ex~actable and -unex~actab~ cell surface ~ b d e d proteins was determined for each fraction. Representative resul~ for two cell fine~ 70Z3 and MOPC-31L are shown in Fig. 2. Most of the cell surface proteins in cell fines 70Z3, WEHI-231 and L10A were located in ~action 2 (the second heaviest~ whi~ most MOPC-315 surface proteins were found in fraction 1 (the heafies0. In all tested cell fines the proportion of NP-40unextractable matefi~ in plasma membrane veeries increased in paralld with the increase in v e r d e buoyant den~ty (see Fig. 2). The plasma membrane pell~ fraction cont~ned mostly (> 90%) NP-40Ansoluble cell surface protons. As shown in Fi~ 2, MOPC-315 cell membrane fractions were characterized by a high degree of unextractable surface prot~n~ whi~ the m~ofity of cell surface proteins in plasma membrane fractions of call fine 70Z3 were NP-40 solubl~ Ex~ac-

p

1

2

3

-

200

-

93

-

68

-

50

-

25

4

Fig. 3. SDS-PAGE analysis of B-cell surface proteins in plasma membrane ~actions of different den~fie~ B cel~ were treated as described in legend for Fig. 2 and proteins of each plasma membrane fraction were analyzed by SDS-PAGE and autoradiography. (A) Pattern of NP-40-soluble proteins in 70Z3 line. p: pellets; 1, 2, 3, 4: plasma membrane fractions as described in legend for Fig. 2. In order to show more clearly the protdn band~ lane 2 is taken from a less exposed film. At the same exposure time as lane p, 1, 3 and 4, no individual protein bands are distinguishable in lane 2. (B) Pattern of NP-40-soluble proteins in MOPC-315 cell fine.

tabifity of surface proteins from fines WEHI-231 and L10A was lower than from 70Z3, but higher than from MOPC-315 (data not shown). The results obt~ned by an~yfis of membrane fractions were in accordance with data obt~ned with whole cell analysis (Fig. 1A-C). The di~ribution of NP-40~oluble cell surface protons in the different membrane fractions for

2~

A

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Fi~ & An~yfis of cell sur~ce protein compofifion in ~asma membrane ~ a ~ n s of different denfifies in cell fine WEHb231. C d ~ were ~eated as described ~ ~gend for Fig. 2. Protons of N P ~ s ~ u ~ e (A) and NP-40-ins~ub~ (B) m~eri~ were an~yzed by SDS-PAGE and autoradiography. (A) P ~ r n of NP-40-soluble proteins, p: pellet; 1 2 3 ~ : ~asma membrane ~action~ ( represents the pell~ ~acfion at a sho~er exposu~ fim~ (C) Wes~rn b ~ t with rat antibody ag~n~ mouse IgM.

the cell fines 70Z3 and MOPC-315 is presented in Fi~ 3. In 70Z3 cells NP-40-extractable surface proteins were found in all five membrane ~ a ~ tions. While fraction 2 contained the highest amount of cell su~ace protNns there were no mNor quafitative differences in protein compofition among these ~actions; the same mNor mem-

brane proteins were found in ~1 four fractions (Fig 3A). In contrast to these findings with 70Z3 cells are those obt~ned with the cell fine MOPC315 (Fig. 3B). In this cell fine most NP-40-soluble call surface antigens were preferenfiMly expressed in only two fractions (fractions 1 and 2). QuMitat i v d ~ both fractions were remarkab~ different.

241

While fraction 2 cont~ned three m~or protein bands of - 80, 95 and 180 kD~ fraction 1 hcked these bands and cont~ned m~or protein bands of different m~ec~ar wdgh~ (72 and 30 kD~. An~yfis of NP-40qns~ub~ proteins ~ the plasma membrane fractions revealed an ~ e a s e in NP40-unextractable proteins with higher denfi~ memb~ne verities. Fractions 3 and 4 of both cell lines contained very ~tfle NP-40-unextra~able call surface proteins. To ~ v e s t ~ a ~ the d~tfibution of a spedfic surface protein among &ff~ent plasma membrane fractions and among N P - 4 0 ~ u N e and NP-4~ins ~ u ~ e mmefi~s ~ the same plasma membrane fraction, we ~u&ed the d~tfibution of a predominant NP-40-s~ub~ cell surface proton of 72.5 kDa (see Fi~ 4A) in the plasma membrane fractions of call ~ne WEHL231. T~s protein is a m~or band in fractions 1 and 2 and less prominent ~ the p d ~ t fraction. ~nce the m ~ e c d a r wright of this protein is fimihr to that of IgM ~ c h i n and WEHL231 has a large amount of surface IgM (slgM~ we ~ m p ~ d to identify this ~asma membrane protein by W ~ r n ~ o t with anti-~ Ab. I m m u n o b ~ a ~ g confirmed the suggestion that the 72.5 kDa protein is a # c h i n of sIgM (Fig. 4C). The same protein band (~so shown by Wes~ ern blot to represent slgM) was found ~ the NP-4~ins~u~e ~ a ~ n s 1 and 2 and in the pellet ~ a ~ n (Fi~ 4B). In the NP-40qnso~ble fraction~ howeve~ it was o ~ y a minor band in fraction 1, but a predominant one in ~action 2. This result showed ~ a t slgM e ~ e d in two forms with a &f~rent &~fibut~n ~ the plasma membran~ Whi~ the N P - 4 ~ s ~ u ~ e sIgM was found in membrane verities of &fferent denfitie~ the NP-40-~s~uNe (membrane skdeton assoda~d) form of slgM was mostly a ~ o d a ~ d with membrane verities of a higher buoyant denfi~ (fraction 2).

nomena of different cell surface antigens such as sIgG. According to our result, the maturation of B calls ~ par~lded by a decrease in the proportion of NP-40~xtractab~ cell surface andgen~ To ev~ua~ the role of actin in this phenomenon we looked at the cellular concentration of actin in different B-cell 5nes. As shown in Fi~ 5, there was an increase in cellular concentration of actin ~om a large pr~B cell to a lymphocytic ~age of B-cell devdopment. This increase in total actin concentration ~om NFS 112, NFS 1135 and 70Z3 to L10A was ~atistically ~gnificant (Krusk~ W~~s ~s0. Further maturation of a B cell to an immunoblast (BALTELM, SJL-4/2) and finally to a matur~ IgA secreting plasma cell (MOPC315), howeveL was asso~ated with a ~gnificant drop in the concentration of cellular actin to ~vds found in pr~B cells. Fig. 6 shows the resul~ of the determination of ~obular actin concentration in different B-cell hnes. There was a sharp increase in the cellular concen~ation of ~obular amin from pre-B cell to lymphocytic stage of maturation and a consequent decrease in mature B cells. A ~milar pat~rn was obt~ned for the di~fibufion of filamentous acfin in the cell ~nes. The dif~rences in ~g act~ / 10 S c ~ l s .~

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Studies of filamentous actin content in whole cells and in different plasma membrane fractions of B-cell ~nes The next question we addressed was whether filamentous actin plays a m~or role in the interaction of cell surface' antigens with the cytoskeleton. It is known (Braun et al., 1982; Braun, 1983) that actin filaments are involved in the capping phe-

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Fi~ 5. Amount of total cellular acfin in different B-call hnes. 8 × 105 B cells were solubil~ed in a 1% Triton X-100 solution. Acfin depolymerizafion buffe~ as described in Idaterials and Method~ was added and the mixture was incubated on ~e for 10 min. An ahquot of Triton X-100 soluble material was analyzed for ~s actin content ufing a DNaseI inhibition assay as described in Materials and Method~ Kruskal Wall~ test was used for ~afistical analyfis ( P < ~001).

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Fi~ 6. Concentration of ~obular amin in different B-call ~nes. Cells were ~eated as described in Mgend for Fi~ 5 except that depolymerizadon buffer was not added to the samples. KruskM Wall~ ~ was used for stafisficM anMys~ ( P < ~001).

total amount of actin among these B-cell lines could nother be explained by differences in fize (they were fimilar in fize - by microscop~ observation), nor by differences in proton content; all had fimilar proton content (estimated per 106 cells by the Bradford method). Thus, no correlation was found between cellular concentration of filamentous actin and degree of cell surface antigen attachment to the cellular matrix. Mature B-call fines had the highest proportion of cytoskdeton-assooated surface protons (Fi~ 1) but had Last amount of cellular filamentous actin (Figs. 5, 6).

Discussion

The m~n finding of this study is the demonstration of changes in the proton compofition and organization in Bqymphocy~ plasma membranes during differentiation from pr~B cells to mature and Ig-secreting plasma cells. The obt~ned resul~ suggest the posfibility to devdop new cell-biologic~ and biochemic~ criteria for the das~fication of B-cell tumor. In more mature B cells a greater number of cell surface proteins ~ associated with the cytoskd~ ton a n d / o r the underling membrane matrix compared with immature B cells. The mature IgA

secreting plasma call MOPC-315 had the highest percentage of NP-40-insoluble surface proteins due to an increased interaction with the cytoskdeton a n d / o r underlying plasma membrane matrix. These are the first experiment~ data that demonstrate plasma membrane changes during differentiation and maturation of B-lymphocytes. One must be cautious in assuming that ~nden~es found with ~ansformed B-call lines will be the same in norm~ B lymphocytes. It was shown, however, that the cell hnes we used, 70Z3 and WEHI-231,.were ab~ to undergo fu~her diffe~ entiation in ~tro in response to extracdlular stimuli, supporting the validity of thor use for such ~udies (P~ge et ~., 1978; Boyd et ~., 1981). In addition, the differences between B-call tumor lines described here could be exploi~d in the development of a nova diagnostic procedure for the das~fication of B-call tumor. An~y~s of call surface membrane proton comportion by SDS-PAGE by u~ng ~ I - c d l surface labded B-cell hnes (Fi~ 1) allowed us to conclude that a subset of surface protons smaller than 70 kDa was uniformly expressed in all studied B-call fines. Protons of this subset were closely a~odated with the cytoskdeton and/or membrane matrix, ~nce they were not ex~actab~ with a nonionic de~rgent. M~ntenance of the same subset of cytoskd~ ton-attached surface protons throughout ~1 stages of B-cell maturation suggesu that these protons play an important role in B-lymphocyte phy~ology. It is pos~ble that these surface protons, by firtue of thor ability to in~ract with the cytoskdeton/membrane matrix, are necessary for the m~n~nance of the B-cell shape and/or motifity at all stages of differentiation. The described properties of the subset of B-call surface proteins ~ss than 70 kDa wa~ant thor fu~her detailed ~udies. Assodation of such proteins with the NP40-unex~actab~ cellular ma~ri~ provides a simple and effident method of purification for subsequent immunization and affinity purification of antibodies. The NP-40-soluble cell surface proteins, howeve~ showed dramatic differences in ~1 cell hnes (Fi~ 1B/C). The mature B-cell hne MOPC-315 had very fitfle NP-40-solub~ call surface protons, whi~ the ~ss mature B-cell hnes exhibited a broad pattern of NP-40-solub~ call

243

surface proteins with ~fferent m ~ e c d a r woghts. The ~ u ~ of B-cell surface membrane orga~zation de~ribed ~ Figs. 3, 4 and 5 were made p o s ~ e by the ~cent devdopment of a plasma membrane ~actionation m~hod that is based on separation by ~opyc~c centrifugation of plasma membrane veeries of ~mi~r ~ze but ~ f ~ n t buoyant den~ties (Takayam~ Friedma~ Trenn and ~ o v s k ~ in p~parafion). This ~actionat~n procedure allowed us to d e m o n s ~ e that a ~ o d ations of B-cell surface protons with NP-40qns ~ u b ~ m~eriE are not due to cytoskd~E structu~s. No m i ~ o t u b d ~ or ~ r m e & ~ e filamen~ were d e ~ e d in ~asma membrane veeries by dec~onmicrosco~c ~ u & ~ (Takayam~ FriedmaK Trenn and ~ o v s k ~ ~ p~parafion). It ~ possib~ that these cytoskd~E ~ r u ~ u r ~ &d not survive con~fions used during p~sma membrane preparation. EvEuation of the r~e of filamen~us actin in o r g a ~ z ~ n of surface protons in B-cell p~sma membran~ r e q ~ e d ad~tionE detMled ~ u d i ~ with independent m~hods of actin detection (Figs. 5,6). No correlation was found b~ween the amount of actin, on a per cell ba~s, and the extra~abih~ of surface protons by nonqo~c de~rgen~ (Fi~ 5). It was stffi pos~bl~ howeve~ that the amount of actin ~ a~o~afion with the p~sma membrane inc~as~ during B~ell m~uratiom w ~ the t o t e amount of cell~ar acfin ~sproportion~ly d e c e a s e . Th~s, it is p o s s i ~ for e x a m ~ that the amount of acfin determined for M O P C - 3 1 5 m a i n ~ reflects the plasmam e m b r a n e - a s s o c ~ d acti~ which wo~d account for lhe ~rge amount cf N P - 4 0 - u n e ~ r a c ~ e cell surface proteins ~ this cell line. To invesfiga~ this p o ~ f f i ~ fu~heG we d ~ m i n e d ~ e actin concen~ation ~ the ~ f f e ~ n t membrane fractions of MOPC-315. The amount cf actin, howeve~ d ~ tected ~ the membrane ~actions of MOPC-315, accounts for ~ss than 4% of the t o t e celldar actin (d~a not shown). According lo SD~PAGE, most, if not all, m e m b r a n ~ a s s o ~ e d a~in ~emed ~ be F~ctinassodated with NP-40-une~ra~ab~ surface protons. No ~rect corrdation was found b~ween the amount cf filamentous actin in membrane v e ~ d ~ and the proportion of N P ~ 0 q n s d u b ~ protons. It is p o s ~ e Ih~ o ~ y a part of Ihe membran~assodated F-actin is ~ v ~ v e d in ~a~l~ation of the

membrane matrix structure by in~racting with the recently described protons of membrane matrix called 'agorin~ (Apgar and Meshes 1986). We have shown prefiously that higher buoyant denfity of plasma membrane verities is associa~d with a higher proportion of surface protein-membrane matrix interactions. That observation is confirmed and expanded her~ We demons~ate here that different stages of B-cell differentiation can be described in ~rms of distribution of surface proteins among the plasma membrane ~actions of different denfities. Thu~ most of the surface proteins in mature MOPC-315 are found in the most dense plasma membrane v e r d e ~action 1 ~ membrane matrix-rich d o m ~ n ) in which practicEly all surface proteins are d o s d y a ~ o ~ a ~ d with the matrix (Fi~ 2). In the ~ss mature B-cell hnes 70Z3, WEHb231, and L10A, howeve~ most of the surface protons are found in ~action 2, which represents phsma membrane verities of a lower buoyant denfity and a ~ e r degree of cell surface-membrane matrix infractions. AnEyfis of lhe ~urface proton organization in different plasma membrane ~actions of these cell ~nes reveEed an inleresting tendenc~ While a fimil~r set of NP-40-soluble surface protons was d ~ e c ~ d in all plasma membrane ~actions of pr~B cell hnes (Fig. 3A), qualitativdy different surface prot o n patterns were found in different plasma membrane ~actions of the mature B-cell fine (Fi~ 3B). These data suggest that random di~fibution of NP-40-solub~ surface protons in the cell membrane of immature B ce~s is changed to organized dom~ns in the mature B-cell plasma membran~ It is no~wo~hy that Emest all NP-40-insoluble cell surface protons cf whole c d ~ were recovered in the membrane ~actions of those cells. This observation suggests lhat most NP-40-insoluble cell surface protons are directly asso~a~d with the membrane matfi~ The inabi~ty to ex~act these protons from plasma membrane ~action and thor recoverability in the cytoskdeton-enfiched whole cell pellet suggest that ~ansmembrane ~nks form between surface protons, membrane matrix proton~ and cytoskd~E proteins. Thus this finding emphas~es the impo~ance of the membrane matrix for organization of cellular membrane protons in the plasma membrane. The observation (Fig. 4) that the same surface

244

protein (slgM) can be tightly a ~ o ~ a ~ d with membrane m~fix ~ one plasma membrane k~cfion, but not ~ anoth~, suggests a mecha~sm for p r e ~ n t i ~ bcalization of surface protdns in ~ ~rent plasma membrane dom~ns. The nature and f u n ~ n cf such plasma membrane dom~ns a ~ not yet known and co~d be bet~r und~smod in future stud,s ev~uafing the co~dation b~ween surface protein reorganization and execution of effector functions by ~ m p h o c ~ .

G . ~ was supposed by a grant from the Deu~che F ~ h u n ~ g e m ~ n s c h a ~ (Tr 214/1).

Rde~n~s Apga~ J.R. and M.F. Mescher: Agofins: m~or ~ructurM protdns of the plasma membrane skd~on of P815 tumor cell~ L Cell Bid. 103, 351-360 (1986L Apgaq LR., S.H. Herrman, LM. Robinson and M.F. Mescher: Triton X-100 extraction of P815 tumor calls: e~dence for a p~sma membrane skd~on ~ru~urm J. Cell Biol. 100, 1369-1378 (1985L Bfik~a& J., F. Marke~ L. Cafl~o~ T. Person and U. Lindberg: Sdective assay of monomeric and filamentous acfin in cell e x t r a ~ ufing inhibition of deoxyfibonudease I. Cell 1L 935-943 (1978). Boyd, A. and J.W. Schrader: The regulation of growth and d ~ r e n t i a t i o n of a murine B call ~mphoma. J. Immund. 12& 2466-2469 (1981~ Braun, J.: Receptor-cytoskd~on interaction in the Dmphocyte. Immunol. Today 1, 21-25 (1983L Braum J., ES. Hochman and E.R. Unanue: Ligand-induced association of surface immuno~obufin with the detergentinsoluble c y t o s k d ~ matrix of the B ~mphocy~. J. Immunol. 12~ 1198-1204 0982). Browm S., W. Lefinson and J. Spudich: Cytoskdetal e~ments of chick embryo f i b r o b l a ~ revefled by detergent extraction. J. Supramol. Stru~. 5, 119-126 (1976). CoopeL T.G.: The Too~ of Biochemi~ry Oohn Wiley&Son~ Inc., New York) pp. 297-299 (1977L Dafidso~ W.F., T.N. Fredfickson, E.K. Rudikoff, R.L. Coffman, J.W. Hartley and H.C. Morse: A unique series of lymphomas r d ~ e d to the Ly-1 + hneage of B lymphocy~ differentiation. J. Immunol. 133, 744-753 (1984). Fox, LE.B., M.E. Docker and D.R. Phillips: An improved method for de~rmining the actin filament content of nonmusc~ cells by Ihe DNasel inhibition as~a~ An~. Biochem. 117, 170-173 (1981L G~ge~ B., D. Rosen and G. Berke: Spati~ rehtionships of microtubul~organi~ng center and the contact area of cyto-

m~c T ~mphoc~ and mrg~ cdl~ J. Call Bid. 95, 137-143 (198~. Gi~ert, M. and A. F d m n : The specifid~ and stabifi~ of ~ e Tri~mex~ac~d cymskeletal ~amewo~k of g~bil fibroma calls. J. Call Sd. 73, 335-345 (1985). Hamm~fin~ U., A.F. C ~ n and J. A b b o t : Onmgery of murine B ~mphoc~es: sequence of B-cell ~ff~entiation ~om surface-immunoglobulin-negative p~ecurso~ ~ ~asma cells. Proc. Nail. Acad. Sd. USA 73, 2008-2012 (197~. Kim, K.J., C. K a n d l o p o ~ o > L a n g e ~ R.M. Merwim D.H. Sachs and R. Aso~ky: Establishment and charac~rizafion of BALB/c ~mphoma fines with B cell properties. J. Immund. 122, 549-554 (197~. K u p p ~ A., S.L. Sw~m C.A. Janeway J~ and S.J. ~ n g ~ : The spedfic direct interaction of h e ~ T c d ~ and antigen-pr~ ~nting Bmells. Proc. Nail. Acad. Sd. USA 83, 6080-6083 (1986L Laemml~ U.K.: Cleavage of structural protdns during ~ e a s ~ m ~ y of the head of bacteriophage T& N~ure 227, 680-685 (197~. La~e~ L.L., N.L. Warne~ J.A. L e d b ~ r and L.A. H~zenberg: QuantitatNe immunofluo~scent an~yfis of sur~ce phenmyp~ of routine B cell ~mphomas and ~asmoc~om~ wi~ monoc~n~ ant~o~. J. Immund. 127, 1691-1697 0981L M ~ h e ~ M.F., Jd.U Jose and S.P. B~k: A ~ m o n t ~ n g matrix associated wilh the ~ m a membrane of murine tumor and ~ m p h d d calls. N ~ u r e 28~ 139-144 (1981L Mus~ns~, S.E, W.E Da~dson and H.C. Morse III: The activation of celluhr oncogenes m human and mouse ~ukemias-~mphomas. Spon~neous and ~duced oncogene expresfion in murine B-~mphoc~e neo~asms. Cancer Invest,, ~ press (1987~ P~gm Chd., EW. Kincade and ~ R~ph: Murine B call ~ukemia line wi~ i n d u d b ~ sur~ce i m m u n o ~ o b d ~ e ~ presfion. J. Immund. 121, 641-647 (197~. P~g< CkL, P.W. Kincade and P. R~ph: Independent contrd of immuno~obufin heavy and fight c h i n expresfion in a mufine pr~B-cell finm N~ure 29L 631-633 (1981L Pfive~ J., A.B. F d m n , S. Penmam M.E D a ~ d s and C.N. Christian: Interaction of ~ e c y ~ s k d ~ £ framework with ace~h~e receptor on the surface of embryo~c muscle cells ~ c ~ m r ~ J. Cell Bid. 9L 231-236 (1982L SchechteL A.L. and M.A. Bothwell: Nerve grow~ ~ctor recepto~ on PC12 cells: e~dence ~ r two receptors with differing c~oskd~M assoc~tion. Cell 2~ 867-874 (1981). Schliwm M.: The Cytoskeletom An ln~oduc~ry Su~ey (Springe~Vefla~ W~m New York) (1986L Tow~m H., Th. Staehelin and J. Gordon: E l e c ~ o p h o r ~ ~ a n ~ of proteins ~om pdyacrylamide gels ~ ~ o c e l l u lose sheets: procedure and some appfications. Pro~ Natl. Acad. Sd. USA 76, 4350-4354 0 9 7 ~ . Woda, B.A. and M.L. McFadden: Ligand-induced assod~mn of rat ~mphocym membrane protdns with the demrgenti n s d u ~ e ~mphocym c y m s k d ~ matrix. J. Immune. 131, 1917-1919 0983). Wdosewick, J. and K.R. Pormr: Smmo ~gh-vol~ge dec~on microscopy of whole cells of the human ~ d linm Wb38. Am. J. Anat. 147, 303-324 (1976~