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.

To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans.In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially. (Blood. 2012; 119(19):4451-4461) IntroductionDendritic cells (DCs) are specialized APCs that play a critical role in the initiation of adaptive immune responses. 1 After antigen exposure, DCs phagocytose antigens in peripheral tissues and migrate via the afferent lymphatic vessels into the draining lymph nodes (LNs) to stimulate T cells. 2,3 During this process, DCs switch their sessile sampling behavior to a highly migratory one, which is characterized by the acquisition of a polarized morphology and increased expression of the chemokine receptor CCR7. Whereas CCR7 signals guide DCs to the LN parenchyma, 4 DCs must pass through a 3-dimensional (3D) interstitial space composed of fibrillar extracellular matrix (ECM) before reaching their destination. To perform this task efficiently, DCs constantly adapt their shape to the given structure of the interstitial ECM and follow the path of least resistance. 5 This amoeboid migration of DCs occurs independently of adhesion to specific substrates and ECM degradation, 6,7 yet its regulatory mechanisms are poorly understood.Cdc42 is a member of the Rho family of small GTPases that function as molecular "switches" by cycling between GDP-bound inactive states and GTP-bound active states. 8 Cdc42 exists in the cytosol in the GDP-bound form and is recruited to membranes, where its GDP is exchanged for GTP because of the action of one or more guanine nucleotide exchange factors (GEFs). Once activated, Cdc42 binds to multiple effector molecules and regulates various cellular functions. Cdc42 is known to act as a master regulator of cell polarity in eukaryotic organisms ranging from yeasts to humans. 8 In addition, a recent stu...

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate–dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D–mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.