Actin-dependent Processes Research Articles

Cortactin Is Overexpressed In Neoplastic Cells of Patients with B-Cell Chronic Lymphocytic Leukemia

AbstractAbstract 2436Cortactin is an ubiquitous actin-binding protein that is encoded by EMS1 gene, localized in chromosome 11q13 region. Cortactin is expressed in nearly all mammalian tissues and it was observed to migrate in two different bands with an apparent molecular weight of 80–85kDa, where p85 isoform originates from the p80 because of tyrosine phosphorylation by various different tyrosine kinases. Cortactin mostly localizes to dynamic actin structures and has been implicated in the regulation of several actin-dependent processes, including lamellipodium protrusion, trafficking of the key invadopodia proteases and intracellular transducer downstream of kinase-mediated cell signalling upon phosphorylation. The identification of two alternative splice variants affecting the F-actin binding domain of human cortactin have been described by van Rossum et al., e.g. SV1-cortactin, lacking the 6th repeat (exon 11), and SV2-cortactin, lacking the 5th and 6th repeats (exons 10 and 11). When compared with cells expressing WT-cortactin mRNA, cells expressing SV1- and SV2-cortactin mRNA differ significantly in their ability to bind and cross-link F-actin, promoting actin polymerization in vitro and cell migration. The protein cortactin was found overexpressed in several tumors such as esophageal squamous cell carcinoma, head and neck squamous cell carcinoma and gastric carcinoma. This overexpression increases tumor aggressiveness, possibly through promotion of tumor invasion and metastasis. This protein was originally identified as a substrate for the protein Src kinase Lyn, that plays a key role in CLL disease pathogenesis and progression.In the present study we investigated the expression of cortactin in B cells from 93 patients with B-CLL and 14 healthy controls. We found that this protein was significantly overexpressed in patients (1.10±1.32 SD) with respect to normal control (0.17±0.17 SD, p<0.01; Student's t-test). We also investigated the correlation between level of cortactin and presence of somatic hypermutations (SHM) in the immunoglobulin heavy-chain variable region (IgVH), since the absence of SHM is relevant in terms of reduced survival and responsiveness to chemotherapy. We found that unmutated patients (n=38, 41%), with unfavourable prognosis, showed higher levels of cortactin (1.42±1.63 SD) with respect to mutated ones (n=55, 59%; 0.93±1.14 SD). The differences in cortactin expression levels between unmutated, mutated and normal subjects was statistically significant (p<0.01, ANOVA test). Moreover, Western blotting data demonstrated that in 44% of our patients, neoplastic B lymphocytes express the protein cortactin with a molecular weight of 80/85kDa never found in normal B lymphocytes, that have a protein with a molecular weight of 70/75kDa (Fisher's exact test, p<0.01). The presence of proteins with different molecular weight was confirmed by two-dimensional polyacrylamide gel electrophoresis (2DE) that allowed to separate proteins according to their molecular weight and to their pI (isoeletrict point). Finally the origin of proteins with different molecular weight was investigated at level of mRNA expression. By semi-quantitative RT-PCR analysis on mRNA using primers that flank the region encoding the entire acting binding domain, in B cells purified from 7 normal controls and 21 patients with CLL we studied the expression of mRNA splice variants. We found that 53% of patients expressed the WT mRNA while none of controls was WT, these latter expressing the SV1 isoform only (100%). Results of our SDS-PAGE, 2DE and RT-PCR analyses suggest that WT mRNA codifies for the p80 protein while the SV1 mRNA codifies for the p70 one and that the p85 and p75 isoforms originate from the p80 and p70 respectively as a consequence of post-transcriptional modifications.These findings might suggest that the overexpression of cortactin in leukemic B cells play a role in the pathogenesis and aggressiveness of the neoplastic clone in B-CLL. Disclosures:No relevant conflicts of interest to declare.

P11. Dissecting biological functions of a Wnt/PCP pathway component Prickle

The Wnt/planar cell polarity (PCP) pathway is one of the non-canonical Wnt signalling pathways, and known to be involved in a variety of biological processes including establishment of organised epithelia in Drosophila and regulation of grouped cell migration in vertebrate development. Prickle (Pk) belongs to the ‘core’ components of the Wnt/PCP pathway. In its N-terminal portion Pk has a PET domain, function of which is not known, and three LIM domains that are thought to be involved in protein–protein interaction. Pk is also shown to localise to the proximal side of the cells to form a complex with Vang in Drosophila PCP. However the precise mechanism by which Pk controls these biological processes remains largely unknown.To dissect and elucidate the biological functions of Pk in further detail, we have constructed a series of Pk deletion variants and tested their activities. We found that expression of a variant that lacks LIM and PET domains induces excess actin-dependent filopodia-like processes in Xenopus animal caps and HEK293 cells. This version of Pk localises to these processes. On the other hand, in animal caps expressing another variant which lacks the middle portion, cells became dissociated from each other, indicating inhibited cell adhesion. This phenotype was partially rescued by co-expression of paraxial protocadherin (PAPC).These results suggest pleiotropic roles of Pk in regulation of cytoskeleton and cell adhesion. The possible molecular interactions and mechanism underlying multiple functions of Pk will be discussed in this presentation.

Primary effusion lymphoma: genomic profiling revealed amplification of SELPLG and CORO1C encoding for proteins important for cell migration

Primary effusion lymphoma (PEL) is associated with Kaposi sarcoma herpesvirus (KSHV) but its pathogenesis is poorly understood. Many KSHV-associated products can deregulate cellular pathways commonly targeted in cancer. However, KSHV infection alone is insufficient for malignant transformation. PEL also lacks the chromosomal translocations seen in other lymphoma subtypes. We investigated 28 PELs and ten PEL cell lines by 1 Mb resolution array comparative genomic hybridization (CGH) and found frequent gains of 1q21-41 (47%), 4q28.3-35 (29%), 7q (58%), 8q (63%), 11 (32%), 12 (61%), 17q (29%), 19p (34%), and 20q (34%), and losses of 4q (32%), 11q25 (29%), and 14q32 (63%). Recurrent focal amplification was seen at several regions on chromosomes 7, 8, and 12. High-resolution chromosome-specific tile-path array CGH confirmed these findings, and identified selectin-P ligand (SELPLG) and coronin-1C (CORO1C) as the targets of a cryptic amplification at 12q24.11. Interphase FISH and quantitative PCR showed SELPLG/CORO1C amplification (>4 extra copies) and low levels of copy number gain (1-4 extra copies) in 23% of PELs, respectively. Immunohistochemistry revealed strong expression of both SELPLG and coronin-1C in the majority of PELs, irrespective of their gene dosage. SELPLG is critical for cell migration and chemotaxis, while CORO1C regulates actin-dependent processes, thus important for cell motility. Their overexpression in PEL is expected to play an important role in its pathogenesis.

A kinome RNAi screen identified AMPK as promoting poxvirus entry through the control of actin dynamics.

Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator.

Mal/SRF Is Dispensable for Cell Proliferation in Drosophila

The Mal/SRF transcription factor is regulated by the level of G-actin in cells and has important roles in cell migration and other actin-dependent processes in Drosophila. A recent report suggests that Mal/SRF and an upstream regulator, Pico, are required for cell proliferation and tissue growth in Drosophila. I find otherwise. Mutation of Mal or SRF does not affect cell proliferation in the fly wing. Furthermore, I cannot reproduce the reported effects of Pico RNAi or Pico overexpression on body size. Nevertheless, I can confirm that overexpression of Pico or Mal causes tissue overgrowth specifically in the fly wing - where SRF is most highly expressed. My results indicate that Mal/SRF can promote tissue growth when abnormally active, but is not normally required for tissue growth during development.

Nitric oxide modulates dynamic actin cytoskeleton and vesicle trafficking in a cell type-specific manner in root apices.

NO is an important regulatory molecule in eukaryotes. Much of its effect is ascribed to the action of NO as a signalling molecule. However, NO can also directly modify proteins thus affecting their activities. Although the signalling functions of NO are relatively well recognized in plants, very little is known about its potential influence on the structural integrity of plant cells. In this study, the reorganization of the actin cytoskeleton, and the recycling of wall polysaccharides in plants via the endocytic pathway in the presence of NO or NO-modulating substances were analysed. The actin cytoskeleton and endocytosis in maize (Zea mays) root apices were visualized with fluorescence immunocytochemistry. The organization of the actin cytoskeleton is modulated via NO levels and the extent of such modulation is cell-type specific. In endodermis cells, actin cables change their orientation from longitudinal to oblique and cellular cross-wall domains become actin-depleted/depolymerized. The reaction is reversible and depends on the type of NO donor. Actin-dependent vesicle trafficking is also affected. This was demonstrated through the analysis of recycled wall material transported to newly-formed cell plates and BFA compartments. Therefore, it is concluded that, in plant cells, NO affects the functioning of the actin cytoskeleton and actin-dependent processes. Mechanisms for the reorganization of the actin cytoskeleton are cell-type specific, and such rearrangements might selectively impinge on the functioning of various cellular domains. Thus, the dynamic actin cytoskeleton could be considered as a downstream effector of NO signalling in cells of root apices.

Dictyostelium discoideum Paxillin Regulates Actin-Based Processes

Paxillin is a key player in integrating the actin cytoskeleton with adhesion, and thus is essential to numerous cellular processes, including proliferation, differentiation, and migration in animal cells. PaxB, the Dictyostelium discoideum orthologue of paxillin, has been shown to be important for adhesion and development, much like its mammalian counterpart. Here, we use the overproduction of PaxB to gain better insight into its role in regulating the actin cytoskeleton and adhesion. We find that PaxB-overexpressing (PaxBOE) cells can aggregate and form mounds normally, but are blocked in subsequent development. This arrest can be rescued by addition of wild-type cells, indicating a non-cell autonomous role for PaxB. PaxBOE cells also have alterations in several actin-based processes, including adhesion, endocytosis, motility, and chemotaxis. PaxBOE cells exhibit an EDTA-sensitive increase in cell-cell cohesion, suggesting that PaxB-mediated adhesion is Ca(2+) or Mg(2+) dependent. Interestingly, cells overexpressing paxB are less adhesive to the substratum. In addition, PaxBOE cells display decreased motility under starved conditions, decreased endocytosis, and are unable to efficiently chemotax up a folate gradient. Taken together, the data suggest that proper expression of PaxB is vital for the regulation of development and actin-dependent processes.

Overexpression of FMNL2 is closely related to metastasis of colorectal cancer

Formin-like 2 (FMNL2) is a member of diaphanous-related formins which can control the actin-dependent processes such as cell motility and invasion. In this study, we investigated the expression of FMNL2 in colorectal cancer (CRC) and its correlation with CRC metastasis. Paraffin-embedded specimens of CRC (including 75 primary CRC tumors and 45 corresponding metastatic lymph nodes) and normal colorectal mucosa (30 samples) were immunostained with a FMNL2 antibody. Thirty-two paired snap-frozen tumor tissues and adjacent normal colorectal mucosa were subjected to real-time reverse-transcription polymerase chain reaction (RT-PCR). Six CRC cell lines (SW480, SW620, SW480/M5, LoVo, LS174T, and HT29) were assayed for FMNL2 expression by Western blotting and real-time RT-PCR. Their invasive abilities in vitro were determined by Boyden chamber assay. The immunohistochemical analysis showed FMNL2 expression was considerably higher in CRC tumors and corresponding metastatic lymph nodes than in normal colorectal mucosa (P < 0.05, respectively). Elevated FMNL2 expression was significantly correlated with lymphatic metastasis of CRC (P < 0.05). Real-time RT-PCR analysis confirmed the results obtained by immunohistochemistry. At mRNA and protein levels, FMNL2 expression was substantially upregulated in cell lines derived from CRC metastases (SW620, SW480/M5, and LoVo) compared to ones derived from primary CRC (HT29, LS174T, and SW480; P < 0.05). In vitro cell invasive assay demonstrated that the former three cell lines had higher invasive ability than the latter cell lines. FMNL2 may play an important role in the metastasis of CRC and may be a useful marker for metastasis of CRC.

The Rac1 inhibitor, NSC23766, depolarizes adhesive secretion, endomembrane cycling, and tip growth in the fucoid alga, Silvetia compressa.

Proper cell morphogenesis is dependent on the establishment and expression of cellular polarity. In the fucoid zygote, cell shape is critical for establishing the developmental pattern of the adult, and is achieved by guiding insertion of new membrane and wall to the rhizoid tip. Selection and growth of the appropriate tip site are accompanied by formation of dynamic actin arrays associated with the actin-nucleating Arp2/3 complex. In eukaryotes, a major pathway for activation of the Arp2/3 complex is via the Rho family GTPase, Rac1, which stimulates the Scar/WAVE complex. To determine whether Rac1 controls actin nucleation in Silvetia compressa (J. Agardh) E. Serrao, T. O. Cho, S. M. Boo et Brawley, we tested the effects of the Rac1-specific inhibitory compound, NSC23766, on actin dependent processes and on actin arrays. We found that NSC23766 disrupted polar secretion of adhesive, polarization of endomembranes, and tip-focused growth in the rhizoid. Similarly, NSC23766 altered actin and Arp2 localization in the growing rhizoid. In contrast, NSC23766 had no effect on selection of the growth site or on cytokinesis. These data suggest that Rac1 participates in nucleation of specific actin arrays in the developing zygote.

RNA Interference in J774 Macrophages Reveals a Role for Coronin 1 in Mycobacterial Trafficking but Not in Actin-dependent Processes

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.

Get to grips: steering local actin dynamics with IQGAPs

IQGAPs are actin-binding proteins that scaffold numerous interaction partners, transmitting extracellular signals that influence mitogenic, morphological and migratory cell behaviour. However, the precise mechanisms by which IQGAP proteins influence actin dynamics and actin filament structures have been elusive. Now that IQGAP1 has emerged as a potential key regulator of actin-cytoskeletal dynamics by recruiting both the actin related protein (Arp)2/3 complex and/or formin-dependent actin polymerizing machineries, we propose that IQGAP1 might coordinate the function of mechanistically different actin nucleators for cooperative localized actin filament production in various cellular processes.

Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion

The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior.

Auxin, actin and growth of the Arabidopsis thaliana primary root

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.

Changes in extracellular amino acid concentrations in the rat hippocampus after in vivo actin depolymerization with latrunculin A

The effect of latrunculin A microperfusion on hippocampal extracellular concentrations of glutamate, aspartate, glycine and GABA, as measured by in vivo microdialysis, was investigated. Latrunculin A (4 μg/ml) was perfused for three consecutive days (8 h a day) to promote in vivo F-actin depolymerization. Intrahippocampal latrunculin A microdialysis induced seizures during the second and third day of perfusion, and the animals started showing spontaneous seizures 1 month after lartrunculin A administration. Hippocampal glutamate levels were significantly increased during the first day of latrunculin A microperfusion without significant changes during the second and third day of perfusion. Aspartate levels were significantly increased during the first and second days of treatment. The rise on glutamate and asparate levels was partially reversed by perfusion of NMDA antagonist MK-801. Glycine concentrations were significantly increased during the 3 days of latrunculin A microdialyis, but no significant effect was observed on baseline GABA levels. One month after latrunculin A microperfusion, no significant differences in glutamate and aspartate extracellular concentrations were detected as compared to controls, however, significant increases in glycine and GABA extracellular concentrations were observed. The immediate increases in glutamate, aspartate and glycine levels indicate a modulatory effect of the F-actin cytoskeleton on extracellular concentrations of glutamate, aspartate and glycine. The chronic elevations in GABA and glycine levels are more likely to be related with long-term epileptogenesis processes. Our results suggest that the in vivo biochemical study of actin-dependent processes seems to be a promising approach to the neuropathology and neuropharmacology of epileptic seizures.

Characterization of Salmonella enterica serovar Typhimurium DT104 invasion in an epithelial cell line (IPEC J2) from porcine small intestine

Salmonella Typhimurium DT104 is an emerging enteric pathogen in swine of increasing medical importance. In this study, the time course and the actin-dependent host signaling processes necessary for invasion of a S. Typhimurium DT104 field isolate were investigated in IPEC J2 epithelial cells derived from porcine small intestine. Internalized bacteria were quantified by a gentamicin resistance assay. DT104 internalization into epithelial monolayers increased steadily between 15 and 120 min after apical inoculation. Internalization was reduced by the Rho GTPase inhibitor mevastatin, the N-WASP inhibitor wiskostatin and the actin-disrupting agent cytochalasin D, but not the Rac1 GTPase inhibitor NSC-23766. Early DT104 invasion of porcine enterocytes appears to be mediated by Rac1 GTPase-independent changes in epithelial actin assembly.

HuGE, a novel GFP-actin-expressing mouse line for studying cytoskeletal dynamics

Analysis of actin remodeling in live cells and tissues has become an increasingly important tool to study actin-dependent cellular processes. To facilitate these experiments in the mouse we have generated a GFP-actin-expressing line (huGE) by knock-in of the GFP-actin gene into the profilin 1 locus. Here we show that GFP-actin is expressed throughout embryonic development and in all tissues except skeletal muscle, in a pattern similar to profilin 1. Particularly high expression of GFP-actin was observed in bone marrow and all blood cells. The GFP-actin fusion protein is functional as shown by its co-localization with endogenous actin in F-actin-rich structures. Therefore, the huGE mouse line provides a novel tool to monitor actin dynamics in mouse embryos and a wide range of organs.

Phosphorylation of WAVE1 regulates actin polymerization and dendritic spine morphology

WAVE1--the Wiskott-Aldrich syndrome protein (WASP)--family verprolin homologous protein 1--is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology.

Drosophila Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis

The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. We have determined that the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, “dumping” of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis.

Interleukin‐6 Induction by Helicobacter pylori in Human Macrophages is Dependent on Phagocytosis

The colonization of the gastric mucosa with Helicobacter pylori is accompanied by elevated levels of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and IL-8. The aim of our study was to determine the mechanisms of IL-6 stimulation in phagocytes upon H. pylori infection. We investigated the secretion of IL-6 by different professional phagocytes from murine and human origin, including granulocyte- and monocyte-like cells and macrophages derived from human peripheral blood monocytes (PBMCs). The influence of viability, phagocytosis, and the impact of different subcellular fractions of H. pylori bacteria were evaluated. IL-6 levels induced by H. pylori were low in cell lines derived from murine and human monocytes and in human granulocyte-like cells. By contrast, macrophages derived from human PBMCs were highly responsive to both H. pylori and Escherichia coli. IL-6 induction was blocked by inhibition of actin-dependent processes prior to infection with H. pylori, but not with E. coli or E. coli lipopolysaccharide (LPS). Using cell fractionation, the most activity was found in the H. pylori membrane. H. pylori LPS exhibited a 10(3)- to 10(4)-fold lower biologic activity than E. coli LPS, suggesting a minor role for toll-like receptor 4 (TLR4)-mediated signalling from the exterior. From these data, we conclude that macrophages may be a major source of IL-6 in the gastric mucosa upon H. pylori infection. The IL-6 induction by H. pylori in these cells is a multifactorial process, which requires the uptake and presumably degradation of H. pylori bacteria.

Dissociation of Recruitment and Activation of the Small G-protein Rac during Fcγ Receptor-mediated Phagocytosis

Rho-family proteins play a central role in most actin-dependent processes, including the control and maintenance of cell shape, adhesion, motility, and phagocytosis. Activation of these GTP-binding proteins is tightly regulated spatially and temporally; however, very little is known of the mechanisms involved in their recruitment and activation in vivo. Because of its inducible, restricted signaling, phagocytosis offers an ideal physiological system to delineate the pathways linking surface receptors to actin remodeling via Rho GTPases. In this study, we investigated the involvement of early regulators of Fcgamma receptor signaling in Rac recruitment and activation. Using a combination of receptor mutagenesis, cellular, molecular, and pharmacological approaches, we show that Src family and Syk kinases control Rac and Vav function during phagocytosis. Importantly, both the immunoreceptor tyrosine-based activation motif within Fcgamma receptor cytoplasmic domain and Src kinase control the recruitment of Vav and Rac. However, Syk activity is dispensable for Vav and Rac recruitment. Moreover, we show that Rac and Cdc42 activities coordinate F-actin accumulation at nascent phagosomes. Our results provide new insights in the understanding of the spatiotemporal regulation of Rho-family GTPase function, and of Rac in particular, during phagocytosis. We believe they will contribute to a better understanding of more complex cellular processes, such as cell adhesion and migration.