GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold–induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Gαi2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein–protein and lipid–lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP3 production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Gαi2. Because both Gαi2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Gαi2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster

DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)–dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation

The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production

A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab′)2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2–Rac axis in PC differentiation and IgG antibody responses

Presentation_1.PDF

<p>A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab′)₂ antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2–Rac axis in PC differentiation and IgG antibody responses.</p

Lobe A-mediated DOCK2 dimerization is required to activate Rac effectively during cell migration.

<p>(A, B) FRET analyses for Rac activation in BW5147α^–β^– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A or Δlobe A). Cells were retrovirally transduced with Raichu-Rac, and were loaded on stromal cells prepared from the lymph nodes. After 4 hours of incubation, images were taken every 30 seconds, and the emission ratio of 527 nm/475 nm (FRET/CFP ratio) was used to represent the FRET efficiency. The FRET/CFP ratios at the plasma membrane were normalized by dividing by the lowest value in the cells, and cells were judged FRET-positive when the ratio was above 1.6. Data indicate the percentage of FRET-positive cells (B) with representative images (A). For each category of cells, 12–19 cells were analyzed. Scale bar, 5 µm. (C) The association of DOCK2 with Rac in BW5147α^–β^– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A or Δlobe A). Cell extracts were incubated with anti-HA affinity matrix in the presence of 10 mM EDTA, and bound Rac was detected with anti-Rac1 antibody.</p

DOCK2 forms homodimer via lobe A of DHR-2 domain in cells.

<p>(A) Following expression of FLAG-tagged WT or mutant DOCK2 (Δlobe A or 3A) in HEK-293T cells in combination with GFP-tagged WT or mutant DOCK2 (Δlobe A or 3A) or GFP alone, cell extracts were immunoprecipitated with anti-FLAG M2 antibody or anti-GFP antibody. Immunoblotting was carried out to detect homodimerization using the relevant antibodies. (B) Extracts from HEK-293T cells expressing FLAG-tagged WT DOCK2 or Δlobe A were pulled down with GST-fusion Rac1. Assays were done in Tris-buffered saline-Tween-20 supplemented with 10 mM EDTA (E) or 10 mM MgCl₂ plus 30 µM GTPγS (M). (C) Following expression of FLAG-tagged WT DOCK2 or Δlobe A in HEK-293T cells, Rac activation was analyzed. In (A-C), data are representative of, at least, three independent experiments.</p