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Seminars in Cell & Developmental Biology 18 (2007) 616–626

Review

Lipid rafts and B cell signaling

Neetu Gupta ∗, Anthony L. DeFranco ∗∗Department of Microbiology & Immunology, University of California,

San Francisco, San Francisco, CA 94143, United States

Available online 24 July 2007

bstract

B cells comprise an essential component of the humoral immune system. They are equipped with the unique ability to synthesize and secreteathogen-neutralizing antibodies, and share with professional antigen presenting cells the ability to internalize foreign antigens, and process themor presentation to helper T cells. Recent evidence indicates that specialized cholesterol- and glycosphingolipid-rich microdomains in the plasmaembrane commonly referred to as lipid rafts, serve to compartmentalize key signaling molecules during the different stages of B cell activation

ncluding B cell antigen receptor (BCR)-initiated signal transduction, endocytosis of BCR-antigen complexes, loading of antigenic peptides ontoHC class II molecules, MHC-II associated antigen presentation to helper T cells, and receipt of helper signals via the CD40 receptor. Here we

eview the recent literature arguing for a role of lipid rafts in the spatial organization of B cell function.2007 Elsevier Ltd. All rights reserved.

eywords: B cell; Membrane raft; Signaling; Tyrosine phosphorylation; Endocytosis; Actin cytoskeleton; Ezrin; Regulation

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6162. BCR signaling and membrane rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6173. Co-receptors for BCR signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6184. Developmental regulation of BCR signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6195. Membrane rafts in BCR-dependent antigen uptake and antigen presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6206. Cytoskeletal regulation of membrane raft dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6217. Membrane rafts and B cell-related disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6238. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624

. Introduction

Immunoglobulin (Ig) gene rearrangements in developing Bymphocytes generate a diverse repertoire of B cells, each with

B cell. The BCR serves key roles in directing the activation ofB cells that make antibodies reactive to infecting microbes andviruses, and also in tolerizing B cells capable of making anti-bodies reactive to self-components. Although BCR engagement

distinct specificity for antigen. This immunoglobulin is ini-ially expressed in a membrane form that serves as an antigeneceptor, the B cell antigen receptor (BCR), on the surface of the

∗ Corresponding author. Present address: Department of Immunology, Cleve-and Clinic, 9500 Euclid Ave., NE40, Cleveland, Ohio 44195, United States.el.: +1 216 444 7455; fax: +1 216 444 9329.

∗∗ Corresponding author. Tel.: +1 415 476 5488; fax: +1 415 502 8424.E-mail addresses: [email protected] (N. Gupta), [email protected]

A.L. DeFranco).

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084-9521/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2007.07.009

s typically required for a mature B cell to leave the quiescenttate and become activated to proliferate and differentiate inton antibody-secreting plasma cell, when the BCR is engagedn an immature B cell, the cell makes one or more responseshat either change the specificity of the cell (“receptor editing”),ead to its rapid death (“clonal deletion”) or reduce its respon-
iveness to the BCR (“clonal anergy”). These responses serveo prevent self-reactive B cells from secreting autoantibodies in
ost cases. B cell activation also requires a variety of other sig-als coming from cytokines and cell-bound stimulatory factors

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mailto:[email protected]

dx.doi.org/10.1016/j.semcdb.2007.07.009

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N. Gupta, A.L. DeFranco / Seminars in Ce

ade by helper T cells. In this review, we discuss the role of lipidafts in the activation of B cells. Our major focus is on the role ofipid rafts in the signaling by the BCR and how this is impactedy other cell surface receptors of B cells that either promote ornhibit B cell activation. In addition, we discuss the role of lipidafts in endocytosis of antigen via the BCR, a process that isssential for interactions with T cells that provide key helperignals for complete activation and differentiation of B cellsnto antibody-secreting plasma cells. Finally, we describe recentnsights into how the actin cytoskeleton might regulate lipid raftynamics, and discuss scenarios wherein the loss of certain com-onents of BCR signaling from lipid rafts might contribute tohe development and/or progression of human diseases.

. BCR signaling and membrane rafts

The BCR is a complex between a membrane-bound Ig (mIg)olecule and a disulfide-linked heterodimer of two polypeptides

esponsible for signaling and endocytosis, called Ig� and Ig�.embrane and secreted forms of Ig are generated by differential

plicing events that incorporate or leave out exons encoding aransmembrane domain and a very short cytoplasmic domain athe C-terminus of the Ig heavy chain. The resulting mIg mustssemble with an Ig�/Ig� heterodimer in order to leave the endo-lasmic reticulum and traffic to the cell surface. Ig� and Ig� eachontain an amino acid motif that is also found in other activatingeceptors of T cells, natural killer cells, and phagocytes, calledhe immunoreceptor tyrosine-based activation motif (ITAM), theonsensus sequence of which is D/ExxYxxL/Ix7YxxL/I. Theost widely accepted model of ITAM signaling holds that recep-

or engagement leads to (1) phosphorylation of both tyrosines ofhe ITAM by Src-family tyrosine kinases, (2) recruitment of theytosolic protein tyrosine kinases Syk or ZAP-70 to the phos-horylated ITAMs by binding of their tandem SH2 domains tohe two phosphoYxxL/I halves of the ITAM, (3) increases inctivity of Syk or ZAP-70, the latter expressed primarily in Tells and natural killer cells, resulting from ITAM binding andyrosine phosphorylation of these kinases, perhaps by Src-familyinases, and (4) phosphorylation of adaptor molecules and sig-aling enzymes by Syk or ZAP-70, and perhaps in some casesy Src-family kinases. The localization of Src-family kinasesn the membrane and their basal activity likely promotes BCRTAM and subsequent phosphorylations at a low level prior toigand binding to the BCR, a process referred to as tonic sig-aling [1]; these signaling reactions are countered by proteinyrosine phosphatases, perhaps including SHP-1 [2].

Ligand-induced clustering of the BCR greatly increasesTAM phosphorylation, Syk recruitment and downstream sig-aling, which represents the way in which the information aboutigand binding is transmitted to the inside of the cell. Exactly howCR binding to antigen increases the biochemical sequence ofvents outlined above is not fully understood, although the clus-ering of BCR ITAMs and their association with active Syk and
rc-family kinases, which also bind to phosphorylated ITAMsia their SH2 domains [3], can keep them in the phosphorylatedtate. This likely amplifies signaling of clustered BCRs beyondhe low level of tonic signaling.
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evelopmental Biology 18 (2007) 616–626 617

It has been hypothesized that lipid rafts, also called “mem-rane rafts”, facilitate amplification of BCR signaling afterigand binding. According to a recent definition, membraneafts are small (10–200 nm in diameter), heterogenous, highlyynamic, sterol- and sphingolipid-enriched domains that com-artmentalize cellular processes. Small rafts can sometimes betabilized to form larger platforms through protein–protein androtein–lipid interactions [4]. Prior to stimulation, the unboundCR is present in the non-membrane raft fraction of the plasmaembrane, whereas BCR clustering rapidly leads to BCR asso-

iation with the membrane raft fraction, as determined byiochemical isolation following cold non-ionic detergent extrac-ion of the cells and sucrose-density gradient fractionation [5].o-localization of clustered BCR molecules with membrane

afts has been directly visualized by fluorescence microscopyoth in cell lines and in primary B cells stimulated with anti-en [6]. In this study, membrane rafts were visualized either bytaining the ganglioside GM1 or by use of a fusion protein con-aining green fluorescent protein (GFP) fused to the N-terminal4 amino acids of Lyn, which contains the N-terminal myristoy-ation and palmitoylation sequences of this Src-family kinase butacks its protein–protein interaction and kinase domains and thusoes not form any known associations with other proteins. Thistudy also demonstrated that Syk was recruited to BCR clustersssociated with membrane rafts and that cellular protein tyro-ine phosphorylation was enhanced at regions of the membranenriched in membrane rafts. These results suggest that BCRignaling is enhanced upon its association with membrane rafts.

Recently these studies were extended by use of fluorescenceesonance energy transfer (FRET), which detects very closeolecular interactions in living cells. With this technique, it was

bserved that the BCR associates with membrane rafts withineconds of being clustered [7]. In these studies, the FRET donoras cyan fluorescent protein (CFP) linked to the C-terminusf Ig� and the FRET acceptor was yellow fluorescent proteinYFP) fused to the first 16 amino acids of Lyn, which is a reporteror the membrane rafts that contain Src-family kinases, similaro the Lyn-based reporter described above. Interestingly, FRETas not seen between Ig� and a membrane reporter contain-

ng the C-terminal isoprenylation motif of Rho, indicating thathe latter is not localized to the membrane rafts that associateith clustered BCRs. Similarly, a membrane reporter derived

rom the N-terminus of Src, which lacks the palmitoylation siteound in Lyn, also did not exhibit an increase in FRET upon BCRlustering. The FRET between the N-terminal Lyn-YFP reporternd the BCR appeared to precede the increase in intracellularalcium induced by BCR signaling, suggesting that it reflects aery early event in the BCR signaling cascade. Interestingly, thisRET was blocked by the Src-family tyrosine kinase inhibitorP2, indicating that phosphorylation of ITAMs by Src-family

yrosine kinases in some way induces association of the BCRith membrane rafts. The mechanism by which this occurs isot yet defined, but since it has been shown that Lyn, Fyn and
lk associate with phosphorylated BCR ITAMs [3], and theseinases are constitutively associated with membrane rafts, anttractive idea is that after these kinases phosphorylate BCRTAMs, they bind to the phosphorylated BCR, and induce its

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18 N. Gupta, A.L. DeFranco / Seminars in Ce

ssociation with membrane rafts. It should be noted that theranslocation of the BCR to membrane rafts, as assessed by bio-hemical isolation of detergent-resistant membrane rafts, wasot blocked by the same Src-family kinase inhibitor [8]. Thexplanation for this discrepancy between the requirement forrc-kinase activity for BCR association with membrane rafts asssessed by biochemical isolation versus FRET is not clear.

An interesting feature of the FRET observed between theCR and the membrane raft reporter is that it peaked after just0 s and decreased to an intermediate value soon thereafter. Thisehavior was not affected by addition of latrunculin A to blockctin polymerization. The decrease in FRET after its initial riseould reflect a change in the nature of the membrane environmentr alternatively could reflect a loss of FRET due to the build up ofultiprotein signaling complexes at the receptor and consequent

rowding out of the FRET reporter. Although FRET studies haveubstantial advantages in terms of avoiding the potential artifactsssociated with detergent extraction methods, and have excellentpatial and temporal resolution, clearly some of the phenomenaemonstrated by FRET need to be explained in terms of whatrotein–protein or protein–lipid associations are responsible forhich changes in FRET.Membrane rafts have been especially associated with activa-

ion of the transcription factor NF-�B by the BCR in B cells andy the TCR in T cells [9]. Activation of NF-�B in response toany receptors typically involves a common final step in which

he I-�B kinase (IKK) complex phosphorylates the inhibitoryubunit I-�B, inducing the latter’s ubiquitinylation and degra-ation and releasing NF-�B to translocate to the nucleus andctivate transcription. Upstream events differ between differ-nt classes of receptors, however, the BCR and TCR uniquelyignal to this pathway via the scaffold molecule caspase recruit-ent domain membrane-associated guanylate kinase protein 1

CARMA1). Signaling-induced conversion of CARMA1 fromn inactive to active conformation or oligomeric state nucleates aarge protein complex between CARMA1, Bcl-10 and MALT1,hich then recruits other components of the NF-�B pathway,

ncluding TRAF2 and TRAF6. BCR signaling induces assem-ly of this signaling complex via protein kinase C�-mediatedhosphorylation of CARMA1 [9]. This complex appears to formt the plasma membrane preferentially in regions of coalescedembrane rafts [10,11]. Extraction of cholesterol with methyl-cyclodextrin to disrupt membrane rafts, however, apparentlyoes not prevent NF-�B activation via CARMA1 [12], suggest-ng that this signaling event can occur outside membrane raftsith reasonable efficiency.

. Co-receptors for BCR signaling

An important feature of lymphocyte antigen receptor sig-aling is the participation of additional receptors on the cellurface that may also interact with the same ligand and con-ribute to lymphocyte activation. These receptors are referred to
s co-receptors since they cooperate with the antigen receptorso promote biological responses. For example, T cells expressither of two co-receptors, CD4 or CD8, which bind to conservedegions of class II or class I MHC molecules, respectively. When
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evelopmental Biology 18 (2007) 616–626

he TCR contacts an MHC-peptide complex that matches thepecificity of the co-receptor on that type of T cell, a trimolecu-ar complex is formed between the MHC-peptide complex, theCR and the CD4 or CD8 co-receptor. The co-receptor helps totabilize the binding of the TCR to its ligand, and in addition itrovides a complementary signaling function. CD4 and CD8 areound to the Src-family kinase Lck, and therefore co-receptorngagement with the MHC brings Lck next to the cytoplasmicTAMs of that TCR, where it can efficiently phosphorylate them.

cells have two types of co-receptors, those that promote BCRignaling analogously to CD4 and CD8 in T cells, and thosehat decrease BCR signaling and for that reason are often callednhibitory co-receptors.

An important positive-acting co-receptor for B cells is com-lement receptor 2 (CR2, also called CD21), which is present onhe cell surface in a complex with two other transmembrane pro-eins CD19 and CD81. When a protein antigen has complementragments derived from C3b attached to it, indicating recogni-ion of its status as foreign by elements of the immune system, Bell activation is enhanced by up to 10,000-fold [13]. In this cir-umstance, the CR2 subunit binds to the complement fragmentttached to the antigen and co-clusters the CR2/CD19/CD81omplex adjacent to the BCR (Fig. 1a). The CD19 subunit playscritical role in promoting BCR signaling by virtue of its adap-

or molecule function. Multiple tyrosines within the cytoplasmicail of CD19 become phosphorylated when the CR2 complex islustered with the BCR and these tyrosines serve as binding sitesor phosphatidylinositol 3′-kinase (PI 3′-kinase), for Lyn, andor Vav, which is a guanine nucleotide exchange factor for Rac.f these, the two binding sites for PI 3′-kinase are especially

mportant for promoting B cell activation [14,15]. Production ofhosphatidylinositol 3,4,5-trisphosphate (PIP3) by PI 3′-kinases essential to many of the BCR signaling reactions. In addition toctivating Akt, PIP3 recruits the PH domain-containing proteinstk and phospholipase C�2 (PLC�2), leading to phosphatidyli-ositol 4,5-bisphosphate (PIP2) hydrolysis. PIP2 hydrolysisy PLC�2 generates the second messengers, inositol 1,4,5-risphosphate (IP3) and diacylglycerol. As in other cell types,P3 induces calcium elevation by acting on IP3-receptors in thendoplasmic reticulum and inducing release of calcium. Oncehe ER has fully released its calcium, it sends an unidentifiedignal to the plasma membrane that opens the store-operatedalcium channels, which in B cells are in some way regulated byhe B cell-specific tetraspanin protein CD20 that also associatesith membrane rafts [16].The other second messenger resulting from PIP2 hydroly-

is is diacylglycerol, a membrane-bound second messenger thatctivates various protein kinase C isoforms and also RasGRP,guanine nucleotide exchange factor for Ras [17,18]. Protein

inase C isoforms are responsible for activation of the tran-cription factor NF-�B, as described above, among other events9].

The CD81 subunit of the CR2 complex is also important
or BCR signaling. CD81 is a tetraspan protein and its func-ion in the CR2 co-receptor complex appears to be to stabilizessociation of the BCR with membrane rafts and in that wayromote prolonged signaling [19]. Interestingly, the CR2 com-

N. Gupta, A.L. DeFranco / Seminars in Cell & Developmental Biology 18 (2007) 616–626 619

Fig. 1. BCR activation and feedback inhibition occur in membrane rafts. (a) Upon binding to complement-coated antigen, the BCR associates with membrane rafts,and comes in proximity to Src-family kinases (SFKs) that phosphorylate ITAM tyrosine residues (red filled circles) in the Ig�/Ig� chains. Phosphorylated ITAMsrecruit Syk which phosphorylates many signaling substrates resulting in B cell activation. The co-receptor CR2/CD21 recognizes the complement component of theantigen and associates with CD19 and CD81, which provide an adaptor function and promote membrane raft association, respectively. (b) Feedback inhibition of Bcell responses occurs upon immune complex-mediated co-ligation of the BCR with the inhibitory Fc�RIIB receptor. In this circumstance, Fc�RIIB is phosphorylatedo ol phoa

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lex does not appear to associate with membrane rafts prior tongagement, as judged by the detergent solubilization approach.

hen the BCR and the CR2 complex are co-clustered, BCR par-itioning into the detergent-insoluble membrane raft fraction isrolonged. The ability of the CR2 complex to promote prolongedssociation of the BCR with membrane rafts has been confirmedy FRET measurements using the N-terminal Lyn-YFP FRETcceptor [7]. This property of the CR2 complex is dependent onhe inclusion of CD81 in the complex, since CD81-deficient Bells fail to promote BCR association with membrane rafts andchimeric form of CD19 that does not associate with CD81 is

imilarly ineffective [20]. Interestingly, co-ligation of the BCRnd the CR2 complex also causes a rapidly inducible palmitoy-ation of CD81. The inducibly palmitoylated CD81 moleculesre strongly enriched in the detergent-insoluble membrane raftraction of cells, suggesting that the inducible palmitoylation ofD81 is important for maintaining co-ligated BCR and CR2 inembrane rafts.The best understood negative co-receptor of B cells is the

nhibitory Fc receptor Fc�RIIB [21]. When antigen is present incomplex with antigen-specific IgG molecules, it can bind both

he BCR and Fc�RIIB and co-cluster them (Fig. 1b). This resultsn phosphorylation of a single tyrosine in the cytoplasmic tailf Fc�RIIB in a sequence also seen in other immune inhibitoryeceptors and referred to as the immunoreceptor tyrosine-basednhibitory motif (ITIM). This phosphorylated ITIM then binds tohe SH2 domain-containing inositol phosphatase (SHIP), which
onverts PIP3 to phosphatidylinositol 3,4-bisphosphate, therebyttenuating most features of PI 3′-kinase signaling. Fc�RIIBppears to localize to membrane rafts upon co-ligation with theCR [22], although how this effect is mediated is not known.
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sphatase that abrogates PI 3′-kinase and Ras signaling, thus dampening B cell

he significance of this association will be discussed in a laterection.

. Developmental regulation of BCR signaling

The participation of membrane rafts in BCR signaling isevelopmentally regulated. During early development of B cellsn the bone marrow, ITAM signaling is required for the transi-ion between pro-B cells, which are rearranging the Ig heavyhain locus to create a functional heavy chain, and pre-B cells,hich express a heavy chain and are rearranging the Ig light

hain loci. Once an Ig heavy chain protein is produced as aonsequence of in-frame VDJ recombination, it pairs with tworoteins expressed only in developing B cell precursors, col-ectively called the surrogate light chain, and with the Ig�/Ig�ignaling heterodimer to form a membrane-bound complexeferred to as the pre-BCR. The pre-BCR is thought to signalonstitutively [1], but there is some evidence for a pre-BCRigand on bone marrow stromal cells that may further enhanceignaling. In any case, it has been shown that the pre-BCR asso-iates with membrane rafts in B cell precursors as it induces tonicignaling [23]. The pro-B cell to pre-B cell transition, which isharacterized by changes in expression of cell surface markers,y ability to proliferate in response to the cytokine interleukin 7,nd by a change in the targeting of the V(D)J recombinase awayrom the Ig heavy chain locus and to the Ig light chain loci, cane promoted artificially by membrane-targeted fusion proteins
ontaining ITAM sequences, which are presumed to generatelow level of tonic signaling [1]. Interestingly, these ITAM-
ontaining fusion proteins are effective whether they are targetedo the liquid-disordered or to the liquid-ordered membrane raft

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raction of the plasma membrane, suggesting that ITAM signal-ng in B cell precursors can occur outside of membrane rafts asell as inside them.Once successful rearrangement has occurred at one of the Ig

ight chain loci, leading to expression of the BCR, the cell iseferred to as an immature B cell if it is in the bone marrownd as a transitional B cell after it has left the bone marrow forhe spleen and before it has matured to a long-lived mature Bell. Studies with B lymphoma cell lines of the immature orransitional phenotype and with primary immature B cells havebserved that BCR clustering in these cells does not result ints co-localization with membrane rafts [24,25], although sub-tantial BCR signaling can be generated in these cells. Thus,t appears that significant BCR signaling can occur outside of

embrane rafts, although in mature B cells there is a correla-ion between strength of signaling and localization in membraneafts, as described above. Recently, it has been found that imma-ure B cells have a lower content of cholesterol in their plasma

embrane than do mature B cells [26]. This is a fascinatingbservation and it suggests that cholesterol content of B cellss regulated during maturation in the spleen, perhaps to alterither the magnitude of BCR signaling or the relative amountsf different signaling reactions. Further studies will be requiredo address this issue, but it was shown that supplementation of

embrane cholesterol in immature B cells up to the level presentn mature B cells, using methyl �-cyclodextrin-cholesterol com-lexes, was sufficient to cause clustered BCRs to localize withembrane rafts in these immature B cells, demonstrating that

he failure of the BCR to associate with membrane rafts wasue to the lower cholesterol content of the plasma membrane inmmature B cells. Whether an increase in the membrane choles-erol content of immature B cells in vivo would alter the outcomef signaling and the fate of these cells remains to be seen.

. Membrane rafts in BCR-dependent antigen uptakend antigen presentation

While the BCR serves to recognize antigen and transducesn activation signal for B cell expansion, it is also responsi-le for delivering the bound antigen to specific compartmentsithin the cell where the antigen is processed into peptides that

re loaded onto MHC class II (MHC-II) molecules, which arehen trafficked to the cell surface for presentation to antigen-pecific helper T cells. Accumulating evidence suggests that thenitial endocytosis of the antigen and MHC-II-associated pre-entation of antigenic peptides to T cells are both coordinatedithin membrane raft domains.Following internalization, the antigen-BCR complexes are

elivered to early endosomes from where they move to late endo-omes. The Ig�/Ig� components of the BCR were shown to beecessary and sufficient for the initial internalization as wells for sorting to the late endosomes [27]. Ig�-mutant receptorsre retained in early endosomes, whereas those containing only
g� were shown to go straight from early endosomes to ter-inal lysosomes and undergo degradation without productive
oading of peptides onto MHC-II [27,28]. The contribution ofg� ITAM residues was further examined more recently in vivo

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y exchanging the ITAM tyrosines for alanines by gene target-ng. The resulting mice (Ig�AA) showed a normal developmentor all B cell subtypes, except for B1 cells which were signif-cantly reduced. Purified B cells from the Ig�AA mice showedighly decreased steady state and ligand-mediated BCR inter-alization. BCR cross-linking resulted in diminished Src-familynd Syk activation, but elevated and prolonged signaling withespect to Ca2+ flux, total tyrosine phosphorylation, and Akt andrk activation. This study concluded that the Ig� component of

he BCR is responsible for setting a threshold for signaling byegulating receptor internalization, which terminates signaling29].

The Ig� chain of the BCR complex also participates in itsnternalization. Proper sorting of the BCR-antigen complexesequires the recruitment of the tyrosine kinase Syk to phospho-ylated ITAMs on Ig� [30,31]. Interestingly, both the ITAMyrosines [29] and the non-ITAM tyrosines Y176 and Y204f Ig� participate in coordinating the internalization signals.eceptors bearing tyrosine to phenylalanine mutations in the

g� chain are still internalized but do not co-localize with MHC-I-rich internal compartments and could not facilitate antigenresentation to T cells [32]. The requirement for both ITAM andon-ITAM tyrosine residues on Ig� for efficient and productiventernalization of the BCR complexes is further supported byxtensive mutational analysis reported in a recent study [33].

While the BCR complex contains sufficient signals for its ownnternalization and sorting, recent reports suggest that B cells canndocytose via two pathways, internalization via clathrin-coatedits and via a clathrin-independent pathway involving mem-rane rafts. A significant amount of clathrin heavy chain wasound to be constitutively associated with membrane rafts in Bells [34,35], and it becomes tyrosine phosphorylated upon BCRngagement in a Src-family kinase-dependent manner [34]. Fur-hermore, antigen uptake is largely dependent on the associationf clathrin with membrane rafts and its phosphorylation at theseites. Chicken DT40 B cells conditionally deficient in clathrineavy chain showed a marked reduction in their BCR-mediatedptake of antigen. Disruption of membrane rafts by treatmentith nystatin caused a reduction similar to that seen with lossf clathrin, whereas treatment with latrunculin A to disrupt thectin cytoskeleton led to a 50% loss in antigen internalization.hese data suggest that clathrin, actin and membrane rafts areeeded for the most complete endocytosis of BCR-bound anti-en, but argue that internalization can still proceed with at leastwo of these three components intact. However, membrane raftsnd the actin cytoskeleton cannot support internalization inde-endently. A complete block in endocytosis of the BCR resultedn increased BCR signaling as assessed by sustained tyrosinehosphorylation and Erk phosphorylation, further supportinghe idea that internalization is a means of signal attenuation asell as a means to promote antigen presentation to helper T cells

36]. Therefore, the view that has emerged from these studiess that BCR ligation drives its association with membrane rafts
lready bearing the clathrin heavy chain and enriched in Src-amily kinases. Both the BCR and clathrin get phosphorylatedn this confined space, the BCR localizes to clathrin-coated pitsnd internalizes along with membrane rafts [34,37].

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In another study, anergic B cells (which are continually bind-ng their antigen) were reported to have an enhanced rate ofCR endocytosis, which was blocked by depleting membraneholesterol with methyl �-cyclodextrin, suggesting that mem-rane rafts play a critical role in BCR internalization in these Bells [12]. Further evidence for at least two pathways of BCRnternalization has recently been presented. In this study, theathway of BCR endocytosis, as indicated by its sensitivity toembrane raft- and actin-disrupting agents and its dependence

n Src and Syk family kinase signaling, was shown to be gov-rned by the nature of the ligand. The internalization of anti-Igntibody/BCR complexes was dependent on Src and Syk fam-ly kinase signaling, the integrity of the actin cytoskeleton and

embrane rafts, and these complexes were delivered to early andecycling endosomes. In contrast, the internalization of a modelntigen/BCR complex was independent of signaling, membraneafts and actin and these complexes trafficked to late endosomes,nd were targeted for proteolytic processing [38,39].

A membrane raft-localized transmembrane protein, theinker for activation of B cells/non-T cell activation linkerLAB/NTAL) [40,41] also appears to play an important role inCR internalization. BCR cross-linking led to co-internalizationf LAB/NTAL along with the receptor and it is the C-terminalail of LAB/NTAL that is responsible for this effect. Mouse Bells deficient in LAB/NTAL showed a reduction in the ligation-ependent uptake of the BCR [42]. To the extent that LAB/NTALs important for BCR internalization, its localization to mem-rane rafts may contribute to the importance of these domainsor BCR-mediated antigen uptake.

Taken together, the available data suggest that BCR endocy-osis is a dynamic and complex process comprised of at leastwo mechanistically distinct pathways for internalization, andncludes additionally regulated steps that control delivery to latendosomes where antigen can be processed and loaded ontoHC-II molecules. Resolution of molecular features of this pro-

ess will depend on getting a closer look at early events thatovern the intracellular fate of the BCR and its association withomponents of membrane rafts.

The endocytosed BCR is delivered to intracellular compart-ents where its bound antigen is proteolyzed into antigenic

eptides. The peptides are then loaded onto MHC-II molecules,nd traffic out to the surface of the B cell from where theyan be surveyed by CD4+ T cells bearing the cognate T celleceptors (TCRs). Biochemical fractionation and fluorescenceicroscopy experiments indicate that not only are the MHC-IIolecules constitutively associated with membrane rafts, they

re also loaded with antigenic peptides in a concentrated mem-rane raft environment [43]. Indeed, our proteomic analysis ofurified membrane rafts from the human B cell line Ramos con-rmed that MHC-II molecules associate with membrane raftsnd showed that this association is unaffected by ligation of theCR [35]. Constitutive association of MHC-II with membrane

afts has also been observed in human tonsil B cells, in trans-
ormed B cell lines, and in human monocytes [44]. While it isnclear where in the biosynthetic pathway MHC-II moleculeset associated with membrane rafts, both microscopic and bio-hemical determinations suggest that half of the cell surface
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HC-II is associated with membrane rafts. The functional rele-ance of MHC-II association with membrane rafts is suggestedy the fact that raft-associated MHC-II/peptide complexes con-entrate in the immunological synapse (IS) and facilitate antigenresentation to T cells. Pharmacological disruption of membraneafts on the antigen presenting B cells abrogated their recruit-ent to the IS and T cell activation. However, this was only true

nder conditions when the numbers of MHC-II/peptide com-lexes were limiting, indicating that the role of membrane rafts iso concentrate rare MHC-peptide complexes at the IS for recog-ition by T cells [45]. Consistent with this hypothesis, confocalmaging of the B cell side of the IS has revealed that mem-rane rafts are rapidly enriched in the IS upon B: T conjugation,nd that this is an actin-dependent phenomenon. In addition,uorescence recovery after photobleaching (FRAP) and fluores-ence loss in photobleaching (FLIP) experiments showed thathe membrane raft proteins in the IS are highly dynamic andapidly exchange with other membrane compartments of the Bell [46].

Thus, it appears that lipid rafts play a significant role inhe entire journey of an antigen, starting with its initial uptakehrough the BCR, trafficking through intracellular MHC-II-oading compartments, and finally its presentation to T cellsn the immunological synapse (Fig. 2).

. Cytoskeletal regulation of membrane raft dynamics

As mentioned above, BCR cross-linking induces fusion ofmaller membrane raft units to generate membrane raft patcheshat ultimately coalesce into stable, micron-sized rafts [6]. Sim-lar membrane raft coalescence also occurs in other cell typespon interaction of a number of cell surface receptors with theirigands. While the exact mechanism of this large-scale coa-escence is unclear, it has been suggested that the actin-basedytoskeleton plays a role in the regulation of membrane raftynamics [47,48].

Use of higher resolution imaging tools such as single flu-rescent molecule video imaging (SFVI), high-speed singlearticle tracking (SPT) using colloidal gold probes, and opti-al trapping, which allows one to move the gold particle-taggedolecules in the living cell membrane, have led to the sugges-

ion that the plasma membrane is divided into submicron-sizedompartments, and that the diameter of these compartmentsaries from 30 to 230 nm. Phospholipid molecules can moveo adjacent compartments by undergoing “hop diffusion”, andhe average residency time for single molecules within theseompartments varies by cell type falling within a broad rangef 1–17 ms. Data from optical trapping and FRAP experimentsnitially led to the membrane skeleton “fence” model that pro-osed that the actin cytoskeleton forms a meshwork underneathhe plasma membrane, and that transmembrane proteins pro-ruding into the cytoplasm collide with the cytoskeleton causinghem to be temporarily confined within the cytoskeletal mesh.
hese interactions create confinement zones. An examinationf the effect of membrane skeleton, extracellular matrix, extra-ellular domains of membrane proteins, and membrane raftomains on hop diffusion subsequently led to the “anchored-

622 N. Gupta, A.L. DeFranco / Seminars in Cell & Developmental Biology 18 (2007) 616–626

Fig. 2. Membrane rafts in BCR endocytosis and antigen presentation. (a) The cross-linked BCR is endocytosed along with bound antigen and membrane rafts. (b)E of anP ciatiom s. Mea

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arly endosomes fuse with late endosomes for antigen processing and loadingeptide-MHC (pMHC) complexes traffic to the surface and are displayed in assoembrane rafts to cognate TCRs in an immunological synapse (IS) with T cell

nd TCR is indicated by lightning signs.

rotein picket model” whereby various transmembrane proteinsnchored directly or indirectly to the membrane skeleton (fence)ct as rows of pickets (the transmembrane proteins serve asosts for the fence, hence termed pickets) that serve as diffu-ion barriers and effectively block free diffusion of phospholipidolecules and non-anchored proteins due to steric hindrance and

ydrodynamic friction [49,50]. Thermal fluctuations causingissociation of actin filaments from membrane attachments areostulated to result in temporary breaks in the barriers, and allowop diffusion of molecules between adjacent compartments.

The use of biophysical methods to observe diffusion ofeceptors in the absence or presence of ligands revealed thatigand-induced receptor oligomers exhibit considerably sloweriffusion than do the monomers, and that their confinementime within a compartment is significantly increased [51]. Basedn these findings it was expected that cell stimulation-inducedembrane raft coalescence would proceed with smaller mem-

rane rafts fusing to form bigger entities, which would thenecome trapped within compartments and not continue to con-olidate. Furthermore, the hydrodynamic friction is expected toe higher in the membrane raft regions due to their orderedature, further limiting their movement. In B cells, however,inding of antigen to the BCR induces coalescence of mem-rane rafts within minutes until most or all of them aggregate atne pole of the cell along with the BCR cap [6].

Interestingly, BCR stimulation leads to a rapid global depoly-erization of actin followed by new actin polymerization. Itas postulated that actin depolymerization and/or remodeling

ould promote an increase in BCR signaling. Indeed, actin
epolymerization was shown to enhance BCR signaling, withncrease in sustained Ca2+ elevation, phosphorylation of Erknd activation of transcription factors including NFAT andF-�B as well as increased BCR and membrane raft clustering.
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tigenic peptides onto MHC-II molecules within the membrane raft milieu. (c)n with membrane rafts. Specific peptide-MHC complexes are presented withinmbrane rafts are identified with grey arrows, and signal transduction via BCR

lockade of actin depolymerization had the opposite effect52]. Apparently, the actin depolymerization breaks downhe diffusion barriers and allows both ligand-clustered BCRomplexes and membrane rafts to coalesce and be mobilizedo one pole of the cell. These data suggest that the actinytoskeleton actively keeps the membrane rafts in a dispersedtate in the absence of BCR stimulation to limit tonic signalingo a low level. Thus, BCR-induced remodeling of the actinytoskeleton may serve to increase the strength and duration ofCR signals facilitating efficient B cell activation.

Recent studies have provided insight into the molecularechanism of coupling plasma membrane rafts with the actin

ytoskeleton. We employed a tandem mass spectrometry andsotope-coded affinity tag (ICAT)-based proteomics approach toxamine changes in the protein composition of membrane raftsuring B cell activation as a means to identify positive and nega-ive regulators of membrane raft coalescence [35]. This approached to the identification of several proteins that change their rel-tive abundance in purified membrane raft fractions in a BCRtimulation-dependent manner. A majority of these proteins arenown to act as modifiers of the actin cytoskeleton, including theRM family member ezrin, the non-muscle myosin Myh9, and

he myosin regulatory light chain. While Myh9 and myosin reg-latory light chain inducibly associated with membrane rafts,zrin was found to dissociate from these domains. Consistentith the latter observation, BCR stimulation resulted in a tran-

ient dephosphorylation of a conserved threonine residue in the-terminal actin-binding domain of ezrin. The phosphorylationf threonine at this site acts as a switch that controls the open and
losed conformation of ezrin and other ERM family members,nd their ability to cross-link the membrane to actin filaments53]. Indeed, BCR ligation causes ezrin to dissociate from theembrane rafts and from actin filaments. The association of

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zrin with membrane rafts in unstimulated B cells is mediatedt least in part by binding to the raft-resident transmembranerotein PAG and this association is decreased following BCRigation. These observations suggest that, in its open confor-

ation, ezrin, together with PAG, constitutes either a diffusionarrier or a tether that extends from the membrane to the actinytoskeleton and limits the mobility of membrane componentsver large distances, and that BCR stimulation induces dephos-horylation of ezrin, its release from PAG and the membrane,nd a loss of the tethers or barriers that keep membrane rafts indispersed state [35].

To test this hypothesis, mutants of ezrin with constitutivectin binding ability were fused to one of two transmembraneomains that would target the chimeric protein to membraneafts or to the non-raft region of the plasma membrane. Thesezrin chimeric proteins were expressed in a B cell line, andheir effect on BCR-induced membrane raft coalescence wasxamined. Both mutant ezrin chimeric proteins were shown tolock large-scale coalescence of membrane rafts as well as BCR
apping [35]. The ezrin chimeras may prevent large-scale mem-rane raft coalescence either by tethering membrane rafts tohe cytoskeleton irreversibly (raft-targeted ezrin) or by creating
ore stable diffusion barriers and trapping them irreversibly

ig. 3. Ezrin and the actin cytoskeleton regulate B cell membrane raft dynam-cs. (a) Membrane rafts are small, dynamic structures dispersed randomly onhe cell surface, and tethered to the cortical actin cytoskeleton by protein pick-ts composed of the raft-associated protein PAG and the linker protein ezrin.n the absence of BCR stimulation, ezrin is phosphorylated (red filled circles)n T567 and exists in its open conformation. The ezrin-based pickets may alsoreate membrane compartments (black rectangle) in which protein and lipidomponents of membrane rafts are trapped with limited diffusion between adja-ent compartments. The Src-family kinases (SFKs) are pre-associated with theembrane rafts, while the BCR is excluded from these domains in the absence

f antigen resulting in low tonic signaling. (b) Oligomerization of the BCR byultivalent antigen results in its association with membrane rafts, increased

roximity to SFKs and greatly amplified signal transduction. Among the effectsf BCR signaling is dephosphorylation of ezrin on T567 with concomitant disso-iation from PAG and actin, resulting in a break in the diffusion barriers, allowingoalescence of individual membrane rafts into bigger entities. At later timesnot shown), ezrin becomes rephosphorylated and again binds to membraneafts. These reconnections may allow for active actin-myosin-based movementf large rafts to one pole of the cell.

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ithin compartments (raft-targeted and/or raft-excluded ezrin)Fig. 3).

Further support for the idea that ezrin-based tethers and/orraps may keep protein components of the plasma membraneelatively immobile over large distances comes from a recenteport that used single particle tracking to demonstrate that theystic fibrosis transmembrane conductance regulator (CFTR) ismmobilized in the plasma membrane via its interactions withhe EBP50-ezrin linker that couples it to the actin cytoskeleton.

utations in CFTR, EBP50 or ezrin that resulted in uncouplingf CFTR from the actin skeleton relieved this immobility [54].herefore, the ability of ezrin and related proteins to tether theembrane to the cortical actin meshwork may be a general-

zed mechanism by which cells regulate the dynamics of theirembrane components including membrane rafts.It is important to note that a number of studies addressing

he significance of membrane rafts in cellular activation haveesorted to the use of pharmacological inhibitors such as methyl-cyclodextrin, which disrupts these domains by depleting themf cholesterol which is an essential component of membraneafts. Methyl �-cyclodextrin can have a generalized disruptiveffect on both the plasma membrane and actin cytoskeleton thats not restricted to membrane rafts, so it is increasingly consid-red a less than ideal tool [55,56]. Thus, the membrane-targetedzrin mutants that we reported may provide a useful new geneticool to allow manipulation of membrane rafts to examine theiroles in B cell activation as well as in other cellular processes.

. Membrane rafts and B cell-related disease

The importance of membrane rafts in B cell activation isurther emphasized by recent reports that membrane rafts areo-opted by gene products of certain pathogens and that inertain disease conditions, critical regulators of BCR signalingose their association with membrane rafts. The LMP2A generoduct of the Epstein Barr virus (EBV) is expressed on the sur-ace of resting B cells during latent infection by this virus. Thentracellular amino terminal region of LMP2A contains eightyrosine residues, two of which (Y74 and Y85) are configurednto an ITAM sequence. Upon phosphorylation of these residues,MP2A binds Lyn and Syk tyrosine kinases and in this way is

hought to deviate these proteins away from BCR signal trans-uction, preventing phosphorylation of key signaling moleculess well as Ca2+ flux [57]. LMP2A constitutively resides inembrane rafts of EBV-transformed human B cell lines, and

nterestingly, blocks the entry of ligand-clustered BCRs intohese domains. The mechanism of this effect is not known.MP2A also inhibits downstream signaling events and inter-alization of the BCR. The molecular mechanisms for blockadef signaling and endocytosis appear to be different as a Y112utant of LMP2A that cannot associate with Lyn, still blocksCR translocation to membrane rafts and associated signalingut does not affect cross-linking-induced BCR endocytosis [58].
his observation suggests that BCR signaling and internaliza-
ion are differentially regulated, and also that EBV has evolvedeparate mechanisms to block these important functions of theCR.

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Another EBV protein that is responsible for the mainte-ance and proliferation of latently infected B cells is the LMP-1rotein. LMP-1 is capable of activating signaling pathwaysesembling those of the TNF receptor family member CD40,ncluding binding to TRAFs, TRADD and JAK3, and acti-ating NF-�B, AP-1 and STAT-mediated transcription. Likeigand-stimulated CD40, LMP-1 localizes to membrane raftsnd recruits TRAF3 into these domains [59]. Targeting the-terminal cytoplasmic domain of LMP-1 to membrane rafts

esults in constitutive signaling. Since the C-terminal domain ofMP-1 recruits TRAF3, this observation suggests that targetingRAF3 to membrane rafts promotes its signaling [60].

Not only does membrane raft localization of viral proteinsontribute to disease, alterations in the membrane raft localiza-ion of certain signaling molecules may also contribute to thenitiation or severity of certain B cell-dependent autoimmuneiseases. A study comparing a group of British patients withhe autoimmune disease systemic lupus erythematosus (SLE) toormal individuals found that a majority of the SLE patientsxhibited lower expression levels of Lyn as well as reducedssociation of Lyn with membrane rafts. These patients alsoad an increased translocation of c-Cbl into membrane rafts.bl contains an E3 ubiquitin ligase activity and is an inhibitorf receptor-mediated tyrosine kinase signaling pathways [61].n B cells, it may induce ubiquitinylation and degradation ofembrane raft-associated Lyn [62]. As Lyn is important for the

unction of inhibitory receptors which decrease BCR signal-ng, as described above, it was suggested that reduced negativeignaling in the membrane raft environment facilitates hyperac-ivation of B cells resulting in uncontrolled antibody responseso autoantigens.

Two independent studies that compared signaling mecha-isms in a Japanese cohort of SLE patients with normal controlslso provide evidence for the hypothesis that alterations inhe sub-cellular localization of BCR signaling pathways mayontribute to development of SLE. Both found that a singleolymorphism in the Fc�RIIB gene, a substitution of threo-ine for isoleucine at amino acid position 232, which lies in theransmembrane region of the protein, is preferentially associatedith the SLE patients and that this mutation causes Fc�RIIB toe excluded from membrane rafts in B cells. The functionalmpact of this change in membrane distribution was diminishednhibition by Fc�RIIB of BCR-derived signals including PIP3eneration, Akt and PLC�2 activation, and Ca2+ mobilization.he T232 form of Fc�RIIB also had less phosphorylation on its

TIM tyrosine residues and recruited lower levels of the inositolhosphatase SHIP [63,64]. Together, the findings concerning theltered distribution of Lyn and Fc�RIIB in membrane rafts indi-ate that, like positive signaling by the BCR in mature B cells,ts feedback inhibition also occurs in a concentrated membraneaft environment.

. Concluding remarks

Membrane rafts participate in many of the cell surface eventsnvolved in B cell activation, including signaling by the BCR,

odulation of that signaling by co-receptors, signaling by CD40,

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ndocytosis of antigen bound to the BCR and its routing toate endosomes to facilitate loading of antigen-derived peptidesnto class II MHC molecules, routing of those peptide/MHC-IIomplexes to the cell surface, and their participation in antigenresentation to T cells. Moreover, in some cases, the involve-ent of membrane rafts in B cell activation is developmentally

ontrolled. Thus, it is likely that membrane rafts play importantoles in B cell activation at multiple stages by controlling theocal concentrations of components that must act together and/oromponents that inhibit the process in question. Understandinghe way in which membrane rafts affect particular events is,owever, hampered by the limited numbers of ways that mem-rane rafts can be manipulated within viable B cells. Mutationalpproaches to target proteins of interest into or away from mem-rane rafts represent an attractive alternative to the cholesterolepletion approach and are beginning to provide some insightnto these questions. Clearly many questions remain regardinghe ways in which these microdomains modulate signaling andrafficking events within B cells and studies in the near futurehould help clarify these issues.

cknowledgements

The authors would like to thank their colleagues for discus-ions. N.G. is the recipient of a K01 mentored research careerevelopment award (DK068292) from NIDDK. Research in theeFranco Lab is funded by the National Institutes of Health

esearch grant from National Institute of Allergy and Infectiousiseases (R01 AI20038).

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Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-

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Lipid rafts and B cell signaling