Cell migration is a fundamental biological process involving membrane polarization and cytoskeletal dynamics, both of which are regulated by Rho family GTPases. Among these molecules, Rac is crucial for generating the actin-rich lamellipodial protrusion, a principal part of the driving force for movement. The CDM family proteins, Caenorhabditis elegans CED-5, human DOCK180 and Drosophila melanogaster Myoblast City (MBC), are implicated to mediate membrane extension by functioning upstream of Rac. Although genetic analysis has shown that CED-5 and Myoblast City are crucial for migration of particular types of cells, physiological relevance of the CDM family proteins in mammals remains unknown. Here we show that DOCK2, a haematopoietic cell-specific CDM family protein, is indispensable for lymphocyte chemotaxis. DOCK2-deficient mice (DOCK2-/-) exhibited migration defects of T and B lymphocytes, but not of monocytes, in response to chemokines, resulting in several abnormalities including T lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. In DOCK2-/- lymphocytes, chemokine-induced Rac activation and actin polymerization were almost totally abolished. Thus, in lymphocyte migration DOCK2 functions as a central regulator that mediates cytoskeletal reorganization through Rac activation.

We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after ~50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular C1-concentrations was always close to the Cl--eqnilibrium potential. These currents are therefore mainly carried by CI-. DIDS only weakly blocked the current (KI = 120 I~M), while another C1--channel blocker, DCDPC (20 IxM) was ineffective.We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca 2+ independent.A similar current was also activated with iso-osmotic pipette solutions containing 300 ~mol/liter GTP~/S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 ixmol/liter) was also ineffective in modulation of this current.The HTS-induced current was completely blocked by 10 Ixmol/liter pBPB, a PLA2 inhibitor. NDGA (4 tzmol/liter) and indomethacin (5 txmol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 I~mol/liter) were variable. In 12 out of 40 cells it either directly activated a CI-current or potentiated the current activated by HTS. The membrane current was decreased at all potentials

following TCR stimulation, lipid rafts containing a variety of signaling molecules are also reorganized and recruited to the APC interface to participate in IS formation (Viola et al., 1999; Bi et al., 2001; Burack et al., 2002). Thus, IS formation is the process of large-scale molecu-

1. Hypotonic stress induces ATP release followed by Ca 2+ oscillations in bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress-induced ATP release.2. Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-mediated Ca 2+ oscillations induced by hypotonic stress.3. ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-treating the cells with Y-27632, a selective inhibitor of Rho-kinase, also suppressed the hypotonic stress-induced ATP release and Ca 2+ oscillations, indicating that Rho-mediated activation of Rho-kinase may be involved in the hypotonic ATP release.4. Hypotonic stress also induced a transient rearrangement of the actin cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress-induced ATP release nor the Ca 2+ oscillations.5. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathways are involved in hypotonic stress-induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs.

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca2+ oscillations and NO production. They did not, however, affect Ca2+ oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a “permeating blocker model,” in which ATP binds with an affinity of 1.0 ± 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl− hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.