The chemotaxis system, but not chemotaxis, is essential for swarming motility in Escherichia coli

The chemotaxis system plays an essential role in swarm cell differentiation and motility. We show in this study that two (Tsr and Tar) of the four chemoreceptors in Escherichia coli can support swarming individually, but sensing their most powerful chemoattractants is not necessary. Conditions that abolish chemotaxis toward serine (presence of serine concentrations that saturate Tsr, or mutations in Tsr that destroy serine binding) have no effect on swarming. Similar results were obtained for the aspartate and maltose chemoreceptor Tar. We also show that although a mutation in the signaling domain of Tsr that inhibits CheA kinase abolishes swarming, nonchemotactic flagellar switch mutants can swarm. Our results suggest that during swarming, the chemoreceptors signal through the chemotaxis pathway and induce swarmer cell differentiation in response to signals other than their known chemoeffectors.     

A role for Cdc42 in macrophage chemotaxis

Grammatical morphemes, such as articles and auxiliary verbs, provide potentially useful information to language learners. However, children with specific language impairment (SLI) frequently fail to produce grammatical morphemes, raising questions about their sensitivity to them. To address this issue, two experiments were conducted in which 3- to 5-year-old children with SLI and with normally developing language (NL) heard sentences asking them to identify a picture corresponding to a named target word. The target occurred in either a grammatical sentence or one with an incorrectly used grammatical morpheme. In Experiment 1, the picture representing the target occurred with three unrelated distractor pictures. In Experiment 2, distractor sets included pictures that were semantically related to the target. In both studies, the SLI group chose fewer correct pictures when the target followed an incorrectly used morpheme. In Experiment 2, the SLI group chose more semantically related than unrelated distractors. These results suggest that children with SLI are sensitive to grammatical morphemes and that their incorrect picture choices may reflect a failure to maintain the target in memory.     

Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases

Adenovirus (Ad) endocytosis via alphav integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

An inhibitor of macrophage chemotaxis produced by neoplasms

The accumulation of macrophages at neoplastic sites may be an important event in immunologically mediated tumor killing. The implantation of syngeneic neoplasms in mice, however, was found to depress the animal's ability to localize macrophages at inflammatory sites. A low-molecular-weight (6,000 to 10,000) factor released by growing neoplasms that inhibits the accumulation of macrophages in vivo and chemotactic responsiveness in vitro was identified. The factor is active in the inhibition of macrophages and is ineffectual at retarding the migration of polymorphonuclear leukocytes. Neoplastic cells may thus abrogate immunosurveillance by releasing products that prevent potentially tumoricidal macrophages from accumulating at sites of developing malignancies.     

Guanine nucleotide exchange factors for the Rho GTPases: a role in human disease?

The Rho family of low molecular weight GTP-binding proteins control important aspects of cell shape, adhesion, movement and growth. The DBL-homology (DH) protein family of upstream regulators of Rho GTPases has recently been identified, and deregulated expression of these proteins can have dramatic cellular consequences. This review examines the possible role of DH proteins and Rho GTPases in oncogenesis, metastasis and development.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Functions of neutrophil motility of human blood: locomotion and chemotaxis in simplified systems

Polymorphonuclear leukocytes (PMN, granulocytes) employ their plasma membranes and subjacent microfilament-rich peripheral cytoplasm for such motile functions as adherence and spreading, random locomotion, chemotaxis (directed locomotion), and phagocytosis. All of these functions are preserved in certain anucleate, granule-poor, cytoplasmic fragments (cytoplasts) derived from PMN. Thus, the sensing, transduncing, and effector capacities involved in these functions remain integrated without control from nuclei or from the other cellular organelles left behind when the cytoplast forms. More recently, we have begun to examine in intact PMN the role of divalent cations, which have been thought to be essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In slide preparations under direct microscopic observation, EDTA (10 mM; to chelate divalent cations) did not impair either random locomotion or chemotaxis, nor did specific antibodies to beta-2 (CD 18) integrins or to other PMN integrins. Motile behavior appeared to benefit from the close approximation of slide and coverslip ("chimneying"). Thus, in "close quarters", PMN can generate the force for locomotion even when adhesion molecules are lacking or disabled. We relate these findings to the reported independence from integrins of PMN in certain experimental and diseases states.     

Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases

The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. Here we have identified a new member of the Dbl family, GEF-H1, by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the lfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules.     

S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells

S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.     

Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation, and the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signalling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signalling pathways to varying degrees. Other signalling pathways could also influence the consequences of Ras signalling. Here we show that when signalling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Cells that lack p21Waf1/Cip1 do not require Rho signalling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signalling is the suppression of p21Waf1/Cip1 induction.     

Bacterial toxins block endothelial wound repair. Evidence that Rho GTPases control cytoskeletal rearrangements in migrating endothelial cells

Pharmacological experiments were conducted to determine the neuronal mechanisms involved in the suppressive effects of the thyrotropin-releasing hormone analog TA-0910 on alcohol intake in alcohol-preferring (P) rats. We previously reported that single intraperitoneal injections of TA-0910 dose-dependently reduced alcohol intake in P rats without altering fluid or total calorie intake; however, after several consecutive, once-daily injections, P rats developed tolerance to the suppressive effects of TA-0910 on alcohol intake and cross-tolerance to like effects of the dopamine D2 agonist bromocriptine, but not to like effects of the serotonin uptake inhibitor fluoxetine. In the present study, rats were injected with vehicle or different doses of the D2 antagonist s(-)-eticlopride (0.01 to 0.05 mg/kg) or the D1 antagonist R(+)-SCH23390 (0.1 to 0.5 mg/kg) and 20 min later with TA-0910 (0.75 mg/kg). Alcohol and water intakes were measured at 2, 4, 6, and 24 hr, and food was measured every 24 hr. Both s(-)-eticlopride and R(+)-SCH23390 produced modest reductions in alcohol intake alone; however, only s(-)-eticlopride antagonized the suppressive effect of TA-0910 on alcohol intake. In related experiments, it was confirmed that the dopamine D3 agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin reduced alcohol intake in P rats, and it was found that tolerance to this effect did not develop during or after seven consecutive once-daily injections. Furthermore, this effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetralin was not diminished in rats made tolerant to the effect of TA-0910 on alcohol intake. These data, those of previous studies, and recent preliminary findings support involvement of dopamine D2, but not D1 or D3 receptors in mediating the suppressive effect of TA-0910 on alcohol intake of P rats.     

Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.     

The macrophage and cancer

An adaptation of a standard activity wheel has been used to determine oxygen uptake of rats prior to and during exercise at 7 different speeds (16-67 m/min). Pre-exercise oxygen uptake was 2.42 +/- 0.10 (S.E.) ml (100 g x min)-1. Oxygen uptake increased linearly with work intensity (running speed). At 16m/min oxygen uptake was 6.44 +/- 0.16 ml (100 g x min)-1 and it increased to a maximal value of 9.51 +/- 0.14 ml (100 g x min)-1 at a running speed of 53.6 m/min. Increasing running speed to 67 m/min did not produce any further increase in oxygen uptake. Some comparisons of exercise intensity between rats of various studies and rats and man can be made from these data.     

Effect of Corynebacterium parvum, methanol-extraction residue of BCG, and levamisole on macrophage random migration, chemotaxis, and pinocytosis

Three parameters of macrophage function: random migration, chemotaxis, and pinocytosis, were studied in the guinea pig after administration of Corynebacterium parvum, methanol-extraction residue of BCG, and levamisole (LMS), a synthetic anthelmintic. Macrophage migration studies were performed with a modified Boyden chamber. Pinocytosis was assessed by the uptake of colloidal 198Au. After ip administration, each of the three immunostimulators induced an increase in macrophage chemotactic responsiveness and, to a lesser extent and duration, in random motility. Kinetic, dose-response, and time course data for the effect of each agent on macrophage movement were explored. LMS was the most effective stimulator of macrophage activation, which occurred earlier and persisted longer than it did with the other agents. Macrophages from animals receiving each of the agents showed enhanced pinocytosis. Measurement of macrophage random migration, chemotaxis, and pinocytosis appeared to provide a rapid and quantitative assessment of several parameters of macrophage function and, when studied with other immunologic parameters, may provide useful tools for the evaluation of potential immunoadjuvants.