Linda Matsuuchi

Gap junction proteins on the move: connexins, the cytoskeleton and migration.

Connexin43 (Cx43) has roles in cell-cell communication as well as channel independent roles in regulating motility and migration. Loss of function approaches to decrease Cx43 protein levels in neural cells result in reduced migration of neurons during cortical development in mice and impaired glioma tumor cell migration. In other cell types, correlations between Cx43 expression and cell morphology, adhesion, motility and migration have been noted. In this review we will discuss the common themes that have been revealed by a detailed comparison of the published results of neuronal cells with that of other cell types. In brief, these comparisons clearly show differences in the stability and directionality of protrusions, polarity of movement, and migration, depending on whether a) residual Cx43 levels remain after siRNA or shRNA knockdown, b) Cx43 protein levels are not detectable as in cells from Cx43(-/-) knockout mice or in cells that normally have no endogenous Cx43 expression, c) gain-of-function approaches are used to express Cx43 in cells that have no endogenous Cx43 and, d) Cx43 is over-expressed in cells that already have low endogenous Cx43 protein levels. What is clear from our comparisons is that Cx43 expression influences the adhesiveness of cells and the directionality of cellular processes. These observations are discussed in light of the ability of cells to rearrange their cytoskeleton and move in an organized manner. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

New views of BCR structure and organization

Recent work has provided new insights into the stoichiometry of BCR subunits, as well as the organization of the BCR before and after engagement by antigen. On resting cells, the BCR may be pre-assembled into oligomeric receptor complexes that generate a basal level of signaling. After antigen binding, the BCR may be organized into larger receptor arrays that reside in lipid rafts - sites where signaling enzymes are concentrated. The critical role of BCR assembly and organization in B cell function is underscored by the recent findings that this process is altered in many B cell tumors.

The Gab1 Docking Protein Links the B Cell Antigen Receptor to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and to the SHP2 Tyrosine Phosphatase

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.

Signal Transduction by the B‐Cell Antigen Receptor

The antigen receptor of B lymphocytes (BCR) plays important roles in recognition of foreign antigens and self-components to allow the immune system to make appropriate antibody responses. The BCR is a complex between membrane immunoglobulin and the Ig-alpha and Ig-beta heterodimer. Site-directed mutagenesis experiments have shown that the mu heavy chain transmembrane domain plays a key role in the association of mIgM with Ig-alpha/Ig-beta. In the absence of complex formation, mIgM is retained in the endoplasmic reticulum, and this function is also specified by the mu chain transmembrane domain. The ability of various mutant mIgM molecules to associate with Ig-alpha/Ig-beta correlates well with their ability to induce signal transduction reactions such as protein tyrosine phosphorylation and phosphoinositide breakdown. Thus, the signaling ability of the BCR appears to reside in the Ig-alpha/Ig-beta heterodimer. The cytoplasmic domains of Ig-alpha and Ig-beta each contain an ITAM sequence, which is defined by its limited homology with subunits of the T-cell antigen receptor and of Fc receptors. Moreover, chimeric proteins containing these ITAMs and surrounding sequences from the cytoplasmic domains of Ig-alpha or Ig-beta exhibit signaling function characteristics of the intact BCR. The Ig-alpha and Ig-beta chimeras are each capable of inducing all of the BCR signaling events tested and thus represent redundant functions. Cross-linking these chimeras leads to their phosphorylation and to binding of the intracellular tyrosine kinases Lyn and Syk. The BCR expressed in the nonlymphoid AtT20 cells, which express the Src-family tyrosine kinase Fyn but not Syk, was not able to trigger vigorous signaling reactions. Introduction of the active form of Syk into these cells restored some signaling events. These results are consistent with a model in which the ITAMs act to initiate the BCR signaling reactions by binding and activating tyrosine kinases.

The gap junction protein Cx43 regulates B-lymphocyte spreading and adhesion

The gap junction protein connexin43 (Cx43) is widely expressed in mammalian cells and forms intercellular channels for the transfer of small molecules between adjacent cells, as well as hemichannels that mediate bidirectional transport of molecules between the cell and the surrounding environment. Cx43 regulates cell adhesion and migration in neurons and glioma cells, and we now show that Cx43 influences BCR-, LFA-1- and CXCL12-mediated activation of the Rap1 GTPase. Using shRNA knockdown of Cx43 in WEHI 231 cells, we show that Cx43 is required for sustained Rap1 activation and BCR-mediated spreading. To determine the domains of Cx43 that are important for this effect, Cx43-null J558 μm3 B cells (which express a wild-type IgM BCR) were transfected with wild-type Cx43–GFP or a C-terminal-truncated Cx43 (Cx43ΔT–GFP). Expression of wild-type Cx43–GFP, but not Cx43ΔT–GFP, was sufficient to restore sustained, BCR-mediated Rap1 activation and cell spreading. Cx43, and specifically the C-terminal domain, was also important for LFA-1- and CXCL12-mediated Rap1 activation, spreading and adhesion to an endothelial cell monolayer. These data show that Cx43 has an important and previously unreported role in B-cell processes that are essential to normal B-cell development and immune responses.

Signal transduction by the antigen receptors of B and T lymphocytes.

B and T lymphocytes of the immune system recognize and destroy invading microorganisms but are tolerant to the cells and tissues of ones own body. The basis for this self/non-self-discrimination is the clonal nature of the B and T cell antigen receptors. Each lymphocyte has antigen receptors with a single unique antigen specificity. Multiple mechanisms ensure that self-reactive lymphocytes are eliminated or silenced whereas lymphocytes directed against foreign antigens are activated only when the appropriate antigen is present. The key element in these processes is the ability of the antigen receptors to transmit signals to the interior of the lymphocyte when they bind the antigen for which they are specific. Whether these signals lead to activation, tolerance, or cell death is dependent on the maturation state of the lymphocytes as well as on signals from other receptors. We review the role of antigen receptor signaling in the development and activation of B and T lymphocytes and also describe the biochemical signaling mechanisms employed by these receptors. In addition, we discuss how signal transduction pathways activated by the antigen receptors may alter gene expression, regulate the cell cycle, and induce or prevent programmed cell death.

Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas

Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igalpha and Igbeta of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igalpha expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).

Association of B lymphocyte antigen receptor polypeptides with multiple chaperone proteins.

The B cell antigen receptor (BCR) is comprised of four different polypeptides, immunoglobulin (Ig) heavy chain, Ig light chain, and the two signaling subunits of this receptor, Ig-alpha and Ig-beta. These four chains must assemble correctly in the endoplasmic reticulum (ER) before the BCR can be transported to the cell surface. The roles of the different chaperone proteins in mediating the assembly of mIg with the Ig-alpha/beta are not fully understood. To gain insights into the roles of chaperone proteins in BCR assembly, we have generated transfected non-lymphoid cell lines that express various intermediate assembled forms of the BCR and used them to examine the interactions of chaperone proteins with subunits of the BCR. We examined the interactions of BiP (GRP78), GRP94 and calnexin with the mu heavy chain, lambda light chain, Ig-alpha and Ig-beta. We report for the first time that Ig-alpha associates with GRP94 and that this interaction increases dramatically when other BCR chains are co-expressed. In contrast, the mu heavy chain interacts strongly with BiP (GRP78) when expressed by itself but this interaction is reduced when the lambda light chain is expressed, with the resulting mu(lambda) complexes interacting with GRP94 and calnexin. Thus, our data are consistent with the idea that there is an ordered association of BCR components with different protein chaperones during BCR assembly.

The role of the gap junction protein connexin43 in B lymphocyte motility and migration.

The gap junction family of proteins is widely expressed in mammalian cells and form intercellular channels between adjacent cells, as well as hemichannels, for transport of molecules between the cell and the surrounding environment. In addition, gap junction proteins have recently been implicated as important for the regulation of cell adhesion and migration in a variety of cell types. The gap junction protein connexin43 (Cx43) regulates B lymphocyte adhesion, BCR‐ and LFA‐1‐mediated activation of the GTPase Rap1, and cytoskeletal rearrangements resulting in changes to cell shape and membrane spreading. We demonstrate here that the actin cytoskeleton is important for the distribution of Cx43 in the B cell plasma membrane and for other cell processes involving the cytoskeleton. Using shRNA knockdown of Cx43 in B lymphoma cells we show that Cx43 is also necessary for chemokine‐mediated Rap 1 activation, motility, CXCL12‐directed migration, and movement across an endothelial cell monolayer. These results demonstrate that in addition to its role in B cell spreading, Cx43 is an important regulator of B‐cell motility and migration, processes essential for normal B‐cell development and immune responses.

The role of Ig-α/β in B cell antigen receptor internalization.

The B cell antigen receptor (BCR) is expressed on the surface of B lymphocytes where it can bind antigen then transmit signals which regulate activation, growth, and differentiation. These signals can induce a number of cytoskeletal rearrangements leading to dynamic cellular processes including internalization of the bound antigen which is then processed and presented to T cells on MHC II. The relative importance of regions within the Igα and Igβ cytoplasmic domains has been well studied in terms of signaling but their roles in BCR internalization and trafficking are less clear. We hypothesize that the Igα and Igβ cytoplasmic domains is important for normal internalization and trafficking of the 4 chain BCR. An Igα and Igβ deficient murine lymphoid cell line was used to express mIgM along with a panel of Igα and Igβ mutants in order to compare their internalization and subcellular localization. Here we show that the Igα and Igβ cytoplasmic domains are each sufficient for internalization, though Igα is dominant in this process. We also show that the internalization signal is contained in a region past the first cytoplasmic tyrosine residue of Igα and Igβ, Y176 and Y195 respectively. We also show that a 4 amino acid motif normally contained within the Igα ITAM is sufficient to rescue aberrant internalization. In terms of receptor trafficking, each cytoplasmic domain is sufficient for trafficking to lysosomal compartments but that a normal rate of trafficking likely requires the tandem effects of both Igα and Igβ.