Studies on the Molecular Basis of

Crosslinked mlgM Interactions with the Cytoskeletal Matrix

Jun Young Park

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Immunology

University of Toronto

@Copyright by Jun Youn; Park (1097)

Acquisitions and Acquisitions et Bibliographic Services services bibliographiques

395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K I A ON4 Canada Canada

Yoiir lile Votre rwference

Our file Notre réfdrencs

The author has granted a non- L'auteur a accordé une licence non exclusive licence aüowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de rnicrofiche/film, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

Abstract

Studies on the molecular basis of

crosslinked mIgM interactions with the cytoskeletal matrix

Master of Science 1997

Jun Young Park Department of ImmunoIogy

University of Toronto

Numerous studies have shown that crosslinked B ce11 antigen receptor (BCR) interacts

with the cytoskeletal matrix. Previous studies have pointed out that the BCR-cytoskeletal

interactions are positively correlated with B cell proliferation and capping, suggesting that the

interactions play a role in B cell activation. To elucidate the function of the BCR-

cytoskeleton interactions, it is necessary to delineate the molecular basis of these interactions.

The experiments reported here addressed the following questions: 1) Which component of the

BCR is required for the interactions of mIgM (surface form of IgM) with the cytoskeleton?

2) What site on the mIgM molecule mediates the interactions with the cytoskeleton upon

crosslinking? By investigating the mIgM-cytoskeleton interactions in the human cervical

carcinoma cell line HeLa S3, 1 report here that neither Ig-cd0 nor any other B cell specific

protein(s), are important in medislting the interactions. Furthermore, by taking advantage of a

IgM molecule in which the cytoplasmic tail of IgM, KVK, is replaced by that of Ig-cl, 1 also

report here that the KVK sequence of mIgM is important for the interactions between mIgM

and the cytoskeleton. The absence of KVK does not, however, completely abrogate the

mIgM-cytoskeletal interactions, suggesting that KVK has a substantial, yet not essential role

for these interactions.

Acknowledgments

1 would like to thank my supervisor Dr. Jenny Jongstra-Bilen, the members of my

cornmittee, Dr. Marc Shulman, Dr. Joan Wither, and Dr. Jan Jongstra, my family, and my

feilow lab members. They have been a constant source of encouragement and support in my

research and writing of this thesis. This work would not have been possibIe without their

help and patience.

Abbreviations

Abs, antibodies RT, room temperature

BAPs, BCR associated proteins SA domains, src homology domains

BCR, B ceIl antigen receptor Syk, splenocyte tyrosine kinase

EGF, epidermal growth factor TM, transmembrane

EGFR, epidermal growth factor receptor

EM, electron microscope

FACS, fluorescence activated ce11 sorting

GAH, goat anti-human Abs

GAM, goat anti-rnouse Abs

GST, glutathione S transferase

HRP, horseradish peroxidase

ITAM, immunoreceptor tyrosine based activation motif

IP3, inositol 1,4,5-trisphosphate

LB, lysis buffer

mlgM, surface IgM

NP-40, nonidet P-40

PI, phosphatidylinositol

PI-3 Kinase, phosphatidylinositol-3 kinase

PI-PLC, phosphatidylinositol-specific phospholipase C

PKC, protein kinase C

PLC, phospholipase C

PTK, protein tyrosine kinase

PTPase, protein tyrosine phosphatase

Ras.GAP, Ras GTPase-activating protein

Table of Contents

Section Page

Introduction 1) The B Ce11 Antiçen Receptor(BCR) Complex 2) Other Molecules Associated with the BCR Complex 3) B Ce11 Activation through the BCR

Materials and Methods 1) Cell lines and Plasmids 2) Preparation of the Plasmids and Sequencing 3) Transfection of J S 8 L Ce11 Line and Selection 4) Antibodies 5) Analysis of mIgM Binding to the Cytoskeleton by Western Blotting (Binding

Assay) 6) Biotinylation and Immunoprecipitation of HeLa Ceils 7) Flow Cytometric Analysis and PI-PLC assay

Results 23 1) The mIgM-Cytoskeletal Interactions Do Not Require Iç-a/p or any other 23

B ce11 Specific Protein 2) The Cytoplasmic Domain, KVK, of the mIgM Molecule Plays a role in the 26

mIgM-Cytoskeletal Interactions

Discussion 34 1) The Molecular Basis of the mlgM-Cytoskeletal Interactions 3 4 2) The Molecular Basis of the Interactions between other Receptors and the 4 1

Cytoskeleton 3) Possible Function of the mIgM-Cytoskeletal Interactions 43

Future Directions 1) Generation of the Tmncated YS"S?VV-Iga Molecule 2) Generation of Mutant IgM

References 48

Figures and Tables 61

Table of Contents : Introduction

Section

1) The B Cell Antigen Receptor(3CR) Complex 1-1) Discovery of the BCR Complex 1-2) The Role of Ig-crJP in mIg Transport 1-3) The Molecular Basis of mIg and the Heterodimer Interactions 1-4) The Role of the Ig-a/P Heterodimer in Signaling

2) Other Molecules Associated with the BCR complex 2- 1) Transmembrane Molecules and BAPs 2-2) Intracellular Molecules Associated with the BCR

3) B Cell Activation through the BCR 3- 1) Biochemical Events 3-2) Structural Events 3-3) "Looking for Connections" 3-4) The BCR-Cytoskeletal Interactions 3-5) Rationale

Page

Table of Contents : Results

Section

1) The mIgM-Cytoskeletal Interactions Do Not Require Ig-a@ or any other B Cell Specific Protein 1-1) Surface Expression of MutA on H6 1-2) MutA Binds to the CytoskeIeton in HeLa S3 crlls 1-3) MutA Interacts with Actin-Based Microfilaments

2) The Cytoplasmic Domain, KVK, of the mIgM molecule Plays a role in the mJgM-Cytoskeletd Interactions 2-1) Surface Expression of mIgM in YS:VV and YS:VV-Igci Transfectants 2-2) The Extent of mlgM Accumulation in the Detergent Insoluble Pellets

is Lower in the Absence of KVK 2-3) The mIgM-Cytoskeletal Interactions are Mediated by Actin Filaments

in the J558L Transfectants

Page

vii

TabIe of Contents : Discussioii

Section Page

1) The Molecular Basis of the mIgM-Cytûskeletal Interactions 1-1) mIgM Itself Interacts with the Cytoskeletai Matrix 1-2) KVK is Important for the mIgM-Cytoskeletal Interactions

2) The Molecular Bmis of the Interactions between other Receptors and 41 the Cytoskeleton

3) Possible Function of the mlgM-Cytoskelefal Interactions 43

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Tables

Table 1

Table 2

Table 3

Fipures and Tables

Page

Page

Introduction

1) The B Cell Antigen Receptor (BCR) C o m ~ l e x

The B ce11 antigen receptor (BCR) is a multi-subunit complex which is expressed on

the surface of B cells. The BCR consists of at least two subunits: the surface

immunoglobulin molecule (mIg), which is formed by the heavy and the light chah of Ig

molecules, and the S-S bonded heterodimer, Ig-aIP. The mIg molecule serves as the

antiçen recognition unit, whereas, the Ig-a@ heterodimer is the signal transducing unit

which links the engagement of the BCR, by antiçen, to downstream signaling. Signals

generated through the BCR mediate the final B cell activation events, such as proliferation,

differentiation, responsiveness to cytokines or apoptosis.

1-1) Discovery of the BCR Camplex

Crosslinking of m I N m I g D with antigen or surrogate antigen, such as antibodies

against mlg receptors, induces rapid sipnaling events leading to B cell activation. Yet, the

çytoplasmic domains of these receptors have only three amino acids, namely KVK (lysine,

valine, and lysine), making it difficult to envision how these mIg molecules can interact

directly with cellular substrate molecules to induce activation.lY This observation led to

the speculation that there may be molecule(s) other than the mIg molecule itself which

functions as a signal transducing unit. The answer to this question was found when Reth

and colleagues discovered the presence of proteins which were CO-immunoprecipitated with

the mIgM molecules from IgM transfectants of J558L plasmacytoma cells (1s-a-, 1ç -~+ ,

IgH-, h1+).3 They found that the bulk of IgM transfectants failed to express mIgM, except

for a very small population, which was isolated by fluorescence activated cell sorting

(FACS). Upon subsequent biochemical assays, Reth and colleagues discovered in this

r n I g ~ + population the presence of 34KDa and 39KDa proteins which co-

immunoprecipitated with mIgM molecules. Tliese glycoproteins, now called Ig-a and Ig-P,

form a disulfide-linked heterodimer and are products of the Mb-1 and B29 genes,

re~pectively.~ Both of these genes were shown to be B ceIl ~pecific,~> fi encoding a

transmembrane (TM) protein with a single extracellular Ig-like domain, a single TM

domain, and a cytoplasmic domain of 6 I or 48 amino acids, for Ig-cc or Ig-j3, respectively,

which has been implicated in B ceIl siçnal transduction (see Introduction section 1-4).

1-2 ) The Role of Ig-alp in mIg Transport

Further studies with the J558L cell line showed that CO-transfection of cells with Ig-

a encoding Mb-1 and IjM expression vectors led to surface expression of rnI@l3

Moreover, transfection of Ig heavy and light chain vectors together with Mb-1 and B29

expression vectors into NIH 3T3 fibroblast or J558L plasmacytoma cells resulted in mIg

expression for each of the five isotypes of membrane Ig, indicating that the Iç-alP

heterodimer is sufficient for the surface expression of al1 Ig molecules.7 In the case of IgD

and IgGZb, however, Ig-a/B was not necessary for their surface expression. For IçD, it was

shown that the molecule can be expressed on the surface as an intact "naked" molecuIe, Le.,

without Ig-aIP,hs well as a glycosyl-phosphatidylinositol linked form.' Currently, it is

believed that an endoplasmic reticulum (ER) resident protein, calnexin, interacts with Ig

molecules to cause the retention of Ig molecules in the ER, and that this retention can only

be overcome when Ig-cc@ binds to Ig moIecules, replacing the retention rnolecule.1°

1-3 ) The Molecular Basis of mlg and the Heterodimer Interactions

Several studies with mutated mouse IgM molecules have investigated the molecular

basis for the interactions between Ig-cr/P and mIg. Williams and colleaçues showed that a

chimeric mIgM molecule ( I ~ M - ~ K ~ ) , in which the linker, TM, and cytoplasmic domains

of the IgM molecule were replaced by the corresponding TM and cytoplasmic domains of

~ - 2 K k (a mouse class 1 MHC molecule) çould be transported to the cell surface of the

J558L and COS-7 cell Iines (monkey kidney cell, Mb-1-, B-29-), independently of Ig-cr/Pnl'

Further studies showed that the I ~ M I H - Z K ~ chimera could not interact with Ig-a/P in the B

cell lines expressing the Mb-1 and B29 genes." chimeric rnolecule, with the external

domain of CDS, and the CH4, linker, TM, and cytoplasmic domains of IgM, was expressed

on the surface of the M l 2 mouse B lymphoma ceIl line ( ~ b - l f , B - 2 9 9 but not on the

surface of COS-7 ~ e l l s . ~ ~ Taken togetlier, these results indicate the linker andlor TM and/or

cytoplasmic domains of IçM molecules are required for the B cell specific surface

expression of IgM by interacting with Ig-alp. Whether these domains play a role by

themselves or in conjunction with each other can not be concluded from these studies. The

TM domain of IgM is quite hydrophilic, containing two polar "sub-regions" in which 10 of

the 26 amino acid residues are serine, threonine, or tyrosine residues (see Fig. 1). A mouse

mutant form of mIgM, MutA (see Fig. l), whose 9 polar TM residues are replaced by

hydrophobic residues, could be expressed on the ce11 surface in the absence of Ig-alf3,ll. 12

suggesting that Ig-dj3 shields the polar residues, allowing the expression of Ig molecules in

the hydrophobic lipid bilayer surface. Interestingly, MutA could still interact with Ig-alP

(see below for more detail). One should note that IgD has less polar residues, (G amino

acids) in its TM domain than IgM, and can be expressed on the surface of a cell without Ig-

aIP.8

The fact that the mouse mutant IgM, MutA, has been shown to interact with Ig-alP13

indicates that it is likely that the cytoplasmic domain andlor non-polar residues of TM

and/or the Iinker region of IgM play(s) a role in the interactions between Ig-alP and rnIgM.

Of these possibilities, the following experiments support a role of the TM domain of IgM in

mediating the interactions with the heterodimer : 1) Studies with a human IgM mutant

molecule, YSW":VV, in which two polar residues within the IgM TM domain, TyrSX7 and

Ser5xx, have been changed to valines, show that the mutant can be expressed on the ceIl

surface, yet cannot associate with the heterodimer.I4 The inability of the human

Y""7SsF:VV mutant to interact with the heterodimer is somewhat puzzling, since the MutA

molecule containing mutations on the YsX7 and SSXX residues (see Fig. 1 ) can still interact

with the heterodimer (see below for more detail). 2) Studies with a mouse mutant IsM

molecule in which only the TM domain of IgM has been replaced by that of the MHC class

II molecule, 1-Aa, show no Ca" mobilization upon IçM crosslinking.ls Since the

heterodimers are involved in ~ a " mobilization via the BCR (see Introduction section 1-41,

these results indirectly point out that the TM domain of IçM is required for Ig-a/p

association with inIgM. The authors did not investigate the physical association of the

mutant with the heterodimer. Further stiidies by the sanle authors have shown that the

initial N-terminal residues of the IgM TM domain (outside of the TTAST polar patch, see

5G9 570 571 Fig. l), Le., the linker proximal N L W residues, are important for the interaction with

the heterodimer.lb This may explain how MutA interacts with Ig-alp. Finafly, there is

evidence that the interactions are not mediated through the cytoplasmic domain of IgM : a

mutant form of mouse IgM, MutB which contains the cytoplasmic KVK, but has extensive

mutations in the linker and TM domains of IgM, can be transported to the surface yet cannot

associate with the heterodimer.17

It is interesting to note that the human IgM mutant molecule, YSuS"H:VV, cannot

associate with Ig-a/P, whereas a mouse mutant IçM molecule, MutA, having Y ~ ~ ~ s ~ ~ ~ to

5x7 588 F A mutations can associate with the heterodimer. Perhaps the replacement of Y ~ ~ ~ s ~ ~ ~

5x7 588 to F A in MutA still preserves a conformational structure that is required for mIgM to

interact with Ig-aIP, whereas the replacement of the same residues with v ~ ~ ~ v ~ ~ ~ in the

human YS%?VV molecule cannot (see Fig. 1). This speculation is further supported by

the fact that a human IçM mutant having the substitution of YSX7 to F5X7 still interacts with

the heterodimer. Interestingly, the mouse form of the Ys7S"s:VV molecule was shown to

interact with Ig-alP, even though the extent of the interaction was reduced compared to the

wild type mouse IgM 1nolecule.1~ Since the only difference between the human and mouse

IgM molecule is the substitution of A572 in the human to T572 in the mouse IgM molecule, it

is likely that this T572 residue plays a role in inediating the mIgM-heterodimer interactions.

Taken together, these resuIts indicate that both the linker proximal and Y5X7S5xx residues are

important in inediating the interactions between IgM and Ig-alp.

1-4 ) The Role of the 1s-alP Heterodimer in Sigiialing

It is clear from the mutational studies that the heterodimer plays a role in signal

transduction as well as the surface expression of Ig molecules. The cytoplasmic tails of the

Ig-a/P heterodimer contain a signaling motif, found in many signaling receptors, known as

an ITAM (imrnunoreceptor tyrosine based activation motif), first described by Reth, with a

consensus sequence of (D or E)XXYXX(I or L)X6-8YXX(I or L), in which X is any amino

acid residue.18a Further studies using the mutant IgM molecules eventually showed a direct

correlation between the ability of the mutants to induce signaling with their ability to

associate with Ig-alP,14- 16. indicatting that the heterodimer plays a crucial role in B ce11

signaling via the BCR.

Direct evidence for the role of 1%-dP in B ce11 siçnaling comes from studies with yet

another set of mutant IgM molecules. Sanchez and colleagues showed that the cytoplasmic

tails of Ig-a and Ig-B could reconstitute caf+ mobilization and phosphotyrosine induction,

when they were expressed as a fusion protein with the external and TM domains of the

human Y5WYVV molecule (see Fig. l).l"he authors also showed that the substitution

of the tyrosine residue within the ITAM motif of the Ig-P cytoplasmic tail by phenylalanine

led to the inability of the Y5uS5m:VV-Ig-P fusion protein to induce Ca++ mobilization. A

chimeric molecule having the external and TM domains of CD8 and the cytoplasmic tail of

Ig-a induced phosphotyrosine induction as well as Ca++ mobilization in the K46 murine B

lymphoma ce11 line. Again, the substitution of an ITAM tyrosine led to the abolishment of

these signaling events via the chimeric molecule.21 Similar results were obtained using a

chimeric molecule with the external and TM domains of FcyR and the cytoplasmic domain

of Ig-a or Ig-p.22 In addition, using CH3 1 , an immature B lymphoma cell line expressing

the CD8 extracellular and TM domains and the cytoplasmic tail of Ig-a or Ig-P, Yao and

colleagues showed that Ig-a and Ig-P can induce ce11 growth arrest and npoptosis i n an

ITAM dependent inanner.23 These results directly pointed out the role of Ig-a@ as the

signaling unit within the BCR and show tliat this ability resides within the tyrosine residues

of the ITAM motif. ExactIy how Ig-a@ links the engagement of the BCR with subsequent

intracellular signaling events is not coinpletely elucidated. Currently, it is believed that the

heterodimer can interact with cytoplasmic effector molecules such as p55B'k, p53/56Ly',

p59Fs", and ~ 7 2 ' ' ~ through its ITAM, functioning as a "scaffold" protein ta bring effector

molecules together 24-26 (see Introduction section 2-2).

2 1 Other MoleciiIes Associated with the BCR Comnlex

2-1 ) Transmembrane Molecules and BAPs

A number of other transmembrane molecules (CD19, CD22, and CD45) and BAFs

(BCR associated proteins) have been shown to interact physically or functionally with the

BCR. The CD19 prorein was shown to CO-cap with mIç27, and to mediate B ce11 activation

by lowering the threshold and amplifying the magnitude of anti-IgM induced B cell

proliferation when sirnultaneousIy crosslinked with mIgM.z8 The CD22 protein is a B cell

restricted adhesion molecule which belongs to the Ig super family, containing an ITAM-like

motif in its cytoplasmic tail.293 30 The cytoplasmic tyrosine residues of CD22 become

phosphorylated after BCR crosslinking and CD22 can be CO-immunoprecipitated with the

BCR complex.31, 32 The BCR-CD22 interactions are disnipted in NP-40 lysis buffer,

suggesting that the interactions are rnediated through Ig-~t//3.~2 CD45 is a protein tyrosine

phosphatase (PTPase) involved in the BCR inediated B cell activation (see Introduction

section 3-1). Brown et al., showed by CO-immunoprecipitation experiments that CD45 was

physically associated with Ig-alP and mIgM/IgD molecules in resting B cells.33 The

authors did not verify whether CD45 interacted with the mIg molecule via Ig-a@ or not.33

Recently, four non-glycosylated ubiquitous membrane proteins known as BAPs have

been shown to be physically associated with the BCR. Two of these proteins (BAP32 and

BAP37) are associated with IgM, while the other two (BAP29 and BAP3 1) are associated

with IgD.347 " The isotype specificity of this association was shown to depend on the TM

residues of rnIg."7 35- 3fi One of the BAPs, BAP32, is a product of the prohibitin gene,

which has been previously shown to inhibit DNA synthesis if its inRNA is micro-injected

into fibroblasts and HeLa ~ e l l s . ~ ~

2-2 ) Intracelliilar Molecules Associated with the BCR

In addition to the above mentioned siirface molecules, there are cytoplasmic

molecules that are associated with the BCR, particularly the src-family protein tyrosine

kinases (PTKs) and Syk (splenocyte tyrosine kinase) PTKs. The association of p72Syk with

the BCR, in digitonin lysates, was discovered by Hutchcroft and c o l l e a g u e ~ . ~ ~

Subsequently, it was shown that p72Syk, from the lysates of anti-IgM stimulated DT40

chicken B lymphoma cells, could interact with Ig-alP via the tyrosine phosphorylated

ITAM motifs." In addition, p72S~k was shown to interact directly with ~ 5 3 / 5 6 ~ ~ " iil

vitro.40 The association of the src-family PTKs with the BCR was first shown by

Yamanashi and colleagues : The analysis of digitonin lysates from the WEHI231 B cell

lyrnphoma line showed the association of the src-family PTK p53/56Ly'1 with mIg41

Subsequently, the src-farnily PTKs p55Bik, ~ 5 3 / 5 6 ~ ~ " , p59Fy" , and p56LCk were found to

be associated with mIgM in splenic B ~ e l l s . ~ ~ ~ 43 3TK:mIç interactions, but not PTK:Ig-

alp interactions, are disrupted by the usage of NP-40 lysis buffer, which disrupts the

interaction between the heterodimer and mIg, indicating that these PTKs are associated with

the BCR via Ig-a/P.26

Further evidence that Ig-a/P was responsible for the association of the PTKs with

the BCR came from the studies of Clark and c o l l e a g ~ e s . ~ ~ Using the cytoplasmic tails of Ig-

a and Ig,-p coupled to glutathione S transferase (GST) protein, they showed that p53/56LYn

and p59Fyn interacted with the ITAM motif within the cytoplasmic tail of Ig-a. This

binding was thought to reflect the association of PTKs with the resting non-phosphorylated

heterodimer (Introduction 3-1). Subsequent studies" revealed that this interaction was

tnediated by the DCSM residues found within the Ig-a ITAM motif (see Fig. 1).

There are other molecules associated with BCR either directly through the

heterodimer or indirectly through the PTKs. Slic, a linker protein implicated in ~ 2 1 ~ s

activation, is recruired to the phosphorylated ITAMs of Ig-a and Ig-P through its SH2

d o ~ n a i n . ~ ~ Upon B ceIl activation via the BCR, Shc gets phosphorylated, and interacts with

the Grb2lmSOS complex.46 PI-3 kinase (phosphatidylinositol-3 Kinase), a heterodimeric

enzyme that phosphorylates inositol phospholipids on the 3' position of the inositol ring$7

interacts with the cytoplasmic tail of Ig-a and Ig-PYZ5 probably through the association with

p53/56Ly" via ifs non catalytic 85KDa subunit." In addition, the kinase can interact with

the GTP bound activated form of p2IbS through its 1 lOKDa catalytic subunit in viiro?g

and with p120Cb' through its non catalytic 85KDa subunit upon BCR crosslinking.5O The

p120Cbl protein interacts with Shc, and the Grb2 linker protein, as well as p59Fyn and

p72Syk.51 Recently, PTP- 1 C (SHP-l), a protein tyrosine phosphatase, has been shown to be

associated with the BCR in the CH12 mouse B lymphoma cell line.52 PTKs themselves can

interact with other effector molecules as well : p53/56LYii can interact with phospholipase

Cy2 (PLCy2), microtubule-associated protein kinase, and p2lr3S-GTPase activating protein

(Ras.GAP) through its N-terminal 27 residues of the unique region, and with PI-3 Kinase

through its SH3 domain.53 Phosphorylated tyrosines within the linker domain between the

two SH2 domains of p72S~k mediate the interactions with PLCy1 .54 Taken together these

findings give rise to a more complex picture of the BCR than a simple Ig-alj3:mIg complex,

especially after crosslinking. It appears that Ig-dj3, which constitutes the "core" BCR

complex with its mIg molecule, functions as a "scaffold to recruit and to organize effector

molecules via the ITAM motif of its cytoplasmic tail, upon BCR crosslinking. This

recruitment of various effector molecules leads to the creation of a "signaling matrix" which

is cornposed of multiple units of the BCR associated with various effector molecules. It is

quite possible that the formation of the "signaling matrix" requires the anchoring of the BCR

in the cytoskeleton to provide a proper spatial alignment of these molecules for high affinity

interactions (See Discussion for more detail).

3) B Ce11 Activation through - the BCR

3-1 ) Biocliemical Eveiits

The biochemical events induced by crosslinking of mIg involve activation of various

proteins and the generation of chemical second messengers eventually leading to phenotypic

changes characteristic of B cell activation. These events include, to name a few: activation

of PTKs, tyrosine phosphorylation of certain protein substrates, activation of phospholipase

C (PLCs), hydrolysis of phospholipids, an increase in intracellular calcium level, activation

of protein kinase C (PKC, a serinelthreonine kinase), and activation of transcription factors.

Two classes of intracellular PTKs, src-family PTKs and p72"k, have been implicated in the

functions of the BCR. The activities of src-family PTKs are regulated through

phosphorylation and dephosphorylation of specific tyrosine residues in their C-terminal

regulatory domain and kinase domain, where the phosphorylation of the tyrosine residues

are inhibitory and stimulatory, respectively.j5 It is believed, at least for the src-family

PTKs, that the phosphorylated C-terminal domain interacts with the SH2 domain of the

PTK in its resting state making the kinase domain inaccessible to its substrates. Thus,

dephosphorylation of the regulatory phosphotyrosine in the C-terminus would lead to a

conformational change in which the kinase domain of the PTKs becomes accessible to its

substrates.j6 CD45 PTPase has been shown to dephosphorylate the C-terminal regufatory

domain of these PTKs in murine lymphoma cell lines, allowing their activation.j7 In the

J558L plasmacytoma B ce11 line which expresses Mb-1 and IgM, intracellular Ca*

mobilization upon BCR crosslinkins requires CD45 expression.

Recentiy, p72Syk has been shown to be critically involved in B cell activation. In a

DT40 chicken B cell line, in which p72Syk expression haî been abolished by homologous

recombination, B cells failed to activate PLCy upon BCR crosslinkingj8 In these cells,

phosphotyrosine induction was not completely abolished, suggesting that src-family PTK

activation occurs prior to the p72Syk activation. It is likely that p53/56L~11 mediates the

activation of p72S4'k, since Lyn-negative DT40 chicken B cells show a reduction in both

tyrosine phosphorylation and the kinase activity of p72S~k.59

p21Ras membrane associated guanine nucleotide-binding protein has been implicated

in B cell signalins as ~ e l l . ~ ~ ) The p 2 ~ R a s protein can exist in an active GTP-bound state and

an inactive GDP-bound state. Its activation is favoured by guanine nucleotide exchange

factors (GNEFs), such as SOS (son of sevenless), and p9gVAV, which stiinulate the release

of GDP from Ras, favouring the active GTP-bound form.". a It is down regulated by

Ras.GAP and neurofibromin, which stimulate the intrinsic GTPase activity of Ras,

favouring the inactive GDP-bound f ~ r m . ~ ~ Activated p21R" associates with Raf-1, a

serinelthreonine kinase, and is required for the activation of Raf-1 in vivo by recruiting Raf

to the plasma membrane.G4> 65 Raf-1 phosphorylates and activates MEK, which in turn

phosphoryiates and activates the mitogen-activated protein (MM) kina~es.~Gs 67 The MAP

kinases were shown to induce phosphorylation of a number of transcription factors itr vitro

including c-jun, c-fos, and c-myc , suggesting a role in gene expre~sion.~8

The PLCy protein has been implicated in phospholipid second messenger

production. Upon BCR engagement, PLCy shows increased catalytic activity and tyrosine

phosph~ryla t ion.~ Activation of PLCy by tyrosine phosphorylation results in the hydrolysis

of phosphatidylinositol (PI) 4,5-bisphosphate, generating the second messengers inositol

1,4,5-trisphosphate (IP3) and diacylglycerol (DAC), which are responsible for the observed

increase in intracellular Caft levels70 and activation of PKC,71 respectively. The increase

in intracelluIar caft level leads to the activation of calcium/calmodulin kinase II

(CaMKinas II), a serinefthreonine kinase, which together with other serinehhreonine kinases

such as PKC, h a been implicated in the regulation of gene t r an~cr ip t ion .~~ Within a few

minutes of mIg crosslinking, transcriptional activation of the c-fos, c-myc, and egr-1 genes

O C C U ~ S . ~ ~ ? 74

At this point, the exact sequence of events following BCR crosslinking is not well

defined, however, PTKs seem to be involved in early activation events of B cells for the

following reasons : 1) PTKs are associated with the BCR complex via the Ig-alP

heterodimer, suggesting they are the early participants of biochemical responses generded

through the BCR.41-42 2) Upon the engagement of the BCR by antigen, one can observe a

rapid (within 5 seconds) appearance of phosphotyrosines.75 3) Inhibition of PTKs using

specific inhibitors can lead to a reduction or even complete abrogation of 3 ce11 activation

events such as ~ a + + increase,7G p21R" activation,77 actin polymeri~at ion,~~ and capping

and internalization,7'. X0 indicating that PTK activation is upstream of these events. Thus,

upon crosslinking of the BCR, activated PTKs phosphorylate the tyrosine residues in their

substrate proteins inducing their activation. This in turn eventually leads to the diverse

downstream B cell activation events.

3-2 ) Structural Events

B lymphocytes contain a cytoskeletal matrix, which forms an extensive interna1

network of protein fibers which encompass the entire cytoplasm, linking the plasma

membrane to the nucleus. The matrix consists of microfilaments, microtubules and

intermediate filaments, which are only found in eukaryotic c e k x l The main constituents of

microfilaments and microtubules are : filamental actin (F-actin) and tubulin, respectiveiy.

Intermediate filaments are made of several classes of ce11 type specific proteins such as

vimentin, desmin, neurofilaments, and cytokeratins. F-actin is the polymeric form of the

43KDa monomeric G-actin protein. The transition of G-actin to F-actin leads to the

transition of sol(liquid) to gel (solid) state of the cytoskeleton, and vice versa. It is believed

that this transition is responsible for spatial re-organization of the cell, replating the shape,

volume, migration, and transduction of signals from the plasma membrane to the nucleus.

The transition requires a group of actin bindinç proteins such as gelsolin and actinin, which

are responsible for F-actin severing and F-actin crosslinking, respectively. The actin-based

microfilarnents are linked to the inner surface of the plasma membrane to form the

membrane skeleton. Studies perforrned using erythrocytes indicate that microfilaments are

attached to membrane receptors such as band 3 or glycophorin by ankyrin and protein 4.1,

via spectrin.82 Deficiencies or defects in any of these proteins are associated with increased

fragility and lysis of erythro~ytes.~"nterior to the membrane skeleton the microfilaments

form an array of interconnected filaments through actin crosslinking proteins such as

actinin, fimbrin, and villin, giving the structural support to a ce11.84 The intermediate

filaments composed of vimentin link the plasma membrane to the nuclear envelope.84

Upon crosslinking of mIg, i3 cells undergo structural events including : 1) Non-

covalent interactions of mIg with the cytoskeletal m a t r i ~ . ~ j - ~ ~ 2) Polymerization of actin,X"

i.e. conversion of G-actin to F-actin. 3) Movement of the antigen bound crosslinked mIg

molecules into a smaI1 rezlon of the cell surface, forming a structure known as "cap"." Al1

of these events are mediated by the extensive network of the cytoskeletal matrix which

allows a controlled and a precise arrangement and movement of molecules within 3 cells.

Eventually, the antigen-mlg complexes are internalized into B cells, and undergo

degradation of the antigen into "antigenic" peptides which will interact with class II MHC

molecules for subsequent antigen presentation to T cells."

3-3 ) "Looking for Connections"

It is increasingly apparent that there is a functional interplay between the

biochemical and structural events mediated by crosslinking the BCR. This is supported by

studies in a variety of ceil types: 1) Actin polymerization is required for anti-CD3 induced

phosphorylation of p56Lck, phosphotyrosine induction, and growth arrest in Jurkat T cellsy2

; 2) Cytochalasin D, which disrupts the microfilament assembly, decreases the rate of CD3

internalization in a inurine Th ceIl type 2 clone,'3 and inhibits the intracellular ~ a + +

increase in T ~ e 1 l . s . ~ ~ 3) Integrin receptor GPIIb-IIIa and signaling molecules such as

pp60c-src, pp63c-yes, ras .GAP," PI3-K," p72Syk,v PLC,98 and PTP- 1 Cw translocate to

the cytoskeletal matrix after thrombin stimulation in platelets. 4) A significant increase in

the specific activities of the cytoskeleton bound enzymes such as phosphoinositide kinase,

diacylglycerol kinase, and PLC, cm occur after epidermal growth factor (EGF) stimulation

in the human A43 1 epidermal carcinoma cells which induces high affinity EGF receptors

(EGFR) to associate with actin filaments.1o0 5) C-Src was shown to translocate to the

cytoskeletal matrix in the A172 human glioblastoma ce11 line upon platelet derived growth

factor and EGF treatment as ~ e l l . ~ ~ l A positive correlation exists between the extent of ceIl

transformation by p6OSrC and its association with the cytoskeletal matrix.l02 6) The

activation of raf kinase depends upon its recruitment by rasGTP to the membrane

skeleton." 7) The ligation of TCR with anti-pan-TCRP antibodies induces an increased

association of the TCRL chain with the cytoskeleton in thyrnocytes or lymph node T

c e l l ~ , ~ ~ ~ via the third tyrosine of the 6 chain ITAM motif. 8) The activation of the T-ceIl

hybridoma DO-1 1.10 with PMA, or with antibodies against the a, P T ce11 receptor (TCR),

induces the translocation of the spectrin-ankyrin-PKCP complex from the cytoplasm into

the cytoskeletal matrix in a manner dependent upon PKC activity.1°4 9) Finally, PKCE

interaction with F-actin has been shown to be correlated with glutamate exocytosis in guinea

pig s y n a p t o s ~ m e s . ~ ~ ~

In addition, several studies indicate a correlation between cytoskeletal events and

BCR induced biochemical events in B cells. Tyrosine phosphorylation was shown to be

essential for actin polymerization, mlg-capping, and internalization.72-74 Both capping and

actin polymerization were positively correlated with B ceIl prol i fera t i~n,~~. indicating

that cytoskeletal reorganization is involved in B cell activation and that this reorganization

can be affected by biochemical events in B cells.

3-4) The BCR-Cytoskeletal Interactions

Non-ionic detergents, such as Nonidet P-40 (NP-401, can dissolve the plasma

membrane but preserve the nuclei and the enveloping cytoskeleton and its associated

proteins in an intact sedimentable form, known as the detergent insoluble pellet.lo7 By

takinç advantage of these detergents, studies have shown that upon crosslinking, the BCR

associates with the cytoskeletal r n a t r i ~ . ~ ~ - ~ ~ After lysis of cells by a non-ionic detergent,

subsequent sedimentation of the lysates at 1,000g separates the lysates to the detergent

soluble supernatant and the insoluble pellet. The soluble supernatant contains the cytosolic

proteins as well as the membrane proteins and lipids. The insoluble pellet contains the

nuclei, enveloped by the cytoskeletal matrix, and any proteins that are associated with the

matrix. Thus, upon crosslinking, BCR molecules translocate from the soluble supernatant to

the insoluble pellet, due to their interactions with the cytoskeletal matrix. Klein et a1.,1°8

indicated that mIgM found in the detergent insoluble pellets of anti-Ig treated cells are not

associated with the nuclei based on the absence of cross-linked mIgM from the nuclear

fraction which was free of cytoskeletal materials. The presence of crosslinked BCR in the

detergent-insoluble fraction does not appear to be due to BCR insolubility for several

reasons: 1) Buffers containing DNAase 1, which disrupts actin filaments,lOg lead to the

destabilization of the cytoskeletal matrix, and to the release of mIgM from the detergent

insoluble pellet.8j In our lab, we have used another method of investigating the interactions

between mIgM and actin filaments by using gelsolin (see Introduction section 3-2), which

severs F-actin filaments from the cytoskeletal matrix. 1 have used this methodology to show

that mIgM interacts with the cytoskeletal matrix via F-actin filaments (see Results section 1-

3 and 2-3). This rnethodology, however, does not show whether mIgM molecules interact

with F-actin directly or not. 2) Addition of anti-IgM after the lysis, does not cause the

mIgM accumulation in the detergent insoluble pellets.12 3) Treatrnent of mIgM containing

pellets from both WEHI23 1 cells and BAL17.7.1 ceIls with 150mM glycine for 15 minutes

at 4"C, pH 2.5, a condition which disrupts the Ag-antibody interactions, does not release

any of the detergent insoluble mIgM or actin.g6: l 2

Studies by others and Our laboratory, using FACS analysis, have demonstrated the

interactions of cross-linked mIgM with the cytoskeletal matrix.87 Detergent solubilization

dissolves the membrane and leaves the remaining nuclei and the enveloping cytoskeleton

intact. These cytoskeleton-nuclei shells are siinilar in size to intact cells and can be

anafyzed by FACS. If the BCR interacts with the cytoskeletal matrix upon crosslinking,

anti-Ig staining of intact cells will be detectable on the cytoskeleton-nuclei shells, by FACS

analysis. Using monoclonal crosslinking and non-crosslinking FITC conjugated anti-mouse

IgD, it was shown that only crosslinking monoclonal Ab's were able to stain the

cytoskeleton-nuclei shells, and thus induce the mIg-cytoskeletal interaction, although both

Ab's stained mIgD molecules of intact cells.12 The most direct evidence for the mIgM-

cytoskeletal interactions comes from studies using electron rnicroscopy (EM)12: Using gold

labeled anti-F(ab1)2 mouse IgM and anti-class II MHC monoclonal antibodies, it was shown

that crosslinked mIgM, but not class II MHC, could be found along the lenhqh of actin

filaments of the cellular detergent-insoluble cytoskeletal preparations by EM. These EM

studies have also shown that the mIgM-cytoskeletal interactions require receptor

crosslinking, since gold labeled F(ab) fragments of anti-mouse IgM do not lead to the

mIgM-cytoskeletal interactions, whereas gold labeled F(ab)'2 fragments do. EM studies

performed in the absence of a detergent, on the membrane skeleton preparations from

mechanically unroofed cellq also showed the mIgM-cytoskeletal interactions, further

supporting that the mIgM-cytoskeletal interactions are not due to the detergent insolubility

of crosslinked BCRs. Taken together, these observations indicate that the mIgM-

cytoskeletal interactions are due to the direct interactions of m1;M with the actin based

cytoskeletal rnatrix and not due to the insolubility of the crosslinked mIgM.

3-5) Rationale

Currently, the function of the BCR interactions with the cytoskeleton is unknown.

Previous studies have pointed out that the BCR-cytoskeletal interactions are positively

correlated with B ce11 proliferation and ~app ing ,~~ t 87 raising the possibility that the

interactions play a role in B ce11 activation. The long-term goal of our laboratory is to

elucidate the function of the mIgM-çytoskeleton interactions durins B ce11 activation, by

generating a mutant BCR which will no longer be able to interact with the cytoskeletal

matrix upon crosslinking. In order to create such a mutated BCR, it is necessary to find out

which component of the BCR is important in mediating the mIgM-cytoskeletal interactions

and to determine the cytoskeletal binding site within that component. The most obvious

candidate is the Ig-alP heterodirner because it is physicaily associated with mIg, and both

Ig-a and Ig-P have longer cytoplasmic tails than mIsM, which have the potential to interact

with intracellular substrates. However, our laboratory has clearly demonstrated that the

mIgM-cytoskeletal interactions occur without any requirernents for Ig-alj312 (see Results

section 2). It is possible then that the interaction is mediated through other B cell specific

protein(s) associating with the BCR (see Introduction section 2). To address this issue, a

transfectant of the human cervical carcinoma ceIl line HeLa S3, expressing a mutant form of

mouse mIgM, MutA (see Fig. l) , was used. The MutA molecule can be expressed on the

surface of cells in the absence of Ig-aIP due to the TM mutations (see Introduction section

1-3), and our laboratory has previously shown that MutA interacts with the cytoskeleton in

the mouse plasmacytoma line J558L.I2 In this study, 1 extended the previous findingsl.2 on

the lack of a role for Ig-alP in mediating the mIgM-cytoskeleton interactions, and found

that no other B ce11 specific protein is important in mediating the interactions. In addition,

to determine the domain of the mIgM molecule which interacts with the cytoskeletal matrix,

1 explored the role of the cytoplasmic tail of mIgM, KVK, in the mIgM interactions with the

cytoskeletal matrix. Although KVK seems very short to be able to interact with other

proteins, it is likely that crosslinking of mIgM may bring several of the KVlK cytoplasmic

tails together into a highly charged repetitive sequence (see Discussion for more detail). In

order to establish the role of KVK in the mIgM-cytoskeletal interactions, another form of a

mutated mIgM molecule, YSflSs5":VV, and a corresponding fusion molecule, YSHSM:VV-

Iga, were used (see Fig. 1). YSUSiw:VV is a mutated form of the human IgM molecule

which can be expressed on the cell surface in the absence of Ig-a@ (see Introduction section

1-3) and has been shown by our laboratory to interact with the cytoskeletal matrix in J558L

cells.12 Y5Ws:VV-Iga has the external and TM domains of the Y"7SSs%VV molecule, and

the cytoplasmic domain of Ig-a which replaces the cytoplasmic tail of IgM, KVK. 1 now

report here that KVK is important yet not essential for mediating the interactions between

mIgM and the cytoskeleton.

Materials and Methods

1) Cell lines and Plasmids

The mouse plasmacytoma cell line J558L (mlg-, Ig-a-, 1g-pf, hl+ , a gift from R.

Tepper, Charlestown, MA) was maintained in RPMl medium with 10% heat-inactivated

FCS (Hyclone Laboratories, Logan, UT), 2mM glutamine, 100U/ml penicillin/streptomycin,

0.1 mM nonessential amino acids (al1 from GIBCO BRL, Life Science Technologies Inc.,

Gaithersburg, MD), and 4 . 8 x 1 0 - ~ ~ P-mercaptoethanol (Sigma Chernical Co., St. Louis,

MO). For J558L transfectants, 1.2 mgml of G418 (GeneticinB, GIBCO BRL) was added to

the media. The hurnan cervical carcinoma HeLa S3 ceII line was maintained in culture in

FI2 media (Life Science Technologies Inc.) with 10% heat-inactivated FCS, 2mM

glutamine, and 100U/ml penicillin/streptomycin. For HeLa transfectants, Spglml

mycophenolic acid, 25pglml xanthine, and 15ygmI hypoxanthine (al1 from Calbiochem, La

Jolla, CA) were added to the above medium. The MutA plasmid, encoding a mutated form

of mouse IgM-heavy chain (see Fig. Ib), and the mouse hl light chain, under the control of

P-globin and cytomegalovirus promoters, respectivelyl1 was a gift from Dr. M. S.

Neuberger, Medical Research Council, Cambrige, UK. The YS:VV plasmid, encoding a

mutated form of human IgM-heavy chain (see Fig. 1 b), under the control of the human Ig

heavy chain p r o r n ~ t e r ~ ] ~ was a gift from Dr. S. Grupp, Boston, MA. The pSV2neo plasmid

coding for the G418 resistance gene ( f ~ e o ) was a sift from Dr. S. Benchimol (OCI, Toronto).

The p523IgM:Iga plasmid, which encodes the YSK7SNS:VV-Iga chimeric molecule (see Fig.

lb) and the K light chain molecule, under the control of spleen focus-forming virus (SFFV)

LTR, as well as having a G418 resistance gene (11c.o ) , lJ was a gift from Dr. Nussenzweig,

The Rockefeller University, New York.

3558L IgM' Mb-l+ transfectant (see Fig. 3b, lane 7) was generated by co-

transfection of plasinids pSVpmS (a gift from Dr. Reth, Max-Planck Institut, Freiburg,

Germany) and pCMV-Mb-lg (a gift from Dr. Grosschedl, University of California, San

Francisco) encoding wild type mouse IgM and Mb- l molecules, respectively.

2) Prenaration of the Phsrnids and Secriiencing

TG2 E.Coli were transformed using puritied plasmids. Plasmids were isolated using

a "miniprep" p r o t o c o l . ~ ~ ~ The plasmids were precipitated in EtOH, and dried using a speed

vac and resuspended in TE (IOmM Tris, ImM EDTA, pH 7.6) containing O.lmg/ml

RNAase A and incubated at RT for I O minutes. After 10 minutes, the plasmid was added to

5ml low Salt buffer (0.2M NaCl, 0.02M Tris, ImM EDTA, pH 7.5) and ran through an

elutip-D column (Schleicher and Schuell). The elutip D column was washed twice with

low-salt buffer, and the plasmid was eluted by addition of high salt buffer (1M NaCI,

0.02M Tris, ImM EDTA, pH 7.5) to the column. The eluted sample was precipitated in

EtOH and dried using a speedvac and resuspended in TE. The concentration of the final

products was determined by rneasuring the 0D260 and their structural integrity was verified

by analysis on a 1% agarose gel stained with EtBr. The presence of TM mutations (YSX7SSXX

+VSX7VSXX) and the cytoplasmic tail of Ig-a in the p523IgM:Iga plasmid, as well as the

presence of TM mutations (Y5wSSw+VSX7V5XX) in the YS:VV plasmid, was confirmed by

sequencing. DNA sequencing was done using the dsDNA thermo cycle sequencing system

from GIBCO BRL, Life Technologies Inc. Primer HpL (5' CTG TGT CTC CTG CAG

AGG 3') was used to sequence the 5' region of the IgM TM domain. Primer JYP4 (5' TGA

TCT CTG CAG TCA TGG CTT TTC CAG CTG 3') was used to sequence the 3' region of

cytoplasmic tail of Ig-a. Primer HpTail (5' GTT CTT CTG TTG GGA TCA 3') was used

to sequence the 3' region of the YM7SSSW:VV molecule downstream of the KVK region. The

mutations in the MutA plasmid were confirmed previously in the laboratory. All the

primers were generated in the laboratory of Dr. C. C. Liew (Toronto Hospital Research

Institute, Toronto, Canada).

3) Transfection of J558L Ce11 line siid Selection

J558L cells were transfected by electroporation. 2 x 1 0 ~ cells were washed twice

with RPMI containing 10 mM HEPES at RT and resuspended in 0.4 ml of the same buffer.

For the generation of cells expressing Y"7Ssm:VV, 21pg of YS;VV plasmid and 2pg of

pSV2neo plasmid were added to the buffer at RT. For the generation of cells expressing

Y"7SSw:VV-Iga, lOOpg of p523IgM:Iga plasmid was added to the buffer at RT. After 10

minutes at RT, the cells were pulsed with 240V at 960pF, incubated at 4°C for I O minutes,

and carefully transferred into 20ml RPMI medium (see above) which had been pre-

equilibrated in a tissue culture incubator for 1 hour. After the transfection, G418 resistant

cells were selected as follows: 24 hours after transfection, cells were plated in 24 weIi plates

at 105, 104, and lo3 viable cells/well. Once the cells reached about 75% confluency, each

well was fed with a selection medium containing 1.2mg/ml of G418 by replacing half of the

existing well volume with the selection medium. Within 12 days, G418 resistant coIonies

could be visualized under an inverted microscope, and 0 . 5 ~ 1 0 ~ cells were taken from

confluent wells and subjected to FACS analysis to test for the level of the surface expression

of mIgM. Transfectant lines were chosen for the binding assays, based on comparable

levels of mIgM expression. Some transfectant lines were generated by ce11 sorting (see

Results section 2-1). Cell sorting was done using a FACSTARPlus (Becton Dickinson) at

the University of Toronto.

4) Antibodies

Abs used for Western blot analysis were as follows: purified goat anti-mouse IgM

Abs (GAM-IgM, Jackson ImmunoResearch Laboratories, West Grove, PA), purified çoat

anti-human IgM Abs (GAH-IsM, Jackson ImmunoResearch Laboratories, West Grove,

PA), rabbit anti-mouse Ig-ci sera (a gift from Dr. J. Jongstra, Toronto, Canada) affinity -

purified using recoinbinant mouse Ig-a coupled to a CNBr activated Sepliadex column,

mouse anti-rabbit actin inAb's 14.3 (a gift froin Dr. D Aunis, Strasbourg, France), 4G10

mouse monoclonal Ab's to phosphotyrosine (Upstate Biotechnology Inc., Lake Placid, NY)

and peroxidase-conjugated secondary Abs: donkey anti-rabbit IgG (Amersham Corp.,

Arlington Heights, IL), rabbit anti-goat IgG (Calbiochem, La Jolla, CA), and goat anti-

mouse IgG (Chemicon International Inc., Teniecula, Ca.). Rabbit anti-mouse IgM serum (a

gift from Dr. C. Paige, Toronto, Canada) was used for immunoprecipitations. Peroxidase

con.jugated streptavidin (Pierce, Rockford, IL) was used to detect biotinylated proteins. For

the FACS analysis of J558L transfectansts, FITC conjugated F(ab')2 fragments of anti-

human IgM Abs (Jackson Immunoresearch Laboratories, West Grove, PA) were used.

FACS analysis of HeLa ceIl transfectants were performed using biotinylated anti-mouse

IgM (biotinylation was performed in our laboratory using the Jackson Ab's, see above),

followed by streptavidin-PE (Sigma Chemical Co.). Cross-linking of the m1gM receptors

was performed using affinity-purified F(abJ)2 fragments of goat anti-mouse IgM Abs

(Jackson Immunoresearch Laboratories) or anti-human IgM Abs (Jackson Immunoresearch

Laboratories) for HeLa transfectants or J558L transfectants, respectively.

5 ) Analysis of m l ~ M Bindin~ to the Cvtoskeleton bv Western Blotting (Bindinp Assavl

Cells were washed twice and resuspended in ice-cold HBSS (50x10~ celldml).

Aliquots ( 2 . 5 ~ 1 0 ~ cells in 50p1) were taken for control and total ceIl extract çample. After 2

minutes of preheating at 37*C, cells were incubated with 30pg/ml of affinity-purified

F ( ~ t b ' ) ~ fragments of anti-mouse IgM or of anti-hurnan IgM Abs (see above) for crosslinking

of mouse or human migM, respectively, for the indicated times. The control and total cell

extract samples were incubated in the absence of Abs. For the duration of mIgM

crosslinking, total cell extract samples were kept on ice. At indicated times, 4 volumes of

1% NP-40 lysis buffer (LB; 50mM Tris, 150mM NaCI, ImM MgC12, and 1% NP-40 by

volume) containing inhibitors (14pg/ml aprotinin, Ipg/ml pepstatin, 2pç/ml Ieupeptin,

1mM PMSF, and 1mM orthovanadate, al1 from Sigma Chemical Co.) were added to 1

volume of cell aliquots and to the total ce11 extract samples (final 0.8% NP-40, 10x10~

cells/ml). After 20 minutes at 4OC, cells were centrifuged for 8 minutes at 1,00Og, except

for total extract samples, to which 5 volumes of Laemmli sample buffer were added, and

immersed in boiling water for 5 minutes. The soluble supernatant contains cytosolic

proteins, as well as membrane proteins and membrane lipids. The insoluble pellet contains

the nuclei, enveloped by the cytoskeletal matrix, and any proteins that are associated with

the cytoskeletal matrix. The pellets were washed twice with LB containing inhibitors,

resuspended in Laemmli sample biiffer112, and immersed in boiling water for 5 minutes.

Sam ples were separated by SDS- 1 0% PAGE, transferred to Immobilon P membranes

(Millipore Corp., Bedford, MA), and revealed with the appropriate Abs. HRP-conjugated

secondary Abs were used to detect bands with Enhanced Cherniluminescence, ECL

(Amersham Corp., Arlington Heights, IL). Dupont ~ e f l e c t i o n ~ ~ films (Dupont, Boston,

MA) were used for autoradiography. To quantitate the amount of mIgM associated with the

cytoskeleton, a fixed amount of pellet was loaded on the gels together with the total samples

representing a range of known ce11 equivalents to generate a standard curve.

Autoradiograms were scanned using a Bio-Rad Imaging Densitometer (mode1 GS-670, Bio-

Rad Laboratories, Richmond, CA).

To confirm that mIgM found in the detergent insoluble pellet is the result of

interactions with actin based microfilaments, the calcium dependent, actin severing protein

gelsolin was used. In these experiments, cells were lysed using LE containing al1 inhibitors

(see above), 5mM ATP, 0.5mM CaCI2, and 1pM purified human recombinant gelsolin (a

gift from Dr. P. Janmey, Boston, MA). The lysates were kept at 4°C for 20 minutes and

treated as above.

In some cases, Western blots of the detergent insouble pellet were stripped by

incubating with lOmM Tris (150mM NaCI) buffer at pH 2.3 for 10 minutes. After

extensive washing (5 times) with the same buffer at pH 8, blots were re-probed with a new

set of antibodies.

6) Biotiiivbtion aiid Iniiniiiionrecipitation of HeLa Cells

HeLa cells were harvested from plates using a cell scraper (Sarstedt, Newton, NC).

Cells were washed three times with ice-cold PBS and resuspended (1x10~ cells/ml) in PBS

with freshly prepared NHS-LS-biotin (Pierce ; final concentration, 100 &ml). After

incubation with end-over-end mixing for 30 minutes at 4OC, cells were washed three times

with PBS and lysed in 1% NP-40 LB with protease inhibitors (see above ; 1 0 x 1 0 ~ cells/ml

LB) for 20 minutes on ice. After centrifugation at 13,000g for 10 minutes,

immunoprecipitations were performed by incubating the detergent soluble supernatant with

rabbit anti-mouse IgM or non-immune sera (I:200) for 2 hours at 4OC, followed by

incubation with protein A-sepharose (Sigma Chemical Co.) as described before.113

7) Flow Cvtometric Analvsis and Pl-PLC assav

Cells were washed twice, resuspended in HFA (HBSS containing 2% FCS and 0.1%

sodium azide, 2 0 x 1 0 ~ cells/ml), and incubated in the presence of 3pgjml of FITC-

conjugated or 10pg/ml of biotinylated Abs for 30 minutes at 4OC. For the cells treated with

biotinylated Abs, additional incubation with streptavidin-PE (1:50; 2 0 x 1 0 ~ cells/ml ; Sigma

Chemical Co.) for 30 minutes at 4OC was performed after removing excess primary

biotinylated Ab's by washing the cells two tiines with cold HFA. Control samples were

incubated without the primary Abs. After the final incubations, cells were washed three

times with HFA, and were analyzed on FACScan (Becton Dickinson, San Jose, CA).

Untransfected parental cells were used as negative controls and for anti-human IgM-FITC

staining, Daudi ( I ~ M + , human B lymphoma) cells were used as positive controls. J558L

transfectants were assayed for possible PI-linked mIgM : cells were washed twice and

resuspended in RPMI containing 1% BSA (Sigma Chemical Co. ; 1 x 1 0 ~ cells/ml). Samples

were divided into two parts and incubated for 1 hour at 37"C, with or without 0.2 unit of

phosphatidylinositol-specific phospholipase C (PI-PLC ; Oxford GlycoSystems, Bedford,

MA). The reaction was stopped by addition of cold RPMI. Samples were then subjected to

FACS analysis as described above. C7A124 (J558L cells expressing PI linked human IgM

molecules lacking the cytoplasmic KVK domain on the ceII surface ; a generous gift from

Dr. S. Grupp) cells were used as a positive control. Daudi (I~M', human lyrnphoma) cells

were used as a negative control.

Resiilts

1') mlgM-Cvtoskeletal - Interactions Do Not Require Ig-a/D or any other B Ce11 Specific

Protein

1-1) Surface Expression of MutA on H6

In order to investigate any possible role of B ce11 specific proteins in mediating

BCR-cytoskeletal interactions, a mouse mutant IgM molecule, MutA (see Fig. l ) , expressing

HeLa S 3 transfectant, HG, was generated in the laboratory, and 1 studied the ability of this

mutant IgM molecule to interact with the cytoskeletal matrix. FACS analysis and

immunoprecipitations were performed, to confirm the surface expression of MutA in the H6

transfectant line (see Fig. 224 2b). FACS analysis, using biotinylated anti-mouse IgM

antibodies, revealed that the H6 transfectants expressed a detectable level of mIgM, i.e.

MutA, molecules, whereas the parental HeLa S3 cells showed no significant staining (see

Fig. 2a). The surface expression level of MutA molecules in the H6 transfectant line

appears significantly lower than that of MU~A' A20 or J558L transfectants.12 The

heterogeneous mIgM expression profile was not altered when high-mIgM expressing H6

cells were sorted and cultured for a brief period of tiine, indicating that the variability in

mIgM expression is not a result of a mixture of clones but rather due to variability in the

stability of mIgM expression amon; Hu cells. Immunoprecipitates from NP-40 lysates

(2x106 cell eq.) of the biotinylated parental HeLa S3 cells and HG transfectants using anti-

IgM (lanes 1, 3, 5, 7) and non-immune sera (lanes 2, 4, 6, 8) were analyzed by Western

blotting for IgM protein (lanes 1 to 4) and biotinylated surface proteins (lanes 5 to 8). As

expected, lysates from parental HeLa S3 sliowed no detectable level of IgM (lanes l),

whereas the lysates from the HG transfectants showed detectable levels of IgM, i.e., MutA

(lane 3). The Western blot for IgM protein revealed two IgM bands, at 82KDa and 75KDa

(lane 3). Previous studies using Ba117 and WEHL231 cell lines also have shown two IgM

bands of 80 and 75KDa, corresponding to surface and intracellular IsM, respectively. l 2 We

therefore, predicted the upper 82KDa band to be the surface IgM. Accordingly, subsequent

Western blotting of anti-IgM immunoprecipitates using streptavidin showed that only the 82

KDa band is biotinylated, and thus, is the surface expressd form of IgM (lane 7). A close

examination revealed the presence of a 75KDa biotinylated band (lane 7) which co-

precipitated with the 82KDa surface IgM molecule. This band does not co-migrate with the

lower 75KDa IgM band in anti-IgM Western blot of the H6 transfectant cells (lane 3), as

determined by two independent immunoprecipitations. In addition to the biotinylated

75KDa protein, two biotinylated proteins of 65 and 3 IKDa were also found to CO-precipitate

with the surface IgM molecule. The 75 and 65KDa proteins may associate with the

transfected mIgM molecules in the H6 transfectant cells. The 3 1KDa biotinylated band

(lane 7) likely represents h light chain encoded by MutA plasmid. Anti-IgM Western blot

of the immunoprecipitates also shows bands between 50 to 55KDa (lanes 1 to 4), most

likely corresponding to the immunoprecipitatinç rabbit IgG Ab's which are cross-reacting

with the anti-goat IgG Ab's used in Western blot analysis (see the Materials and Methods

section 4 for more detail). Longer exposure of the anti-IçM Western blot showed that these

50 to 55KDa bands were clearly present in lanes 3 and 4 as well. Other

immunoprecipitation experiments also showed these cross-reactive rabbit IgG bands.

1-2 ) MutA Binds to the Cytoskeletoii in HeLa S3 cells

Once surface expression of mIgM was confirmed, the cytoskeletal binding assay was

performed, and the extent of mIgM-cytoskeletal interaction determined by Western blot

analysis of detergent-insoluble cytoskeleton-rich pellets using densitometry, as described in

the Materials and Methods. Fig. 2c illustrates one representative of three experiments

performed, and Table 1 shows the quantitative summary of these results. As expected, IgM

could not be found in the detergent insoluble pellet from the HG transfectants whose mIgM

has not been crosslinked, i.e. control sample (lane 9). However, upon crosslinking, using

30pg/ml of F(abt)2 anti-mouse IgM, a significant portion of the IçM protein translocated to

the detergent insoluble pellet (lanes 1 to 5) at 37OC. This accumulation could be observed

as early as 30 seconds (lane 1). Table l a shows tliat 18(f5)%, 32(B)%, and 31(1s)% of

82KDa surface IgM protein interacts with the cytoskeletal matrix upon crosslinking at 2, 5,

and 10 minutes, respectively. Similar results were obtained at 4 O C as well (data not shown).

As mentioned above, only the upper IgM band could be found in Western blots of the

detergent insoluble pellet probed for IgM, consistent with the fact that only the upper IgM

band is the product of the surface expressed form of IgM (see Fig. 2b) which can be

crosslinked. Treatment of H6 transfectants with a varying concentration of F(ab')2 anti-IgM

for 5 minutes showed that the maximal binding was achieved with 3Opg/ml antibodies:

60pdml and 10pgfml of F(ab1)2 led to 24% and 15% of total mIgM to interact with the

cytoskeleton, respectively. 15% binding with 10pç/ml of anti-IgM is probably due to an

insufficient crosslinking. The maxiinal 30% mIgM binding to the cytoskeleton was also

obtained in mIgM transfectants of A20 (I~G', I ~ - ~ / P + ) cells using the same MutA

plasmid,'2 indicating that the mIgM-cytoskeletal interactions are not modulated by B cell

specific proteins and are not affected by surface IgM expression level.

1-3) MutA Interacts with Actin-Based Microfilaments

In order to exclude the possibility that the accumulation of the detergent insoluble

mIgM was due to the formation of an insoluble immune complex by mIgM crosslinking, an

F-actin severing protein, gelsolin, was used as described in the Materials and Methods.

Gelsolin is a 90KDa F-actin binding protein which severs F-actin filaments to short

oligomers and depends on Ca++ for its activation.l]' By addition of gelsolin to a ce11 lysate

prior to centrifugation, proteins which are associated with F-actin and thus sediment into the

detergent insoluble pellets together with F-actin, will, after severing the F-actin filaments,

remain in the detergent soluble supernatants, upon centrifugation. Therefore, we predicted

that if IgM is interacting with F-actin, tlien upon the gelsolin treatment, it will remain with

the oligomers of actin in the detergent soluble fraction after the centrifugation, thus leading

to a reduced amount of IgM in the deterçent insoluble pellets as compared to non-gelsolin

treated samples. To test tliis hypothesis, the transfectants were lysed, after mIgM

crosslinking, in NP-40 lysis buffer in the presence of 1 [LM gelsolin. Fig. 2c shows that

mIgM and F-actin were released from the detergent insoluble pellets of H6 Lysates upon the

addition of gelsolin (see Fig. 2c, lanes 6 to 8). Table Ib shows that 38(e)% and 56(u)% of

the cytoskeletal bound mIgM molecules were released by the gelsolin treatment, at 2 and 5

minutes respectively, indicating that mIgM molecules in the detergent insoluble pellets were

interacting with the cytoskeleta1 matrix via F-actin. The reduced amounts of IçM in the

detergent insoluble pellets of the gelsolin treated samples were not due to a proteolytic

activity : Staining of samples with coommassie brilliant blue and analysis of the detergent

soluble supernatant by Western blotting showed that proteins, including IgM, were not

being degraded (data not shown). As to the nature of the remaining mIgM molecules after

the gelsolin treatment in the detergent insoluble pellet. Considering the fact that actin is one

of the most abundant molecules in lymphocytes,'lb it is quite likely that a small percentage

of F-actin filaments remaining after the gelsolin treatment are sufficient to mediate the

mIgM-cytoskeletal interactions. Indeed, one can still observe a low level of F-actin after the

gelsolin treatment (see Fig. 2c, lanes 6 to 8). It is likely that mIgM molecules interact with

this portion of actin filaments which are resistant to gelsolin cleavage (see the Results

section 2-3 for more detail).

In conclusion, the above results indicate that neither the Ig-a@ heterodimer nor any

other B-cell specific protein(s) are required for the mIgM-cytoskeletal interactions. The

results presented here confirm Our previous findings that the mIgM-cytoskeletal interactions

do not require the Ig-a/P heterodimer.12 At this point, however, one cannot conclude

whether this interaction is mediated by mIgM binding directly to F-actin or, more likely,

indirectly via ubiquitous "linker" molecule(s).

2) The Cvtoplasmic Domain. KVK, of the mlgM Molecule Plays n role in the mIgM-

Cvtoskeletal Interactioiis

As described in the Introduction, al1 of the polar residues in the TM dornain of MutA

have been converted to non-polar residues except for S584 (see Fig. la). Despite these

mutations, the molecule still interacts with the cytoskeletal matrix upon crosslinking'2 (see

Results section 1-3), indicatinç that the polar residues of the TM domain are not required for

the mIgM-cytoskeletal interactions. Since the cytoplasmic tail of IgM, KVK, is still intact

in MutA, we postulated that KVK may play a role in mediating the mIgM-cytoskeletal

interactions. In order to investigate the role of KVK in the mIgM-cytoskeletal interactions,

another form of a mutated mIgM molecule YsX7S5%VV, and a corresponding fusion

molecule, Y5flS5x?VV-Iga, having the external and TM domains of Y5wSSw:VV and the

cytoplasmic domain of Ig-a, were used (see Fig. 1 ) . Ig-a was used as the fusion partner to

replace the cytoplzismic tail of IgM, KVK, based on the following considerations : 1) Under

NP-40 lysis condition in which the interaction of Ig-a/P with mIgM is disrupted, one does

not observe any detectable level of Ig-a molecules in the cytoskeletal fraction of cells upon

crosslinking of mIgM, even though m1gM is found.12 2) Using the transfectants of IgM in

J558L cells12 and HeLa S3 cells (see Fig. 2 and Table I), which are Ig-alP negative, we

have shown that Ig-alP is not required for the mIgM-cytoskeletal interactions. Taken

together, these observations indicate that Ig-a has no intrinsic affinity for the cytoskeletal

matrix. Thus, we predicted that by comparing the extent of the mIgM-cytoskeletal

interactions between Y5Ww:VV and Y5flS5wVV-Iça, we will be able to elucidate the role

of KVK in mediating the mIgM-cytoskeletal interactions.

2-1) The Surface Expression of mlgM in YS:VV and YS:VV-Iga Transfectants

J558L cells were transfected witb plasinids encoding the Y5N7Ss":VV or

Y5flS5w:VV-Iga molecules as described in the Materials and Methods. Six transfectant

lines expressing YS"SSh?VV, designated as YS:VVs, and five transfectant lines expressing

Y5uS5m:VV-Iga, designated as YMs, were chosen, based on comparable levels of surface

IgM expression, for further biochemical analysis (see Fig. 3a). Of the transfectant lines

shown in Fig. 3a, three transfectant lines of the YS:VVs and two transfectant lines of YMs

were derived by sorting the high expressors using FACS (YS:VV26, YS:VV47, YS:VV52,

YM 1 O3 0, and YM46).

2-2 ) The Exterit of mIgM Accumulation in the Detergent Insoluble Pellets is Lower in

the Abserice of KVK

Cytoskeletal binding assays were performed on the transfectants to test the ability of

their mIgM molecules to interact with the cytoskeletal matrix upon crosslinking. Cells were

incubated with or without 30yg/ml of F(abi)2 anti-human IgM for a varying period of time,

and subsequently lysed in NP-40 lysis buffer. The detergent insoluble pellets were then

analyzed by Western blot analysis for the presence of Y5WW:VV molecules by using anti-

human IgM antibodies for YS:VVs, and for Y"7Ss?VV-Iga molecules by using anti-mouse

Ig-a antibodies for YMs. Figures 3c and 3d show typical examples of Western blots

analysed by densitometry for two of the transfectant lines studies: YM32 and YS:VV47,

respectively. As expected, two IgM bands, at 85KDa and SOKDa, and a doublet at 93KDa,

were found in the unfractionated total samples from YS:VV47 (see Fig 3d, lanes 8 to 12)

and YM32 (see Fig. 3c, Lanes 5 to 9), respectively. The 93KDa doublet from YMs could

also be detected by Western blotting using anti-human IgM antibodies, confirming the

chimeric character of the Y5"SSW:VV-Igct molecule (see Fig. 3b, lanes 1 to 3; Fig 3e, lanes

3 to 5). For Western blot analysis of YSwSs5?VV-Iga molecules, anti-mouse Iga antibodies

were mostly used instead of anti-human IçM, since the former gave a consistently better

resolution and separation of the 93KDa doublet. Anti-human IgM and anti-mouse I ç a

Western blot analysis of the total lysates of parental untransfected J558L cells did not show

any bands in the 75 to lOOKDa range (see Fig. 3b, lanes 4 and 5). Just as seen with the

HeLa cell results (see Results section 1-2), non-crosslinked mIgM did not translocate to the

detergent insoluble pellet (see Fig. 3c, lane 4; Fig. 3d lane 7). Previous experiments with a

Y5flSSM:VV transfectant of J558L cells received from Dr. S. Grupp showed that the upper

band was the surface expressed IsM, Le. mIçM, and bound to the cytoskeleton.l()~ l 2 In

accordance with these results, only the upper IgM bands of the YS:VVs and YMs

translocate to the detergent insoluble pellets upon crosslinking as can be seen by Western

blot analysis, suggesting that the molecules corresponding to the upper bands are surface

expressed (see Fig. 3c and d, lanes 1 to 3). Additional evidence supporting the notion that

the upper 93KDa band is the surface expressed form of Y5s7S5n:VV-Iga molecule cornes

from the following experiment illustrated in Fig 3e. Anti-human IgM Western blot of total

unfractionated samples show the 93 KDa doublet (lanes 3 to 5). Again only the upper

93KDa band is present in the detergent-insoluble pellets following mIgM cross-linking (lane

1). The re-probing of the same blot after striping (see Materials and Methods section 5)

using anti-phosphotyrosine antibodies reveals that this cytoskeletai-bound mlgM is highly

tyrosine phosphorylated (lane 6). Some background tyrosine phosphorylation can be

observed in the unstimulated total unfractionated samples (lanes 8 to 10) probably due to an

insufficient stripping of the antibodies used for the previous anti-human IgM Western

blotting or an artifact of reprobing where anti-mouse IgG interacts with hurnan IgM

molecules or a basal level of YSo7SSw:VV-Iga tyrosine phosphorylation. Since the surface

Ig-a molecule, associated with mIgM on the B ce11 surface, undergoes tyrosine

phosphorylation upon mIgM cross~ ink in~ , '~~ ' ' these results support the notion that the upper

93KDa molecule from YMs is the surface expressed form of Y5flSs5":VV-Iga. Saken

together, these results confirm that the 93KDa doublet is the product of the Ysx7SSVVV-Iga

molecule and that the upper protein band of the 93KDa doublet, found in the cytoskeleton of

mIgM cross1 inked YMs, is the membrane form of Y"7SSw:VV-Iga.

Table 2 summarizes the amount of mIgM found in the detergent insoluble pellets of

different YS:VV and YM transfectant lines upon mIgM crosslinking. Six transfectant lines

of YS:VVs (YS:VVS, YS:VV43, YS:VV 18, YS:VV47, YS:VV52, and YS:VV26) showed

the average amount of the surface Y5h7Sw'i":VV molecules interacting with the cytoskeleton

was 41(11x)% and 6 l ( ~ ) % , at 2 minutes and 10 minutes, respectively (see Table 2a). One

of the transfectant lines, YS:VV26, showed a lower extent of the mlgM-cytoskeletal

interactions with the average values of 15(+3)%, and 33(+6)%, at 2 minutes and 10 minutes,

respectively (see TabIe 2a). Five transfectant lines of the YMs (YM906, MM3 16, YM32,

YM46, YM1030) showed the average of 26(rt25)% and 39(+35)% of mIgM interacting with

the cytoskeleton, at 2 minutes and 10 minutes, respectively (see Table 2b). Taken together,

these data show a high degree of variability in the ability of mIgM to interact with the

cytoskeleton among the YS:VV transfectant lines and to a greater extent in YM transfectant

lines. For YM transfectant lines, in three of them (YM32, YM316, and YM906) the

average amount of the surface Y587S5M:VV-Igu molecules detected in the cytoskeleton-rich

pellets was 1 l(+_i)% and 18(&4)%, at 2 minutes and 10 minutes, respectively. However, one

of the transfectant lines, YM 1030, showed a high level of the mIgM-cytoskeIetal

interactions similar to that of YS:VVs, and another transfectant line, YM46, showed an

intermediary level of the interactions (see Table 2b). Possible explanation for these

variabilities are discussed in Discussion (see section 1-2) and below.

During the course of analysis, 1 observed variability in the amount of actin recovered

in the detergent insoluble pellets among some of the transfectants (see Table 2a and b).

Interestingly, YM46, and to a lesser extent, YM 1030, consistently had more F-actin than

any other YM transfectant lines analyzed. Since the mIgM-cytoskeletal interactions are

mediated via F-actin, it is possible to speculate that the high level mIgM-cytoskeletal

interactions exhibited by these transfectant lines are due to the increased amount of F-actin

in these transfectant lines. This observation led to the standardization of % mIgM in the

cytoskeleton-rich pellets : The amount of mIgM in the detergent insoluble pellet was

expressed as a ratio of % mIgM over % F-actin. It is important to note that these ratios are

derived from values within each experiment and not from the average values generated.

The mIgM over actin ratios from an individual Western blot of the detergent insoluble

pellets probed for IgM and actin were quantified using densitometer and were summarized

in Table 2c, and 2d. The standardization of the amount of the detergent insoluble mIgM to

that of the F-actin amount, resulted in a drastic reduction in the extent of the mIgM-

cytoskeletal interactions in MM46 transfectant line, but did not change significantly the

extent of the mIgM-cytoskeletal interactions in YM1030 (see Table 2d). The increased F-

actin content of YM46 and 1030 may be due to a different cytoskeletal structure within

these transfectant lines, leading to an increased interaction between mIgM and the

cytoskeleton. Previously, we have noticed that the mIgM-cytoskeletal interactions,

measured by the same assay, are higher in WEH123 1 cells, compared to BAL17.17.1 cells,

which has a less F-actin content than WEH123 1 cells.I2 This explanation, however, does not

answer why YM1030 demonstrates a high affinity interaction between mIgM and the

cytoskeleton, even when the interaction is expressed as % mIgM over % F-actin ratio (see

Table 2d) (see Discussion section 1-2 for more detaif). The difference between YS:VVs and

YMs in the ability of their mI2M to interact with the cytoskeleton are summarized in Fig. 4.

The results show a statisticaily significant difference for the extent of the mIgM-cytoskeletal

interactions between YSs7Sss?VV and Y5X7SSw:VV-Iga molecules at 10 minutes (P=0.04),

indicating that KVK, the cytoplasmic tail of the IgM molecule, plays a role in mediating the

mlgM-cytoskeletal interactions. Since the replacement of KVK did not aboiish the mIgM-

cytoskeletal interactions, it is likely that KVK is not the only site for mediating these

interactions (see Discussion for more detail).

2-3) The mlgM-Cytoskeletal Interactions are Mediated by Actin Filaments in the

J558L Transfectants

Just as in the HeLa experiments, one crucial question is whether mIgM molecules

found in the detergent insoluble pellets of YS:VV or YM transfectants are indeed the result

of the mIgM-cytoskeletal interactions, or due to insolubility upon crosslinking of mIgM. To

clarify this issue, gelsolin was added to the cell lysates, and the amount of mIgM remaining

in the detergent insoluble pellets assessed by densitometry. Fig. 5a shows the analysis of

such an experiment iising YM316 (similar results were obtained using other YM

transfectant lines, see below). Lanes 3 to 5 represent the samples treated with gelsolin. No

detectable F-actin levels were found upon gelsolin treatment (lanes 3 to 5). This is

expected, since severing of F-actin by çelsolin will cause a substantial decrease in the

average size of the actin filaments, leading to the decreased amount of sedimentable actin.

Note that lanes 1 and 2 have half of the cell equivalents used for lanes 3 to 5. Thus with the

treatment of gelsolin, the amount of YM7Ssw:VV-Iga molecules associated with the

cytoskeletal rnatrix was decreased from 21% to IO% at 10 minutes (see Fig. 5a, lanes 2 and

4), corresponding to 52% of the cytoskeleton-associated Y587S?VV-Iga molecules beinç

released from the cytoskeleton at 10 minutes. Based on the amount of mIgM and F-actin

remaining after the gelsolin treatment, the amounts of mIgM and F-actin released from the

detergent insoluble pellets were calculated as seen in Table 3 . For YMs, YM3 16, YM906,

and YM32 were used for gelsolin experiments, producing a similar release Ievel of the

cytoskeletal bound Y5W?VV-Iga molecules upon the gelsolin treatment with an average

value of 54(*13)% (see Table 3a). Gelsolin experiments were performed on YM1030 as

well but will be discussed later (see Discussion section 1-2). Of YS:VVs only YS:VV43

was examined. Three independent experiments were performed, producing an average

release value of 54(+9)% for Y5"S5":VV molecules at 10 minutes (see Table 3b). As with

the HeLa experiments, not al1 the cytoskeletal associated mIgM molecules were released

from the cytoskeletal matrix upon the gelsolin treatment. We predicted that even after the

gelsolin treatment, there may be a significant amount of F-actin remaining to interact with

the remaining mIgM molecules. This small amount of F-actin may not be visible in Fig. Sa

which compares F-actin in the detergent insoluble pellet samples with the total cellular actin

which is mostly G-actin. To test the amount of F-actin remaining after the gelsolin

treatment, a fixed amount of gelsolin treated sample was cornpared with various amounts of

non-gelsolin-treated sample using YM3 16 and YM32. Fig. 5b shows such a result obtained

from YM3 16, with 18% of F-actin remaining even after the gelsolin treatment. Between the

YM and YS:VV transfectant lines tested, I I % of F-actin remained after the gelsolin

treatment (see Table 3a, b). Considering that actin is one of the most abundant proteins in

lymphocytes, '~~his gelsolin resistant F-actin might still be enough in quantity to mediate

the mIgM-cytoskeletal interactions.*

In conclusion, my results comparing the extent of the cytoskeletal interaction

between Y5WW:VV and Y5h7S5":VV-Iga molecules indicate that KVK is important for

mIgM-cytoskeletal interactions. 1 also show that F-actin filaments are involved in

mediating the interactions between mIgM and the cytoskeleton. However, since cornplete

abrogation of mIm-cytoskeletal interactions was not achieved by the replacement of KVK,

the results point out that KVK has a substantial, yet non-essential role in mIgM interactions

with the cytoskeleton.

- -

* Our laboratory sliowcd tliat lOOiig of actiii csist i n Bo117.17.1 lysatcs corrcsponding to los celIs. This

aniount eqiials to 13s101' total ccllular nctin iiiolcculcs pcr ccll (scc bclow), wliicli l a d s 10 1 . 4 ~ 1 0 ~ aclin

nioleculcs forniirig F-actin Lilnnients pcr ccll. Siiicc Uic gclsolin Lrcatnicnt libcrritcs about 90% of F-actin

(scc Table 3), 1.4~10' actiii niolccuIcs will rcniain as F-actin filiiiiiciits dtcr Uic gclsolin Ircatnicnt. Tlic

csprcssion or niIg niolccolcs is hoiiglit to bc 10' iiiolcciilcs pcr ccll (Rctli ct al. (1991) Tlic B-ccll aitigcn

rcccptor con-iplcs, I~~ii~trr?olo,gy ï i ~ l a y , 12, 196-20 l), and about j0'%1 of nilgM is rclcascd by gclsoliii wliicli

diiis corrcspoiids to 5 x 1 0 ~ iiiolcciilcs of nllgM. Tnkcii togetlicr tlicsc valucs siiggcsi diat tiiere are siill

cnoiigli actiii iiiolcculcs foniiiiig F-actin filoiiieiits, in tlic gclsolin trcatcd dctergent insoluble pcllets, to

intcrnct with nilgM nioleculcs. Calculntions wcrc pcrfornicd as following : 1 Dalton = 1 g/iiiol, :. (100sIO-

9g)s(43,000~iiiol) = ~ . 3 s i O - ~ ~ n i o l or sctiii i n 105 cclls = 2 . 3 ~ 1 0 - ' ~ niol of aciin per ccll. N = 6 ~ 1 0 ~ ~

niolcciilcs pcr mol, :. 2 .3s10- '~ niol= 1 . 4 ~ 1 0 ~ niolcciilcs of total actiii pcr ccll = 14s1O6 molcculcs of total

iictiii per ccll.

Discussioii

1) The Molecular Basis of the mkM-Cvtoskeletal Interactions

Numerous studies have demonstrated that upon crosslinking, mIgM interacts with

the cytoskeletal matrix.12. 8j-g8 Yet the role of these interactions during B cell activation is

not well understood. We postulated that the BCR "anchor" to the cytoskeletal matrix may

play a crucial role in the regulation of B cell activation, by forming a template upon which a

"signaling matrix" can be built. With a long term goal to elucidate the nature of the ligand

induced mIgM-cytoskeletal interactions during B ceIl activation, 1 investigated the

molecular basis of these interactions by focusing on two questions: 1) Which component of

the BCR is required for the interactions of mIgM to the cytoskeleton? 2) What is the site on

the mIgM molecuie which mediates the interactions with the cytoskeleton upon

crosslinking? The answer to the latter question is critical in developing strategies to disnipt

these interactions for future functional studies.

1-1) mlgM Itself Interacts with the Cytoskeletal Matrix

Since the BCR is a multi-subunit complex, it was necessary to find out which

component of the BCR is important for the mIgM-cytoskeletal interactions. I started my

study by investigating a possible role of B ceIl specific protein(s) in mediating these

interactions. 1 studied the ability of the MutA molecule to interact with the cytoskeletal

matrix in a HeLa S3 transfectant (see Results section 1-2). Upon crosslinking, mIçM

interacted with the cytoskeletal matrix in a HeLa S3 transfectant (see Fig. 2c and Table 1).

These data conclusively indicated that other B-ce11 specific protein(s) are not required for

mIgM interaction with the cytoskeleton, supporting the previous findings from our

laboratory that the Ig-a/P heterodimer is not required for the mIgM-cytoskeletal

interaction^.^^ It thus appears that these interactions must involve only the mIgM molecule

itself, either directly or indirectly through ubiquitous intermediary protein(s). The possible

identity and the nature of these protein(s), are discussed in section 2. It is likely that these

ubiquitous intermediary protein(s) are cytoskeletal protein(s) such as a-actinin or ankyrin

(see section 2). In particular, a-actinin has been suggested as a possible link between mIgM

and the cyt~skeleton."~ However, ubiquitous membrane proteins known as BAPs have also

been proposed as mediators of dg-cytoskeletal interactions because of the internalization

sequences found in these m01ecules.l~~~ I l y

1-2) KVK is Important for the mIgM-Cytoskeletal Interactions

The next question 1 addressed was : which part of the mIgM molecule is important in

mediating the interactions with the cytoskeleton? 1 focused on the cytoplasmic tail, KVK,

of the IgM molecule as a possible candidate based on the following reasons : ])The MutA

molecule has extensive mutations in its TM domain, yet it can still interact with the

cytoskeleton upon crosslinking. This indicates that either the linker, linker proximal TM

domain, or the cytoplasmic tail of mIgM is important for these interactions. 2) The

cytoskeleton binding domains of ICAM- 1, L-selectin, and P 1432 integrins contain highly

charsed sequences including a common element KXK where X is 1, S, or E,

re~pectively.12~-1~~ Although KVK seems too short to be able to interact with other

proteins, it is likely that the crosslinking of mIgM brings several of the KVK cytoplasmic

tails together in a highly charged repetitive sequence, increasing its avidity with respective

substrate(s).

Nussenzwei; et aI.,l4 have created chimeric constructs with the extracellular and TM

domains of a human IgM mutant, YSuS5?VV, and the cytoplasmic tail of Ig-a or Ig-P (see

Fi;. la), to study the signaling capacities of the Ig-a and Ig-P cytoplasmic tails. Our

laboratory lias previously shown that in a Y*~S~?VV+ J558L transfectant, the majority of

the surface Y5Ww:VV molecules interact with the cytoskeletal matrix upon crosslinking.l2

In addition, Ig-a has been demonstrated not to bind to the cytoskeleton (see results section

2). Thus, we reasoned that the comparison of transfectants expressin2 YW7SS5?VV, which

contain KVK, with transfectants expressing YN7S5":VV-Iga, which jack KVK, would allow

us to study the role of KVIC i n the rnIçM-cytoskeletal interactions. Using transfectant lines

of Y5wSM?VV and YW7S5VVV-lga, I have shown here that the replacement of KVK leads

to a significant decrease of the mIgM-cytoskeletal interactions, without completely

abrogating the interactions. It thus appears that KVK plays a substantial yet not essential

role in mediating the mIgM interactions with the cytoske1eton (see Fig. 4).

Three issues were raised regarding my results : 1) 1s i t possible that the differences

observed in the extent of mIgM-cytoskeletal interactions between the YS:VV and YM

transfectant lines are due to the different surface level expression of YswSsM:VV and

YsflS%VV-Iga molecules'? As one can see in Fis. 3a, the overall surface expression level

of mIgM is quite similar among the transfectant lines while the YM316 and 906 relatively

express lower levels of mIgM. YMs can thus be categorized into two classes based on their

mIgM expression level: lower expressors of mIgM (YM316 and 906), and higher mIgM

expressors (YM1030, 32, and 46) expressing similar or slightly higher levels of mIgM as

compared to that of the YS:VV transfectants. Both low (YM3 16 and 906) and high mIgM

expressors (YM46 and 32) of YMs showed reduced rnIgM-cytoskeletal interactions

compared to YS:VVs (see Table 2c and d), indicating that the observed reduction in mIgM-

cytoskeletal interactions of YM transfectants compared to that of YS:VV transfectants is

independent of mIgM expression level. The nature of the high extent of mIgM-cytoskeletal

interactions observed in YM1030 is unknown at this point. However, some possible

explanations are given later in this section (see below). 2) Our laboratory has previously

shown that the cytoskeleton binding of mIgM requires crosslinking of mIgM.I2 1s it

possible that the atnount of anti-IgM used in my mIgM-cytoskeletal binding assays yielded a

sub-optimal IeveI of mIgM crosslinking for the YM transfectant lines, leading to a reduce

mIgM-cytoskeletal interaction? To address this question, varying concentrations of F(ab')2

anti-human IgM (IOpdml, 30pg/ml, 6OpJm1, and IOOpg/ml) were used to crosslink mIgM,

and the extent of the mIgM-cytoskeletal interactions was determined. For YS:VV47,

lO@ml of F(ab) '~ anti-human IgM led to less than 15% of the total mIgM molecules to

interact with the cytoskeletal matrix (see Fig. 3d, lane 4). Maximum binding was achieved

by 30pg/inl, and an increased amount of anti-IgM beyond this concentration did not

increase the extent of the inIgM-cytoskeletal interactions (see Fis. 3d, lanes 2, 5, and 6).

The same results were obtained for YM32 (data not shown), indicating that the reduced

ability of Y5HSSw:VV-Iga to interact with the cytoskeletal matrix was not due to reduced

crosslinking caused by an insufficient amount of anti-IgM. 3) A ceIl line described by

Wienands and Reth" was found to express a PI anchored form of IgD on its surface (see

Introduction section 1-2). Since PI linked molecules do not contain the TM and the

cytoplasmic domains, it is possible that the variability in the m1m-cytoskeletal interactions

among the transfectant lines was due to the expression of mIgM in a PI-linked form. To

exclude the possibility that my transfectants may express mIgM in P1-linked form,

transfectants were incubated with PI-PLC prior to FACS analysis. PI-PLC is known to

cleave PI linkage and thus will strip PI-linked surface molecules from the cell surface.8

YS:VV43, YS:VV47, YS:VV52, Y S:VV26, YM 1030, and YM46 were tested as described

in the Materials and Methods (section 7). The results were clearly negative. Incubation

with PI-PLC had no detectable effect on the surface expression level of mIgM molecules

(data not shown), In addition, Western blot analysis of the total unfractionated samples

from the different YM transfectant lines showed a doublet of similar molecular weight of

93KDa. Thus no reduction in the inolecular weiçht of the upper 93KDa protein which

could account for PI linkage, was obsewed in any of the YM transfectants.*p 124 Taken

to~ether these results indicate that mIçM molecules are not PI linked in my transfectants and

that, in particular, the abnormal extent of the mIgM-cytoskeletal interactions obsewed in

YM1030 and YS:VV26 compared to the rest of YM and YS:VV transfectant lines,

respectively, is not due to a PI linkage.

* PI liiikagc is tliouglit 10 cliiiiiiiatc TM alid tlic cyloplasinic doinain of inIg, Icadiiig lo ri rcductiori iii Uic

iiiolccular wciglit of i1i1g.I~~ Siiicc tlic TM aiid cyloplnsriiic doiiiaiiis of Y5"S5":W-lg~x coiitaiii 86 aiiiino

acids, Pl liiikagc will Icnd 10 about 8.6KDa rcdiiciioii in ils iiiolcculnr ivcighl.

What complicated this study was the variation in the mIgM-cytoskeletal interaction

capacities of the Yîvi1030 and YS:VV26 transfectant lines compared to the rest of the YM

and YS:VV transfectant lines, respectively. It is unlikely that the low level mIgM-

cytoskeletal interactions in YS:VV26 is due to a mix-up of the transfectant lines, Le.,

YS:VV26 is in fact a YM transfectant line, since Y5Wm:VV molecules have a different

molecular weight than Y5WS?VV-Iga molecules. In addition, al1 the YS:VV transfectant

Lines were probed for the presence of Ig-a proteins, and none of them showed a cross

reactivity with anti-Iga, indicatinç there was no mix-up of the transfectant lines between

YS:VV and YM (data not shown). For YM1030, phosphotyrosine induction patterns

indicated the presence of a functionally intact Ig-a cytoplasmic tail, further supporting that

Y5uS5m:VV-Igu molecules were not PI linked in YM1030. Gelsolin treatment of YM1030,

after 10 minutes of anti-IgM stimulation, led to a release of 60% of the cytoskeletal bound

Y5wS5u:VV-Igu molecules, indicating that the interactions are F-actin mediated (data not

shown). This value is similar to that of Y5S7S5":VV and Y5N7S5w:VV-Iga molecules

released upon the gelsolin treatment in other transfectant lines (see Table 3). One possible

explanation, for the higher mIgM-cytoskeleton interaction capacity observed in YM1030, is

that it has a mutated "linker" protein(s) which facilitates the interaction of Y5WsX:VV-Iga

with the cytoskeletal matrix, circumventing the usual requirements of KVK in the mIgM-

cytoskeleton interactions. It would be interesting to see if any protein(s) can be CO-

immunoprecipitated with Y5W?VV-Iga molecules from YM1030 in a manner that is

unique to this transfectant line. It is also possible that a structural mutation(s) exist in the

TM or cytoplasmic domain of Y""7s":VV and Y5flSsw:VV-Iga molecules in YS:VV26 and

YM1030, respectively. This possibility can be tested by mRNA sequencing of transcripts

from transfectant lines.

There are three possibilities as to why the mIgM-cytoskeletal interactions were not

completely abrogated in Yw7S5m:VV-Ig molecules despite the absence of KVK. 1) Other

element(s), than KVK, may be sufficient to mediate the interactions. As shown in Fig. la,

the Y*7S"s:VV-Iga molecule has the cytoplasmic tail of Ig-a replacing the KVK.

However, on close inspection, one can observe that KVK is not entirely replaced : KVK is

changed to KRW. It is possible that this KVK to KRW mutation can lead to reduced but not

abrogated YYW:VV-Iga cytoskeleton interaction compared to that of Y5x7S5?VV. KR

residues of the Y5WYVV-Iga molecule would leave the overall positive charge of KVK

intact, and this may account for persistent albeit reduced ability of Y5W~?VV-Iga to

interact with the cytoskeleton. Studies performed on adhesion molecules have shown that

positively charged residues are a common element found in the a-actinin binding sites of

these molecules (see below for more details). Perhaps both the sequence specificity and the

overal1 positive charge of KVK are important in mediating the mIgM-cytoskeletal

interactions, and thus, both elements need to be eliminated, in order to achieve a complete

abrogation of the mIgM-cytoskeletal interactions. It is also possible that the mIgM-

cytoskeletal interactions may involve a larger region of mIgM than just KVK. The

interactions may require the amino acids upstream of KVK which are located at the junction

of the TM and cytoplasmic domains (see Fig. la). It is interesting to note that the Klein,

Kanehesia, and Delisi algorithm, having 17 ainino acids as the membrane-spanning segment

instead of 9 amino acids used by Kyte-Doolittle algorithrn, predicts a structural mode1 of the

mIgM cytoplasmic tail containing YSTTVTLFKVK instead of just KVIS.12jl 126 2) It is

possible that the cytoplasmic tail of Ig-a, within the context of the YM7Ss":VV-Iça

molecule, rnay mediate the mIgM-cytoskeletal interactions. The Ig-a tail may now exhibit

ability to interact with the cytoskeletal matrix, by the virtue of being expressed as a

homodimer rather than being complexed with Ig-P to form a heterodimer. The

homodimeric tail may have an increased affinity toward certain substrate(s) which may link

the Y"7SsS""YVV-Iga molecule to the cytoskeletal matrix. As described in the Introduction,

Ig-ci can interact with a variety of effector molecules which has been shown to interact with

the cytoskeletal matrix (see Introduction sections 2-2 and 3-3). An alternative explanation

can be derived from the cooperative functions between Ig-a and Ig-P. It has previously

been shown that the Ig-a and Ig-P cytoplasmic tails mediate distinct functions.l4. 22 It is

possible that one function of 1;-P may be to inhibit the ability of Ig-a to interact with the

cytoskeleton. Thus, the absence of Ig-P would abolish the inhibition imposed by the Ig-P

rnolecule on the Ig-a molecule to interact with the cytoskeletal matrix. It is interesting to

note that the TCRC chain can associate with the cytoskeleton via its third ITAM motif, in a

tyrosine-phosphorylation dependent manner (see Introduction section 3-3).103 3) The lack

of the complete abrogation of mIfl-cytoskeletal interactions in YM transfectant lines may

be due to use of anti-IgM rather than an antigen. Studies have shown that anti-Ig lead to B

ceIl blastogenesis, whereas T ceIl dependent A y ~ a n n o t , ' ~ ~ suggesting anti-Ig leads to

stronger activation signals, than Ags, by overcoming the activation threshold required for B

cell blastogenesis. In addition, mutant forms of the mouse IgM molecule ex i~ t , '2~ which

can undergo Ca'+ mobilization and phosphotyrosine induction upon crosslinking by anti-

IgM, but not by Ags, indicating anti-IgM can overcome certain signaling defects associated

with mutations in IgM. Thus, it is possible that anti-IgM which was used to crosslink

Y5Ws?VV-Iça molecules, could lead to association of the molecules with the cytoskeleton

despite the mutations. Since Y 5 W m : W are specific for phosphorylcholine (PC), the

possibility could be tested by using PC conjugated ovalbumin to crosslink mIgM and seeing

if Y~7Ssm:VV-Igcr shows a complete abrogation of the mIgM-cytoskeletal interaction.

Previous studies using YsWSsMVV and Ysx7Ssos:VV-Iga molecules showed that the

molecules are capable of internalization, yet incapable of Ag presentation.128 Furthermore,

it was shown that the kinetics of internalization were similar in both m0lecules.l2~ This

result is interesting, since it suggests that mIgM-cytoskeletal interactions are not correlated

with mIgM internalization, and raises the possibility that different elements are regulating

the receptor-cytoskeletal interaction and receptor endocytosis. However, one should note

that the internalization experiments were performed using A20 cells, whereas my study was

performed using J558L cells. It is possible that in J558L cells, only the YsuSsW:VV

molecule will internalize. Also it is possible that the extent of the mIgM-cytoskeletal

interaction which occurs in the absence of KVK may be sufficient to induce internalization.

At this point it is difficult to draw a definite conclusion on the relationship between the

mIpM-cytoskeletal interactions and mIçM internalization. It is interesting to note however

that the phosphotyrosine induction has been shown to mediate BCR internalization,74 yet

internalization of BCR can still occur in the absence of the Ig-alP heterodimer,l28 which is

thought to directly mediate phosphotyrosine induction (see Introduction section 1-4).

Perhaps not ail BCR mediated phosphotyrosine induction is generated through the Ig-alP

heterodimer.

2) The Molecular basis of the Interactions between other Recentors and the

Cvtoskeleton

Over the past years, numerous studies have been performed to elucidate the

component of receptors that mediate their interaction with the cytoskeletal matrix. In

particular, extensive studies have been perforrned to study the nature of the interactions

between adhesion molecules and a-actinin. In vitro studies have revealed that the following

peptides derived from the cytoplasmic domains of the corresponding adhesion molecules,

interact with a-actinin :

RQRKIKKYR ( ICAM-l)i22 VRAAWRRL ( ICAM-2)12', FAKFEKEKMN ( Pl integrin )123 HLSDLREYRRFEKEKLK ( Ps integrin )12' KKSKRSMNDPY ( L-selectin )12"

Notably, al1 the or-actinin binding sites of the above mentioned molecules, with the

exception of ICAM-2, contain the sequence KXK, which is expressed as KVK in mIgM,

and was shown to be important in mediating the mIgM-cytoskeletal interactions in this

study. It is possible that the mIgM-cytoskeletal interactions are mediated via a-actinin,

particularly when one considers the study of Gupta and colleagues which suggested the

interaction between a-actinin and mIgM.Il7 The authors showed that anti-Ig

immunoprecipitations of non-activated rat B cells contained a 112KDa protein which could

be precleared from anti-Ig immunoprecipitates by the pre-administration of anti-cc-actinin.

However, the authors did not to distinguish whether the I12KDa protein was associated

with the membrane bound (Le. surface) or with intracellular Ig, or show that anti-cc-actinin

would immunoprecipitate surface Ig, making it difficult to conclude whether mIgM is

interactins with a-actinin or not. In addition, three lines of evidence suggest that KXK does

not always mediate the interaction with a-actinin : 1) Interaction with a-actinin can occur

independently of KXK as is the case for ICAM-2. 2) a-Actinin does not interact with a

random peptide, GKKEKPEKKVKKSDC, which contains KXK. 122 3) KXK can interact

with a molecule other than a-actinin as is the case for CD45 and Fodrin via EENKKKNRN

residues of the CD45 cytoplasmic domain.13" Based on these results, one can not, at this

time, conclude whether the mIgM-cytoskeletal interactions are rnediated by a-actinin or by

other cytoskeletal proteins.

The vitamin D-binding Gc protein was suggested as a possible mediator of the

mlgM-cytoskeletal interactions, since the protein was found to interact with mIg and actin

in non-stimulated peripheral blood lymphocytes and CO-capped with crosslinked rr11g.l~~

However, studies have shown that B cells are positively stained for the membrane Gc

p r ~ t e i n l ~ ~ and that the Gc/G-actin complexes are extracellular in origin,l32 indicating that

the observed mIg/actin/Gc complexes are likely extracellular in origin. The CO-capping of

mIg and Gc was probably due to the Gc protein interacting directly with mIg or indirectly

through a molecule such as the Gc r e ~ e p t 0 r . l ~ ~

It is also possible that mIg may interact directly with F-actin. Binding of Ig isoiated

from the anti-Ig treated mouse peripheral lymphocytes or P3 myeloma cells to myosin

affinity columns, was used to suggest that Ig binding to the cytoskeleton involved actin.

However, these experiments did not explore the presence of other proteins in

Ig/actin/m yosin complexes. I 3 l The on1 y surface receptor whose site of direct F-actin

interaction known is EGFR, which interacts with F-actin directly through its

DDVVDADEYLIPG cytoplasmic residues. l *"bis iack of data, regarding the surface

receptor interactions with F-actin, makes it difficult to conclude whether mlgM is

interacting with F-actin directly or not.

Studies performed with other surface receptors show that no consensus sequence

exists among receptors that interact with a particular cytoskeletal protein. For example,

CD44 interacts with ankyrin via NGGNGTVEDRKPSEL residues, whereas the studies

performed on Na+,K+-ATPase showed that instead a cluster of four amino acids, ALLK, is

important in mediating the interactions between this protein and ankyrin. 1347 '35 This

apparent lack of a consensus sequence suggests that there is no common mechanism for

surface receptor-cytoskeletai interaction, and that different surface receptors may utilize

different mechanisms of binding to interact with the cytoskeletal matrix. The only common

element in the cytoskeletal binding regions of the above mentioned proteins is the presence

of charged residues, indicating that receptor-cytoskeletal interactions either require or are

enhanced by electrostatic interaction. It is also possible that a few key hydrophobic residues

CO-operate together with the charged residues to enhance the strength of the receptor-

cytoskeletal interactions. Suzuki et al.,136 showed that a peptide derived from the F-actin

binding site of myosin, IRICRKG, had an increased strength of interaction with F-actin

compared to peptides composed of I N or CRKG, suggesting that the interaction is enhanced

by the presence of both hydrophobic and charged residues. Perhaps both charged and

597 594 hydrophobic residues of mIgM, such as lysines of KVK and L ' F , respectively, are

functioning CO-operatively to mediate mIgM-cytoskeletal interaction.

3) Possible Function of the rnleM-Cytoskeletal lnterrctions

The long term goal of the laboratory is to determine the role of the mIgM-

cytoskeletal interactions during B ce11 activation. Previous studies have pointed out that

BCR-cytoskeletal interactions are positively correlated with B ceIl proliferation and

capping, X7 raising the possibility that these interactions play a key role in B cell

activation. It is possible to envisage that ligand induced inIgM-cytoskeletal interactions

may serve as an important regulator of B ceIl activation by forming a template upon which a

"signaling matrix" can be built. The following observations support the idea of "siçnalinç

matrix" formation on the cytoskeleton : 1) As mentioned previousiy, the BCR can complex

with a large number of cytoplasmic effector molecules which can interact with the

cytoskeleton (see Introduction section 2-2). 2) PTKs, ~ 5 3 1 5 6 ~ ~ " and p72S~k, which

associate with the BCR, via the Ig-a@ heterodimer, translocate to the membrane-skeleton

upon BCR crossIinking.137 3) Tyrosine phosphorylated substrates and p21R" were

localized underneath the mZg caps.I38> 139

Therefore, once established, this "signalins niatrix" would allow : 1) Transmission

of signals to the cytoskeleton, leading to its re-organization. The PLCy protein bas been

shown to interact with F-actin via its SH3 domain.140 Activated phosphorylated PLCy

recognizes PI 4,5-bisphosphate141 which regulates actin polymerization by interacting with

actin binding proteins such as gelsolin and profilin.142-144 It has been suggested that PI 4,5-

bisphosphate levels in the plasma membrane may be altered by activation of receptors,

leading to the g-owth of new actin filament structures close to the site of action of

extracellular ~ignals.14~ In addition, increases in intracellular calcium concentration may

activate calcium dependent actin filament-severing proteins such as gelsolin, leading to the

reorganization of F-actin filaments.ll5 2) Regulation of the "signaling matrix" itself,

providing a spatial and a temporal regulation to the matrix. Many of the molecules involved

in BCR-induced early siçnaling transduction such as ras.GAP, PI3-K, ~72Syk, ~53/56L~n,

PLC, and PTP-IC, have been shown to associate with the cytoskeleton (see Introduction

sections 3-3 and 3-4). The actin-containing microfilament system may provide a matrix to

permit the orsanization of signaling molecules, bringing their targets and regulators to

juxtaposition. 3 ) Transmission of the signals to the nucleus via the re-organized cytoskeletal

matrix, leading to gene transcription. No study has investigated how the initial signaling

events which occur proximal to the plasma membrane can be transmitted to the nucIeus to

initiate gene transcription. It is likely that certain effector molecules, once activated, travel

to the nucleus via the cytoskeletal rnatrix. Ito and colleagues have shown that the

translocation of activated PKC was dependent upon actin filaments. 146

In this study, 1 have shown that the cytoplasmic tail of the mIgM molecule is

important in mediating mIgM-cytoskeletal interactions. Once the cytoskeletal bindinç

domain of mIgM is determined more accurately (see Future Directions), the challenge will

be to determine the effects of the abrogation of the "anchoring" ability of mIg on activation

events, such as phosphotyrosine induction, Ca++ mobilization, capping, internalization, and

antigen presentation.

Fritrire Directions

1) Generation of Truncated Y~n7$snn:VV-lga Molecule

It is possible that the reduced ability of YM7S5YVV-Iga to interact with the

cytoskeleton is due to the steric hindrance caused by the Ig-a tail. To resolve this issue, I

will generate a truncated Y5WS"?VV-Iga molecule which contains only N-terminal end

KRW residues of the Ig-a cytoplasrnic tail, fused with the Y5Vti?VV external and TM

domains (YSMSS?VV-KRW). J558L cells expressing the truncated YS:VV-Iga molecule

(Ysx7S5M:VV-KRW) will be generated by transfection*, and the mIgM-cytoskeletal

interactions will be analyzed for transfectants. Three possible outcomes can be envisioned

from the studies using Y"7S5XYVV-KRW molecules : i) A cornplete abrogation of the

mIgM-cytoskeletal interactions, indicating that the reduced yet present ability of

Y5"S":VV-Igct molecules to interact with the cytoskeleton was due to the presence of the

Ig-a cytoplasmic taii for the reasons described previously (see Discussion section 1-2). ii)

No change in the mIgM-cytoskeletal interactions, indicating the role of KRW in mediating

the interaction and/or of another region which mediates the mIgM-cytoskeletal interactions

together with KVK. iii) Increased mIgM-cytoskeletal interactions, indicating the reduced

ability of Y5WW:VV-Iga to interact with the cytoskeleton was due to a steric hindrance

caused by the cytoplasmic tail of Ig-a. This also implies that K5p5V5p6K597+K9NR596W597

mutation does not reduce ability of mutant IgM to interact with the cytoskeleton, indicating

that either the positively charged residues are important in mediating the ml@-

cytoskeleton interactions or soine elements other that KVK is inediating the interactions (see

Discussion section 1-2).

*Al1 tlic trnnsfcctioiis wilI bc pcrfoniicd oii singlc ccll dcrivcd sub-cloiics of Uic rcspcctivc cc11 line. This is

to rcdiicc any poiciitial variability in irilgM-cytoskclctal iiiteractioiis due to cloiiiil variations.

25 Generstion of illutant IgM

Based on the outcorne of the studies iising the YsuS"?VV-KRW molecule, further

IgM constmcts will be generated. 1) If Y5uS5M:VV-KRW leads to the complefe abrogation

of the mIgM-cytoskeletal interactions, the wild type human IgM molecule with the KRW

cytoplasmic tail will be generated, using site directed mutasenesis. In addition, the wild

type human IgM molecule with KRWKVK cytoplasmic tail will be generated to test the

gain of function due to KVK. Plasmids will be transfected into A20 cells, and the

functional significance of the mIgM-cytoskeletal interactions will be studied. 2) If the

mIsM-cytoskeletal interaction level remains the same or increases upon using YSnSSmVV-

KRW molecules, a new set of fusion molecules will be generated using Y5flSsM:VV and

CD3c. Rozdzial et al., showed that the CD3< chain with deletions in its cytoplasmic

domain (CD66 157) lost its ability to interact with the cytoskeletal matrix.1°3 A constmct

encoding the external and TM domains of YSX7SSXX:VV and the cytopiasmic domain of

6D66: 157 (YSK7S%VV-CD3C) will be generated by site directed mutagenesis and PCR. In

order to study the gain of function due to KVK, a construct encoding the e~ternal and TM

domains of CD36 and the cytoplasmic domain of IgM, KVK, wiI1 be generated by site

directed mutagenesis by introducing KVK-stop codon at the junction of the TM and

cytoplasmic domains of CD3C. By using these constructs 1 will be able to further

investigate the role of KVK in the mIgM-cytoskeletal interactions. J558L cells will be

transfected and the extent of the mIgM-cytoskeletal interactions will be compared among

Y5°7S5M:VV, Y5H7SSxK:VV-CD3c, and CD3C-KVK.

References

1. Cheng H. L., Blattner F. R., Fitzmaurice L., Mushinski J. F. and Tucker P. W. (1982) Structure of genes for membrane and secreted murine IgD heavy chains. N d w e 296, 410- 415

2. Rogers J., Early P., Carter C., Calame K., Bond M., Hood L. and Wall R. (1980) Two mRNAs with different 3' ends encode membrane-bound and secreted forms of immnoglobulin CL chah. Cd/ 21, 303-3 12

3. Hombach J., Tsubata T., Leclercq L., Stappert H. and Reth M. (1990) Molecular components of the B-cell antigen receptor complex of the IgM class. N n t m 343, 760-762

4. Hombach J., Lottspeich F. and Reth M. (1990) Identification of the çenes encoding the IgM-a and Ig-j3 components of the IgM antigen receptor complex by amino-terminal sequencing. Ei~r. .J. Znzmind 20: 2795-99

5. Sakaguchi N., Kashiwamura S., Kirnoto M., Thalmann P. and Melchers F. (1988) B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties. M O J. 7, 3457-64

6. Hermanson G.G., Eisenberg D., Kincad P. W. and Wall R. (1988) B29: a member of the immunoglobulin gene superfamily exclusively expressed on B-lineage cells. Proc. N d t ~ Acnd. Sci. U.S.A. 85, 6890-6894

7. Venkitaraman A. R., Williams G. T., Dariavach P. and Neuberger M. (199 antigen receptor of the five immunoglobulin classes. Nnti~re 352,777-781

1) The B-cell

8. Williams G. T., Dariavach P., Venkitaraman A. R., Gilmore D. J. and Neuberger M. (1993) Membrane immunoglobulin without sheath or anchor. Mokec. Znzmrrn. 30, 1427-1432

9. Wienands J. and Reth M. (1992) Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunogIobulin D. Nnfirre 356, 246-248

10. Grupp S. A., Mitchell R. N., Schreiber K. L., McKean D. J. and Abbas A. K. (1995) Molecular mechanisms that control expression of the B lymphocyte antigen receptor complex. ,J. i k p . M d . 181, 161-168

11 . Willimas G. T., Venkitaraman A. R., GiImore D. J. and Neuberger M. (1990) The sequence of the p transrnembrane segment determines the tissue specificity of the transport of immunoglobulin M to the cell surface. ,/. IGp. M d 171, 947-952

12. Hartwig J. H., Jugloff L. S., Groot N. J., Gmpp S. A. and Jongstra-Bilen J. (1995) The ligand-induced membrane IgM association with the cytoskeletal matrix of B ce11 is not mediated through the Ig ap heterodimer. .J. lr~~mrrir. 155,3769-3779

13. Patel K. J. and Neuberger M. (1993) Antigen presentation by the B cell antigen receptor is driven by the alP sheath and occurs independently of its cytoplasmic tyrosine. Cell 74, 939-946

14. Sanchez M., Misulovin Z., Burkhardt A., Mahajan S., Costa T., Franke R., Bolen J . B. and Nussenzweig M. (1993) Signal transduction by immunoglobulin is mediated through Iga and Igp. ,J. Exp. Med 178, 1049-1055

15. Parikh V. S., Bishop G. A., Liu J. K., Do B. T., Ghosh M. R., Kim D. S. and Tucker P. W. (1 992) Differential structure-function requirement of the transmembrane domain of the B cell antigen receptor. ./. Exp. Med. 176, 1025-1 03 1

16. Michnoff C. H., Parikh V. S., Lelsz D. L. and Tucker P. W. (1994) Mutations within the NH2-terminal transmembrane domain of membrane immunoglobulin (Ig) M alters Iga and 1gp association and signal transduction. ,l. Biol. C h m . 269, 24237-24244

17. Neuberger M. S., Patel K. J., Nelms C. J . , Peaker G. and Williams G. T. (1993) The mouse B-cell antigen receptor: definition and assembly of the core receptor of the five immunoglobulin isotypes. Imm~rnologicnl Review 132, 147- 16 1

18. Stevens T. L., Blum J. , Foy S. P., Matsuchi L. and DeFranco A. L. (1994). A mutation of the 11 transmembrane that disrupts endoplasrnic reticulum retention. ./. Immun. 152, 4397- 4406

1 8a. Reth, M. (1989) Antigen receptor tail clue. Nalzrve 338, 383-384

19. Grupp S. A., Campbell K., Mitchell R. N., Cambier J. C. and Abbas A. K. (1993) Signaling-defective mutants of the B lymphocyte antigen receptor fail to associate with Ig-a and Ig-Ply. .J. Biol. C'hem. 268, 25776-25779

20. Blum J. H., Stevens T. L. and DeFranco A. L. (1993) Role of the p immun~globulin heavy chain transmembrane and cytoplasmic domains in B cell antigen receptor expression and signal transduction. .J. Biol. Chetn. 268, 27236-27245

2 1 . Flaswinkel H. and Reth M. (1994) Dual role of the tyrosine activation motif of the Ig-a protein during signal transduction via the B cell antigen receptor. ,!&BO ,J. 13, 83-89

22. Choquet D., Ku G., Cassard S., Malissen B., Korn H., Fridman W. H. and Bonnerot C. (1994) Different patterns of calcium signaling triggered through two components of the B lymphocyte antigen receptor. .J. Biol. Ct~em. 269, 6491-6497

23. Yao X. R., Flaswinkel H., Reth M. and Scott D. (1995) Immunoreceptor tyrosine-based activation motif is required to signal pathways of receptor-mediated growth arrest and apoptosis in murine B lymphorna cells. ./. Iriinirtir. 155, 652-661

24. Johnson S. A., Pleiman C. M., Pao L., Schneringer J., Hippen K, and Carnbier J. C. (1995) Phosphorylated immunoreceptor signaling motifs ( ITAMs ) exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases. ./. In7n11tn. 155, 4596-4603

25. CIark M. R., Campbell K. S., Kazlauskas A., Johnson S. A., Hertz M., Potter T., Pleiman C. and Cambier J. C. (1992) The B cell antigen receptor complex : association of Ig-a and Iç-P with distinct cytoplasmic effectors. Science 258, 123-126

26. Lin J. and Justement L. B. (1992) The MB-lm29 heterodimer couples the B ceIl antigen receptor to multiple src family protein tyrosine kinases. ,J. Inzmii. 149, 1548-1 555

27. Pesando J, M., Bouchard L. S. and McMaster B. E. (1992) CD19 is functionally and physically associated with surface immunoglobulin. J. &p. Med 170, 2 159-2164

28. Carter R. H. and Fearon D, T. (1992) CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 256, 105-107

29. Engel P., Mojima Y., Rothstein D., Zhou L. J., Wilson G. L., Kehrl J. H. and Tedder T. F. (1993) The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils,. and monocytes. J. Immun. 150, 4719-4732

30. Wilson G. L., Fox C. H., Fauci A. S. and Kehrl J. H. (1991) cDNA cloning of the B cell membrane protein CD22: a mediator of B-B cell interactions. ./. Exp. Med 173, 137-1 46

3 1 . Peaker C. J. G. and Neuberger M. S. (1993) Association of CD22 with the B cell antigen receptor. Eur. .I. In~mzrnol. 23, 1358-1 363

32. Leprince C., Draves K. E., Geahlen R. L., Ledbetter J. A. and Clark E. A. (1993) CD22 associates with the human surface IgM-B-ceIl antigen receptor complex. Proc. Nnbz. Acad. Sci. U.S.A. 90, 3236-3240

33. Brown V. K., Ogle E. W., Burkhardt A. L., Rowley R. B., Bolen J. B. and Justment L. B. (1994) Multiple components of the B cell antigen receptor complex associate with the protein tyrosine phosphatase, CD45. .J. Uiol. C'hem. 269, 17238- 17244

34. Terashima M., Kim K. M., Adaclii T., Nielsen P. J., Reth M., Kohler G. and Lamers M. C. (1994) The IgM antigen receptor of B lymphocytes is associated with prohibitin and prohibitin-related protein. .!&BO ./. 13, 3782-3792

35. Kim K. M., Adachi T., Nielsen P. J., Terashima M., Lamers M. C., Kohler G. and Reth M. (1994) Two new proteins preferentially associated with membrane immunoglobulin D. M O ./. 13,3793-3800

36. Adaclii T., Schamel W. W. A., Kim K. M. Watanabe T., Becker B., Nielsen P. J. and Reth M. (1996) The specificity of association of the IgD molecule with the accessory proteins BAP3 I/BAP29 lies in the IgD transmembrane sequence. FMBO ,J. 15, 1534-1541

37. Nuell M. J., Stewart D. A., Walker L., Friedman V., Wood C. M., Owens G. A., Smith J. R., Schneider E. L. Dell'orco R., Lumplin C. K. Danner D b. and McClung J. E. (1991) Prohibitin, an evohtionarily conserve intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells. Molec. Cell Biol. 11, 1372- 138 1

38. Hutchcroft J. E., Harrison M. L. and Geahlen R. L. (1991) Association of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor. ./. Biol. Chem. 267, 8613- 86 19

39. Kurosaki T., Johnson S. A., Pao L., Sada K., Yamamura H. and Cambier J C. (1995) Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling. .I. Exp. M d . 182, 18 15-1 823

40. Sidorenko S., Law C. L., Chandran K. A. and Clark E. A. (1995) Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53156Lyn and a 120-kDa phosphoprotein. Proc. Nabi. Acnd Sci. U.S.A. 92,359-63

41. Yamanashi Y., Kakiuchi T. Mizuguchi J., Yamamoto T. and Toyoshima K (1991) Association of B cell antigen receptor with protein tyrosine kinase Lyn. Science 251, 192- 194

42. Burkhardt A. L., Brunswick M., Bolen J. B. and Mond J. J. (1991) Anti- immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc. NCI/II. Acad. Sci. US. A. 88, 74 10-74 14

43. Campbell M. and Sefton B. M. (1992) Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases. Molec. CeZI Biol. 12, 23 1 5-232 1

44. Clark M. R., Johnson S. A. and Carnbier J. C. (1994) Analysis of Ig-a-tyrosine kinases interaction reveals two levels of binding specificity and tyrosine phosphorylated Ig-a stimulation of Fyn activity. EMUO ./. 13, 19 1 1- 191 9

45. D'Ambrosio D., Hippen K. L. and Cambier J. C. (1996) Distinct mechanisms mediate SHC association with the activated and resting B cell antigen receptor. Lw. .J. Immunol. 26, 1 960- 1965

46. Saxton T. M., van Oostveen I., Bowtell D., Aebersold R. and Gold M. R. (1994) B cell antigen receptor cross-linking induces phosphorylation of the p21raS oncoprotein activators SHC and mSOS I as well as assembly of complexes containing SHC, GRB-2, mSOS 1 and a 145-kDa tyrosine-phosphorylated protein. .J. It~inrrar. 153, 623-636

47. Witman M., Downes C . P., Keeler M., Keller T. and Cantley L. (1991) Type 1 phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3- phosphate. Natln.e 332,644-646

48. Yamanashi Y., Fukui Y., Wongsasant B., Kinoshita Y., Ichirnori Y., Toyochima K. and Yamamoto T. (1991) Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-ceIl antigen receptor-mediated signaling. Proc. Nnh. Acad Sci. I/. S. A. 89, 1 1 18- 1 122

49. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M., Waterfield M. D. and Downward J. (1994) Phosphotidylinositol-3-OH kinase as a direct target of Ras. Nntirre 370, 527-332

50. Kim T. J., Kim Y. T. and Pillai S. (1995) Association of activated phosphatidylinositol 3-kinase with p120cb1 in antigen receptor-ligated B cells. ./. Biol. Chem. 270, 27504-27509

51. Panchamoorthy G., Jukazawa T., Miyake S., Soltoff S.,, Reedquist K., Druker B., Shoelson S., Cantley L. and Band H. (1996) p12~cb1 is a major substrate of tyrosine phosphorylation upon B ce11 antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3- kinase. .J. Biot. Chern. 271, 3 187-3 194

52. Pani G., Kozlowski M., Cambier J. C. Mills G. B. and Siminovitch K. A. (1995) Identification of the tyrosine phosphatase PTPlC as a B cell antigen receptor-associated protein involved in the regulation of B ceIl signaling. ./. fip. M d . 181, 2077-2084

53. Pleiman C. M., Clark M. R., Gauen L. K., Winitz S., Coggeshall K. M., Johnson G. L., Shaw A. S. and Cainbier J.C. (1993) Mapping of sites on the Src family protein tyrosine kinases p55blk, p59b'11, and p561~fi which interact with the effector molecules phospholipase C-y2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase. Molec. CeIl Biol. 13, 5877-5887

54. Law C., Chandran K. A., Sidorenko S. P. and Clark E. A. (1995) Phospholipase C-y1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk. Molec. Cc// Biot. 16, 1305-13 15

55. Kmiecik T. E. and Shalloway D. (1987) Activation and suppression of pp6OC-SrC transforming ability by mutation of its primary sites of tyrosine phosphorylation. Ce11 49, 65-73

56. Cooper J. A. and Howell B. (1993) The when and how of Src regulation. Cell 73, 105 1- 1054

57. Ostergaard H. L., Shackelford D. A., HurIey T. R., Johnson P., Hyman R., Sefton B. M. and Trowbridge 1. S. (1989) Expression of CD45 alters phosphorylation of the kk-encoded tyrosine protein kinase in murine lymphoma T-cell Iines. Proc. Nalt~. Acad Sci. U3.A. 86, 8959-8963 .

58. Takata M., Sabe H., Hata A., Inazu T., Homma Y., Nukada T., Yamamura H. and Kurosaki T. (1994) Tyrosine kinases Lyn and Syk regulate B ce11 receptor coupled Ca2+ mobilization through distinct pathways. M O ,/. 13, 1341-1 349

59. Kurosaki T., Takata M., Yamanashi Y., Inazu T., Taniguchi T., Yamamoto T. and Yamamura H. (1994) Syk activation by the Src-family tyrosine kinase in the B ce11 receptor signaling. .I. Exp. M d . 179, 1725-1729

60. McMahon S. B. and Monroe J. G. (1995) Activation of the p21raS pathway couples antigen receptor stimulation to induction of the primary response gene egr-1 in B lymphocytes. ,/. fip. Med. 181,417-422

61. Bowtell D., Fu P., Simou M. and Senior P. (1992) Identification of murine homologues of the Drosphiln Son of sevenless gene:potential activators of rus. Proc. Nafil. Acad Sci. USA. 89,65 1 1-65 1 5

62. Gulbins E., LangIet C., Baier G., Bonnefoy-Berard N., Herbert E., Altman A. and Coggeshall K. M. (1994) Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells. ./. Ininlrrn. 152, 2123-2129

63. Alan H. (1992) Signal transduction through srnaIl GTPases-a tale of two GAPs. C d 69, 389-39 1

64. Koide H,, Satoh T., Nakafuku M. and Kaziro Y. (1993) GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc. Ndt7. Acnd. Sci. U.S.A. 90, 8683-8686

65. Stokoe, D., Macdonald S. G., Cadwallader K., Syrnons M. and Hancock J. F. (1993) Activation of Raf as a result of recruitment to the plasma membrane. Science 264, 1463- 1467

66. Kyriakis J. M., App H., Zhang Z., Banerjee P., Brautigan D. L, Rapp U. R. and Avnich J. (1 993) Raf- 1 activates MAP kinase-kinase. Natirre 358, 4 17-42 1

67. Davis Alessandrini A., Crews C. M. and Erikson R. L. (1992) Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product. Psoc. N d t ~ . Acad. Sci. IlS. A. 83, 8200-8204

68. Davis R. J. ( 1 993) The mitogen-activated protein kinase signal transduction pathway. ./. Biol. Cl?ent. 268, 145 53- 14556

69. Hempel W. M., Schatzman R. C. and DeFranco A. L. (1992) Tyrosine phosphorylation of phospholipase C-y7 upon cross-linking of membrane Ig on murine B lymphocytes. .J. Iulnlmiin. 148, 302 1-3027

70. Ransom J. T., Harris L. K. and Cambier J. C. (1986) Anti-Iç induces release of inositol 1,4,5-trisphophate, which mediates mobilization of intracellular Ca++ stores in B lymphocytes. ./. Immurr. 137, 708-7 14

71. Ku Y., Kisliimoto A., Takai Y., Ogawa Y., Kimura S. and Nishizuka Y. (1981) A new possible reçulatory system for protein phosphorylation in human peripheral lymphocytes. II. Possible relation to phosphatidylinositol turnover induced by mitogens. ./. I m m m 127, 1375-1379

72. Hunter T. and Karin M. (1992) The regulation of transcription by phosphorylation. Ceil 70, 375-387

73. Klemsz M. J., Justment L. B., Palmer E. and Cambier J. C. (1989) Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin binding ligands. ./. Intmiiil. 143, 1032- 1039

74. Seyfert V. L., McMahon S., Glenn W., Cao X., Sukhatme V. P. and Monroe J. G. (1 990) Egr-1 expression in surface Ig-mediated B ce11 activation. ./. Imminz 145, 3647-3653

75. Saouaf S. J., Mahajan S., Rowley B., Kut S. A., Fargnoli J., Burkhardt A. L., Tsukada S., Witte O. N. and Bolen J. B. (1994) Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-ce11 antigen receptor surface engagement. Proc. Nah. A c d . Sci. US. A. 91, 9524-9528

76. Lane P. J. L., Ledbetter J. A., McConnell F. M., Draves K., Deans J., Schieven G. L. and Clark E. A. (1991) The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. ./. I t r~mu~. 146, 715-722

77. Kawauchi K., Lazarus A. H., Ropoport M. J. Hanvood A., Camber J. C. and Delovitch T. L. (1994) Tyrosine kinase and CD45 PTPase activity mediates p2Iras activation in B cells stimulated througli the antigen receptor. .J. Irtlintm. 152, 3306-33 16

78. Melained I., Downey G. P. and Roifman C. M. (1991) Tyrosine phosphorylation is essential for microfilament assembl y in B lymphocytes. Biocheni. Biophys. Res. Conm. 176, 1424- 14.29

79. Shimo K., Gyotoku Y., Arimitsu Y., Kakiuchi T. and Mizuguchi J. (1993) Participation of tyrosine kinase in capping, internalization, and antigen presentation through membrane immunoçlobulin in BAL17 B lymphoma cells. IXBS Le!/. 323, 171-174

80. Pure E. and Tardelle L. (1992) Tyrosine phosphorylation is required for ligand-induced internalization of the antigen receptor on B lymphocytes. Proc. Nah. Acnd. Sci. U.S.A. 89, 114-1 17

81. Stryer L. (1988) Microfilaments, intermediate filaments, and microtubles form dynamic cytoskeletons. In Biochenistry, p. 938. Freeman and Compariy, New York.

82. Luna E. J. and Hitt A. (1992) Cytoskeleton-plasma membrane interactions. Sciet~ce 258, 955-964

83. Palek J. and Hanspal M. (1992) Biogenesis of normal and abnormal red blood cell membrane skeleton. Sen~itrars i t ~ Heninfology 29, 305-3 19

84. Darnell J., Lodish H. and Baltimore D. (1990) Actin, myocin, and intermediate filaments. In Moleculor Cd1 Biology, p. 859. Freeman and Company, New York.

85. Woda B. A. and Woodin M. B. (1984) The interaction of lymphocyte membrane proteins with the lymphocyte cytoskeletal matrix. J. Immirn. 133, 2767-2772

86. Braun J., Hochman P. S. and Unanue E. R. (1982) Ligand-induced association of surface immunoglobulin with the detersent-insoluble cytoskeletal matrix of the B lymphocyte. ./. Itnmwl. 128, 1 198-1 204

87. Albrecht D. L. and Noelle R. J. (1988) Membrane Ig-cytoskeletal interactions. 1. Flow cytofluorometric and biochemical analysis of membrane IgM-cytoskeletal interactions. ./. Irnmw. 141, 3915-3922

88. Woda B. A. and McFadden M. L. (1983) Ligand-induced association of rat lymphocyte membrane proteins with the detergent-insoluble lymphocyte cytoskeletal matrix. J. Inimut?. 131, 1917-1919

89. Melamed I., Downey G. P., Aktories K. and Roifman C. M. (1991) Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. ./. ltnn~~rn. 147, 1 139- 1 146

90. Braun J., Fujiwara K., Pollard T. D. and Unanue E. R. (1978) Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. ./. Cd1 Biol. 79, 409-41 8

91. Lanzavecchia Antonio (1985) Antigen-specific interaction between T and B cells. Noiure 314, 537-538

92. Parsey M. and Lewis G. K. (1993) Actin polymerization and pseudopod reorganization accompany anti-CD3-induced growth arrest in Jurkat T cells. ./. Inlmlrt~. 15 1, 1881- 1893

93. DeBell K. E., Conti A., Alava M. A., Hoffman T. and Bonvini E. (1992) Microfilament assembly modulates phospholipase C-mediated signal transduction by the TCWCD3 in murine T helper lymphocytes. .J. It~?ntrrrr. 149, 2271-2280

94. Valitutti S., Dessing M., Aktories K., Gallati H. and Lanzavecchia A. (1995) Sustained signaling Ieading to T ce11 activation results from prolonged T ce11 receptor occupancy. Role of T ceIl actin cytoskeleton. .J. Exp. M d 181, 577-584

95. Fox J. E. B., Lipfert L., Clark E. A., Reynolds C. C., Austin C. D. and Bmgge J. S. (1993) On the role of the platelet membrane skeleton in mediatinç signal transduction. ./. Biol. Chem. 268,25973-25984

96. Zhang J., Fry M. J., Waterfreld M. D., Jaken S., Liao L., Fox J. E. B. and Rittenhouse S. E. (1992) Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. .l. Biol. Chent. 267, 4686-4692

97. Tohyama Y., Yanagi S., Sada K. and Yamamura H. (1994) Translocation of p 7 2 ~ ~ k to the cytoskeleton in thrombin-stimulated platelets. ./. Biol. Chem. 269, 32796-32799

98. Grondin P., Plantavid M., Sultan C., Breton M., Mauco G. and Chap H. (1991) Interaction of pp60C-srC, phospholipase C, inositol-lipid, and diacyglycerol kinases with the cytoskeletons of thrombin-stimulated platelets. ,/. Biol. Chem 266, 15705-1 5709

99. Li R. Y., Gaits F., Raçab A., Ragab-Thomas J. M. F. and Chap H. (1994) Translocation of an SH2-containing protein tyrosine phosphatase (SH-PTP1) to the cytoskeleton of thrombin-activated platelets. FEBS Leu. 343, 89-93

100. Payrastre B., van Gergen en Henegouwen P. M. P., Breton M., den Hartigh J. C., Plantavid M., Verkleij A. J. and Boonstra J. (1991) Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor. ./. Ce/! Biol. 1 15, 12 1 - 128

101. Weernink P. A. 0. and Rifksen G. (1995) Activation and translocation of c-Src to the cytoskeleton by both platelet-derived growth factor and epidermal growth factor. ./. Biol. Chen]. 270, 2264-2267

102. Hamaguchi M. and Ganafusa H. (1987) Association of p60~v1'c with triton X-100- resistant cellular structure correlates with inorphological transformation. Proc. N L I ~ I . Acad S'ci. [/.S.A. 84(8):23 12-6

103. Rozdzial M., Malissen B. and Finkel T. (1995) Tyrosine-phosphorylated T ceII receptor chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. /rmii~i i ty 3, 623-633

104. Gregorio C. C., Repasky E. A. Fowler V. M. and Black J. D. (1994) Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase CP. .J. CeII Bioi. 125, 345-358

105. Prekeris R., Mayhew M. W., Cooper B. and Terrian D. (1996) Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the reçulation of synaptic function. ,/. Ce11 Biol. 132, 77-90

106. Goroff D. K., Stall A., Mond J. J. and Finkelman F. D. (1986) IH vilro and i t ~ vivo B lymphocyte-activating properties of monoclonal anti-6 antibodies. J. I m m m 136, 2382- 2392

107. Hartwig J. H. and Shevlin P. (1986) The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages. J. (311. Biol. 103, 1007- 1020

108. Klein D. P., Galea S. and Jongstra J. (1990) The lymphocyte-specific protein LSPl is associated with the cytoskeleton and CO-caps with membrane IgM. .J. I m w i . 145, 2967- 2973

109. Blikstad I., Markey F., Carlsson L., Persson T. and Lindberg U. (1978) Selective assay of nomomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease 1. CeIl 15, 935-943

110. Shaw A. C., Mitchell R. N., Weaver Y. K., Campos-Torres J., Abbas A. K. and Leder P. (1990) Mutations of immunoglobulin transmembrane and cytoplasmic domains: effects on intracellular signaling and antigen presentation. CeIl 63, 361-392

111. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Analysis of recombinant DNA clones, In Molecrilar Clot~irlg n Lahoraiory Matlual, p. 368. Cold Spring Harbor Laboratory, New York.

112. Laemrnli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriaphage T4. Ndrrre 227, 680-685

113. Jongstra-Bilen J., Janmey P. A., Hartwig J. H., Galea S. and Jongstra J. (1992) The lymphocyte-specific protein LSPl binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain. ./. Ckll Biol. 118, 1443-1453

114. Den Hartigh J. C., Van Bergen en Henegouwen P. M. P., Verkleii A. J. and Boonstra J. (1992) The EGF receptor is an actin-binding protein. ,/. CPIlBiol. 119, 349-355

115. Janrney P. A. and Matsudaira P. T. (1988) Functional comparison of Villin and Gelsolin. ./. 13iol. Clhem. 263, 16738- 16743

116. Barber B. H. and Delovitch T. L. (1979) The identification of actin as a major lymphocyte component. .J. lnttnttri. 122, 320-325

117. Gupta S. K. and Woda B. A. (1988) Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve a-actinin. ./. Inlmiin. 140, 176-182

118. Itin C., Kappeler F., Linstedt A. D. and Hauri H. P. (1995) A novel endocytosis signal related to the KKXX ER-retrieval signal. EhBO .f. 14, 2250-2256

119. Chen W., Goldstein J. L. and Brown M. S. (1990) NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. ./. Bioi. Cheni. 265,3 1 16-3 123

120. Pavalko F. M., Walker D. M., Graham L., Goheen M., Doerschuk C. and Kansas G. S. (1995) The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via a- actininxeceptor positioning in microvilli does not require interaction with a-actinin. ./. C d Bioi. 129, 1155-1 164

121. Pavalko F. M. and LaRoche S. (1993) Activation of human neutrophils induces an interaction between the integrin P2-subunit (CD18) and the actin binding protein a-actinin. ,/. In~mrin. 151, 3795-3807

122. Carpen O., Pallai P., Staunton D. E. and Sprinçer T. A. (1992) Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and a- actinin. ./. CeIl Biol. 118, 1223- 1234

123. Otey C. A., Vasquez G. B., Burridge K. and Erickson B. W. (1993) Mapping of a- actinin binding site within the P l integrin cytoplasmic domain. ./. Bioi. Chem. 268, 21 193- 21 197

124. Mitchell R. N., Shaw A. C., Weaver Y. K., Leder P. and Abbas A. K. (1991) Cytoplasmic tail deletion coverts membrane immunoglobulin to a phosphatidylinositol- linked form lacking signaling and efficient antigen internalization functions. ./. Biol. Chem. 266, 8856-8860

125. Pleiman C. M., Chien N. C. and Cambier J. C. (1994) Point mutations define a mIgM transmembrane region motif that determines intersubunit signal transduction in the antigen receptor. .J. 11nmrrn. 152, 2837-2844

126. Klein P., Kanehisa M. and DeLisi C. (1985) The detection and classification of membrane-spanning proteins. Biochi~n. Biophys. Acta 815, 468-476

127. Noelle R. and Snow E. C. (1990) Cognate interactions between helper T cells and B cells. lnlm. kday 1, 361-368

128. Mitchell R. N., Barnes K. A., Grupp S. A., Sanchez M., MisuIovin Z., Nussenzweig M. C. and Abbas A. K. (1995) Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes. .J. L;Lx-p. Me8 181, 1705-1714

129. Heiska L., Kantor C., Parr T., Critchley D. R., Vilja P., Gahmberg C. G. and Carpen 0. (1996) Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM- 2) a-actinin. .J. Biol. Chem. 42, 26214-26219

130. Iida N., Lokeshwar V. B. and Bourguignon L. Y. W. (1994) Mapping the fodrin binding domain in CD45, a leukocyte membrane-associated tyrosine phosphatase. J. Biol. Chem. 269,28576-28583

131. Flanasan J. And Koch G. L. E. (1978) Cross-linked Ig attaches to actin. Ndiim 273, 278-28 1

132. Petrini M., Emerson D. L. And Galbraith R. M. (1983) Linkage between surface immunoglobulin and cytoskelton of B lymphocytes may involve Gc protein. Nuhrre 306, 73-74

133. Esteban C., Geuskens M., Ena J. M., Mishal Z., Macho A., Torres J. M. And Urie1 J. (1992) Receptor-mediated uptake and processing of vitamin D-binding protein in human B- lymphoid cells, ./. Biol. Chenr. 267, 10177-10183

134. Lokeshwar V. B., Gregien N. and Bourguignon Y. W. (1994) Ankyrin-binding domain of CD44(GPS5) is required for the expression of hyaluronin acid-mediated adhesion function, .J. CeII Biol. 126, 1099- 1 109

135. Jordan C., Buschel B., Koob R. and Drenckhahn D. (1995) identification of a bindiny motif for ankyrin on the a-subunit of Na',KS-ATPase. .J. Biol. Chem. 270,29971-29975

136. Suzuki R., Nishi N., Tokura S. and Morita F. (1987) F-actin-binding synthetic heptapeptide having the amino acid sequence around the SH1 cysteinyl residue of myosin. .J. LM. Cheni. 262, 1 141 0-1 1412

137. Jugloff L. S. and Jongstra-Bilen J. Cross-linking of the IgM receptor induces rapid translocation of IgM-associated Iga, Lyn and Syk tyrosine kinases to the membrane skeleton. .J. Itnn111r1. In Press

137a. Gold M. R., Matsuuchi L., Kelly R. B. and DeFranco A. L. (1991) Tyrosine phosphorylation of components of the B-ce11 antigen receptors following receptor crosslinking. Proc. Nultr. Acnd Sci. ü.S.A. 88, 3436-3440

138. Takagi S., Daibata M., Last T. J., Huinphreys R. E., Parker D. C. and Sairenji T. (1991) Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoçlobulins in B cells. .J. Er-. n/kd 174, 381-388

139. Graziadei L., Raibowol K. and Bar-Sagi D. (1990) Co-capping of ras proteins with surface immunoglobulins in B lymphocytes. Nnllirc. 347, 396-400

140. Bar-Sagi D., Rotin D., Batzer A. and Mandiyan V. (1993) SH3 domains direct cellular localization of signaling molecules. C'dl 74, 83-9 I

141. Goldschimidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G. and Pollard T. D. (1991) Regdation of phospholipase C-yl by profilin and tyrosine phosphorylation. Sciewe 251,1231-3

142. Lassing 1. and Lindber U. (1985) Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin. Nnfure 314, 472-474

143. Yu F. X., Sun H. Q., Janmey P. A. and Yin H. L. (1992) Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin. J. Biol. Chent. 267, 1 46 1 6- 1462 1

144. Janmey P. A. and Stossel T. P. (1987) Modulation of gelsolin function by phosphatidylinositol4,5-bisphosphate. N d w e 325, 362-364

145. StosseI T. P. (1 989) From signal to pseudopod. .J. Biol. C'hem. 264, 18261-1 8264

146. Ito M., Tanabe F., Sato A., Ishida E., Takami Y. and Shigeta S. (1989) Possible involvement of microfilaments in protein kinase C translocation. Biochem. Biophys. Res. Ckotnnt. 1 60, 1 344- 1 349

Mouse IgM :

MutA :

Linker TransMembrane Cytoplasmic

m : polar amino acid :mutated amino acid

Figure 1. a) A partial amino acid sequence of mutant IpM molecules. The C-terminal ends of the linker regions are shown, together with the transmembrane and cytoplasmic domains. For the Y5W": W-Iga molecule, 60 amino acid residues of the cytoplasmic tail of Ig-a is fused to the TM domain of Y5uS5?VV. The ITAM motif of the Ig-a tail is shown within the indicated box. The numbers indicates the position of the mIgM heavy c h a h arnino acid residues.

MutA

GPT AMP

R1 B c

promoters E : Ig heavy chain enhmcer

Ig-a cytoplasmic sequences S :switch region

Ig sequences

Figure 2. b) Structure of YS:VV, YS:VV-Iga (p523IgM:Iga), and MutA plasmids. In the YS:VV plasmid, genomic DNA encoding the human Ig heavy chain molecule with Ig heavy chain promoter and enhancer is used. In the YS:VV-Iga plasmid, spleen focus-forming virus (SFFV) LTR is used as promoters for both Y5X7S5XX:VV-Ig~ and K light chain molecules. Genomic DNA encoding the human Ig heavy and light chain molecules was used. (The nature of the enhancer, its position and that of the switch region is uncertain.) In the MutA plasmid, P-globin and cytomegalovirus promoters are used for mouse IgM heavy chain and mouse hl light chain molecules, respectively. For hl light chain, cDNA was used. For the Ig heavy chain, modified genomic DNA with the deletions of the switch region and the intron between VDJp and Cpl is used. (The position and nature of the enhancer is uncertain.) The plasmids are not drawn to scale. Approximate Iength of each plasmids are 16, 18, and 14 kilobases for YS:VV, YS:VV-Iga and MutA, respectively. L- VDJp corresponds to rearranged IgM heavy chain variable region; Cpl-4, IgM heavy chain constant region exons; M, IgM Iieavy chain membrane exons; E, IgH enhancer; S, switch region; L-VJK and L-VJX, K and h light chain variable regions, respectively; CK and Ch, K and h light chain constant regions, respectively; NE0 is neomycin resistance gene; AMP is ampicillin resistance gene; GPT is guanine phosphoribosyltransferase gene.

Figure 2

a) FACS Analysis

Hela

Relative Fluorescence Int ensiiy

anti-IgM

HeLa H6 HeLa - -5i-C

H6 -CII -

1 2 3 4 5 6 7 8

streptavidin

c)

gelsolin : anti-p :

tive : (min.)

rn.w. 101 - (KDa)

82 -

90- " .., .,. ,. , ., i

dm- , ,

Figure 2. a) The MutA transfectant HeLa cell line H6 and the parental HeLa cells were stained with IOpg/ml biotinylated anti-mouse IgM antibodies followed by streptavidin-PE (open curves) or by streptavidin-PE alone (shaded curves) and analyzed by FACScan. b) Immunoprecipitates from NP-40 lysates of biotinylated HeLa cells (parental) and H6 transfectants (2x10~ ceIl eq.) using anti-IgM (lanes 1, 3, 5, and 7, 1:200 dilution) and non- immune sera (lanes 2,4,6,8,) were analysed by Western blotting for the presence of the IgM protein (lanes 1-4), using GAM-IgM (1:2,000 dilution) followed by HRP conjugated rabbit anti-goat IgG (1 : 10,000 dilution), and for biotinylated proteins (5-8), using HRP conjugated streptavidin (1:5,000 dilution). c) Western blot analysis of the detergent insoluble pellets of HG transfectants from the cells incubated with(+) or without(-) the F(ab')2 fragments of purified GAM-IgM (30pg/ml), and with(+) or without(-) the gelsolin treatment, for the indicated times at 37OC. To quantitate the amount of mIgM associated with the cytoskeleton, a fixed amount of pellet (7x10~ ce11 eq.) was loaded on the gels (lanes 1 to 9) together with the total samples representing a range of known cell equivalents (7x10~ ceIl eq. as 100%) to generate a standard curve (lanes 10 to 13). Western blotting was performed using GAM-IgM (upper panel), as described above, or mouse anti-rabbit actin (lower panel, 11300 dilution), followed by HRP conjugated goat anti-mouse IgG (1:5,000 dilution). Notice the faint yet detectable level of F-actin in the samples treated with gelsolin (lanes 6- 8, lower panel).

Table 1.

% of mIgM Bindinp in H6

ntd, not deteçtable, below detection level.

T h e (min.)

anti-IgM AVG.

n = number of experimcnts f = S.D.

Effect of Gelsolin on ~ I P M of H6

% of mIgM in the cytoskeletal matrix 2

+ 1 8 t5 (n = 3)

n = nuinber of experimcnts *, (100% - % oof IgM in thc cytoskclctitl rnatrix) = % IgM rclcoscd I = S.D.

Time ( iiiin.)

Gelsolin AVG.

?

Table 1. The level of mIgM accumulation in the cytoskeleton of HeLa transfectant H6. The summary of the MutA-cytoskeletal interactions (a), and the effect of geisolin on the MutA-cytoskeletal interactions (b), in HG transfectants. ii) The binding assay was performed as described in the Materials and Methods (section 5) for the indicated time period. b) The amounts of MutA in the detergent insoluble pellet with(+) or without(-) the gelsolin treatment at 2 and 5 minutes. The gelsolin treatment was performed as described in the Materials and Methods (section 5)

5

+ 32 t5 (II = 4)

10

+ 3 1 rC8 (n = 3)

% of mIgM in the cytoskeletal matrix

10

- n/d

% of mIgM released* 2

38 12 (n = 2)

2 5

56 k3 (n = 2)

1 6 f 3 (il = 2)

5

+ 10 I I ( n=2)

-

29 14 (n = 2)

+ 1 3 s (n = 2)

Y f l 316

r n s

m 1838

YH 32

M 46

YSIW 52

YS: W 5

YSiW 18

YSIW 43

YS:W 26

WIW 47

Figure 3. a) FACS analysis of the J558L transfectants. YMs and YS:VVs correspond to the transfectants expressing Y5uSSm:VV-Iga and YsS7SSSa:VV molecules: respectively. Cells were incubated with or without (background) 3prr/ml of FITC conjugated anti-hurnan 1-M.

1 1 2 3 4 J C s o J anti-igM anti- Igû!

Figure 3. b) Western blot analysis of total ce11 lysates from YM3 16 J558L transfectants (lanes 1 to 3), untransfected parental J558L cells (lane 4 and 5), WEHI231 mouse B- lymphoma cells (lane 6), and J558L IgM+Mb-I+ transfectants (lane 7; see Materials and Methods section 1). 3x105, 1x1 05, and 0.5~105 cell eq. of YM3 16 were used for lanes 1, 2, and 3, respectively. 3x105 ceIl eq. of untransfected parental J558L cells was used for lane 4. 4x1 05, l x l ~ j , and 10x105 cell eq. of corresponding total lysates (see above) were used for lanes 5, 6, and 7, respectively. Samples were separated by SDS-10% PAGE (lanes 1 to 4) or SDS-12% PAGE (lanes 5 to 7). As expected no IgM or Ig-a bands can be seen in parental untransfected J558L cells (lanes 4 and 5). 31 to 34KDa Ig-ci bands can be seen from the total samples of Ig-a' ce11 lines (lanes 6 and 7). The blots were developed using GAH-IgM (left panel, 1:500 dilution), followed by I-IRP conjugated rabbit anti-goat IgG ( 1 :20,000 dilution), or rabbit anti-mouse Iga (left panel, 12,000 dilution), followed by HRP conjuçated donkey anti-rabbit IgG (1 :5,000 dilution).

anti-lgM +

time : 2 (min.)

anti-lgM : + + + + + + - Total% time : 2 5 10 5 5 5 10 '100 60 30 l 5 8 (min.)

anti-lgM : + - ,--Total% 7 anti-lgM : + - ,-Total% time : 10 10 40 30 20 time :fO (min (min.) 10 40 30

101- mw.

(KDa) 82- rn

Figure 3. c) and d) Western blot analysis of the detergent insoluble pellets of YM32(c) and YS:VV47(d) from the cells incubated with(+) or without(-) the F(ab')2 fragments of anti- human IgM for the indicated period of time at 37OC. To quantitate the amount of mIgM associated with the cytoskeleton, a fixed amount of pellet (2x10~ cell eq.) were analyzed together with the total samples representing a range of known cell equivalents (2x105 cell eq. as 100%). The blots were developed using rabbit anti-mouse Igcl (c, upper panel, 1:2,000 dilution), followed by HRP conjugated donkey anti-rabbit IgG (1:25,000 dilution), or GAH-IgM (e, upper panel, 1:1,000 dilution), followed by HRP conjugated rabbit anti- çoat IgG (1 :20,000 dilution), and mouse anti-rabbit actin (lower panels) as described before (Fig. 2c). For YM32, 30pglml of anti-human IgM F(ab'l2 were used for stimulation. For YS:VV47, 10pç/rnl (lane 4), 30pg/ml (lanes 1-3), 6Opg/inl (lane S), and 1 0 0 @ n l (lane 6) of anti-human IgM F(ab')2 were used for stimulation. e) YSX7SSXX:VV-Iga molecules in the detergent insoluble pellets are tyrosine phosphorylated. Western blot analysis of YM32 was performed with GAH-IgM (left panel) as described above. The same blot was re-probed (see Material and Methods section 5) with 4G10 mouse anti-phosphotyrosine (right panel, 1 : 1,000 dilution) followed by HRP conjugated goat anti-mouse IgG (1:5,000 dilution). The upper 93KDa IgM band from stimulated cells (lane 1) is clearly tyrosine phosphorylated (lane 6). 2 . 5 ~ 1 0 ~ ce11 eq. (lanes 1 and 2) were analyzed together with titrated amounts of the total cell extract samples having 2 . 5 ~ 1 0 ~ cell eq. as 100% (lanes 3 to 5). 30pg/ml of anti-human IgM F(abt)2 was used for stimulation.

Table 2.

4 - Y"@~YVV cvtoskeleton interactions and actin percenta~es

1 the cytoskeletal

(n=2)

AVG. 41 ki8

% Actin in the % Y5S7S5":VV in % Actin in the cytoskeletal the cytoskeletal cytoskeletal

matrix matrix matrix

Y S H ~ ~ : V V - I P C ~ cytoskeleton interactions and actin percentayes -

Time ( min. ) YM 906 F

1 AVG.

matrix matrix

% Y507S5?VV-Iga in the cytoskeletal

matrix 2

12 +i (t1=2)

1 1 f5 (n=3)

11 k6 (n=3)

26 M (n=2)

69 rt6 (11=2)

26 rt25

% Actin in the cytoskeletal

matrix 2

7 *l (n=3 )

11 f3 ( 11=3 )

4 (n=2 )

22 356 ( n = 2 )

15 rti ( n=2 )

12 f 7

18 1 4 (n=3)

15 +5 (n=3)

42 +i (n-2)

98 +2

12 17 ( ii=3 )

7 rtl ( n=2 )

17 +4 ( n = 2 )

20 12

C) The ratio of Y ~ K ~ ~ Y V V over F-actin

Time f min. 1 1 2 1 1 O 1

The ratio of Y S X ~ S ~ Y V V - I ~ over F-actin

AVG. 1 4.3 G . 6

Table 2. The Ievels of mIgM accumulation in the cytoskeleton of the J558L transfectants stimulated with 30pgIml of F(ab')2 anti-human IgM at 37OC for the indicated time periods. The summary of % mIgM accumulation in the detergent insoluble pellets for YS:VV (a), and YM transfectant lines (b). The summary of the extent of mIgM accumulation in the detergent insoluble pellets normalized to F-actin values in YS:VV (c), and YM transfectant lines (d). Individual experimental values, not the average values, are used to generate the ratios.

5.9 9 . 7 I

The Ratio of mIpM over F-actin

rnYS:W

f3 Chimera U 7

2 minutes p = 0.1

10 minutes p = O.W*

* = Statistically Significant

Figure 4. The ratio of mIgh4 over F-actin at 2 and 10 minutes. A Significant difference exists for the extent of the mIgM-cytoskeletal interactions between YSR7S5Q:W and Y5WSS:VV-Iga molecules at 10 minutes. The two tailed Mann-Whitney nonparametric (unpaired) t test was performed to obtain the p values.

anti-lgM: + + + t - t-'Total%-, tive: 2 10 2 10 10 30 15 8 3 (min.)

Figure 5. a) Western blot analysis of the detergent insoluble pellet of YM316 from the cells treated with(+) or without(-) 30pgIml of F(ab');! fragments of anti-human IgM and with(+) or without(-) gelsolin treatment, for the indicated time periods at 37OC. 4x10~ cell eq. were used for lanes 3 to 5 , together with titrated amount of the total ce11 extract samples having 4x 105 cell eq. as 100%. 2x 105 ce11 eq. were used for lanes 1 and 2. The blots were developed using goat anti-mouse Igu (upper panel) or mouse anti-rabbit actin (lower panel), as described before (Fig 3). b) Determination of F-actin remaining after the gelsolin treatment. 1 . 2 ~ 1 0 ~ (100%) cell eq. of the detergent insoluble pellet of the gelsolin treated sample (lane 6) was compared with 3x 105 (lane 1, 25%), 1 .5xlo5 (lane 2, 13%), 7 . 5 ~ 10"lane 3, 6%), 4x 104 (lane 4, 3%), and 2x 1 o4 (lane 5, 1.5%) cell eq. of the detergent insoluble pellets from the samples which were not gelsolin treated. The blot was developed using mouse anti-rabbit actin as described before (Fig. 2c).

Table 3.

a) The effects of Gelsolin on YMs

The effect of Gelsolin on YS:VVs

Gelsolin AVG.

YS:VVs % of YY3mnx:VV iii 1 '%) of YW7SMN:VV 1 %) F-actin

YMs

N = iiiiinbcr ol'cxpcriinciiis

f = S.D.

Gelsolin AVG.

Table 3. The mIgM accumulation in the cytoskeletal pellets is due to the interactions between mIgM and F-actin filaments. The effects of gelsolin on YS:VVs (a), and YMs (b), after 10 minutes of anti-IgM stimulation. For YMs, YM3 16, YM906, and YM32 were used to obtain the data. For YS:VVs, YS:VV43 was used.

'%, of Y-sx7Sw:VV-lga in the cytoskeleton

'%) of Y"7Ssn:VV-Iga relessed*

54 *i3 (n=3)

- 24 hi i

(n=3)

% F-actin released#

86 *G (n-2)

4-

12 *8 (ii=3)

the cytoskeleton released*

54 19 (N=3)

- 64 rt3 (N=3)

releasedM

92 rts (N=3)

+ 30 i7 (N=3)

APPLIED IMAGE. lnc 1 1653 East Main Street - A. - Rochester, NY 14609 USA -- -- -, Phone: 7161482-0300 -- --

A - Fax: 71 61288-5989

O 1993. Applied Image. Inc.. All Righls Aeserved