Assessing the Quality of Models of the Ambient Solar Wind.

In this paper we present an assessment of the status of models of the global Solar Wind in the inner heliosphere. We limit our discussion to the class of models designed to provide solar wind forecasts, excluding those designed for the purpose of testing physical processes in idealized configurations. In addition, we limit our discussion to modeling of the 'ambient' wind in the absence of coronal mass ejections. In this assessment we cover use of the models both in forecast mode and as tools for scientific research. We present a brief history of the development of these models, discussing the range of physical approximations in use. We discuss the limitations of the data inputs available to these models and its impact on their quality. We also discuss current model development trends.

RhoA, RhoB and RhoC have different roles in cancer cell migration.

Rho GTPases are well known to regulate cell motility through activation of a variety of downstream effector proteins, including enzymes, adaptor proteins and actin nucleators. The three closely related Rho GTPases RhoA, RhoB and RhoC all have the potential to interact with the same downstream effectors, yet they have substantially different effects on cell shape and migratory properties. Here I review the different ways in which RhoA, RhoB and RhoC expression is regulated in cancer and how they play distinct roles in cancer progression. I describe their main effectors known to contribute to cell motility. Recent results from our laboratory and others indicate that RhoA, RhoB and RhoC can be activated by specific stimuli and act through different effectors to control distinct aspects of cancer cell migration and invasion. This suggests that they each make unique contributions to cancer by participating in different protein complexes.

Pulmonary inflammation promoted by type-2 dendritic cells is a feature of human and murine schistosomiasis.

Schistosomiasis is a parasitic disease affecting over 200 million people in multiple organs, including the lungs. Despite this, there is little understanding of pulmonary immune responses during schistosomiasis. Here, we show type-2 dominated lung immune responses in both patent (egg producing) and pre-patent (larval lung migration) murine Schistosoma mansoni (S. mansoni) infection. Human pre-patent S. mansoni infection pulmonary (sputum) samples revealed a mixed type-1/type-2 inflammatory cytokine profile, whilst a case-control study showed no significant pulmonary cytokine changes in endemic patent infection. However, schistosomiasis induced expansion of pulmonary type-2 conventional dendritic cells (cDC2s) in human and murine hosts, at both infection stages. Further, cDC2s were required for type-2 pulmonary inflammation in murine pre-patent or patent infection. These data elevate our fundamental understanding of pulmonary immune responses during schistosomiasis, which may be important for future vaccine design, as well as for understanding links between schistosomiasis and other lung diseases.

A model of localised Rac1 activation in endothelial cells due to fluid flow.

Endothelial cells respond to fluid flow by elongating in the direction of flow. Cytoskeletal changes and activation of signalling molecules have been extensively studied in this response, including: activation of receptors by mechano-transduction, actin filament alignment in the direction of flow, changes to cell-substratum adhesions, actin-driven lamellipodium extension, and localised activation of Rho GTPases. To study this process we model the force over a single cell and couple this to a model of the Rho GTPases, Rac and Rho, via a Kelvin-body model of mechano-transduction. It is demonstrated that a mechano-transducer can respond to the normal component of the force is likely to be a necessary component of the signalling network in order to establish polarity. Furthermore, the rate-limiting step of Rac1 activation is predicted to be conversion of Rac-GDP to Rac-GTP, rather than activation of upstream components. Modelling illustrates that the aligned endothelial cell morphology could attenuate the signalling network.

Cyclin' round the cell with Rac.

New insight into how integrins stimulate cell cycle progression is provided by a recent paper showing that Rac is activated in endothelial cells plated on fibronectin, but not on laminin, and regulates translation of Cyclin D1 mRNA.

Rho family proteins: coordinating cell responses.

Rho GTPases hit the headlines several times in 1990-1992: the proteins regulating their GTP-GDP cycle were identified and they were found to be key signal transducers, mediating growth factor-induced changes to the actin cytoskeleton and activating the phagocyte NADPH oxidase. Since then, they have been implicated in numerous cellular processes, from cell migration to cell survival, transcriptional regulation and vesicle trafficking. An explanation for why they affect so many aspects of cell behavior might lie in their ability to interact with a number of downstream targets, so that they can coordinately activate several molecular processes required for a particular cellular response.

Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering.

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.

Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells.

Transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 were found to be potent mitogens for purified rat Schwann cells, each stimulating DNA synthesis in quiescent cells and also increasing their proliferation rate. Half-maximal stimulation of DNA synthesis occurred at approximately 0.1 ng/ml TGF-beta 1 or TGF-beta 2. Mitogenic stimulation by TGF-beta 1 and TGF-beta 2 was enhanced by forskolin, which activates adenylate cyclase, at concentrations up to 0.5 microM forskolin. However, at 5 microM forskolin, the synergistic interaction between forskolin and TGF-beta 1 was abolished. These results are in contrast to the observed synergy between forskolin and another Schwann cell mitogen, glial growth factor (GGF). Both 0.5 and 5 microM forskolin were found to enhance the stimulation of DNA synthesis by partially purified GGF (GGF-CM). As well as being functionally distinct, TGF-beta 1 and GGF-CM activities were also physically separable by chromatography on a Superose 12 gel permeation column. Thus, TGF-beta 1 and beta 2 are rat Schwann cell mitogens, and Schwann cells are one of the few normal cell populations to respond mitogenically to TGF-beta.

A role for Cdc42 in macrophage chemotaxis.

Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)-induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1-induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished. We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.

Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

Rho-related proteins: actin cytoskeleton and cell cycle.

The past year has produced an abundance of data on the function and regulation of Rho-related GTP-binding proteins. In mammalian cells, it has been shown that Rho is required for contractile ring assembly at cell division, as well as for regulating extracellular factor induced actin reorganization. In addition, many new regulators and/or potential targets for Rho, Rac and Cdc42 have been characterized, including several oncogene products, protein kinases and signal transducing proteins in mammalian cells, and genes defined by cell cycle or bud emergence mutations in yeast. These provide further connections between Rho-related proteins, signal transduction pathways and changes in actin organization during cell cycle entry and progression.

Regulation of macrophage adhesion and migration by Rho GTP-binding proteins.

The Rho family proteins Rac and Rho are believed to be key regulators of cell migration through their effects on the cytoskeleton and cell adhesion. However, recent studies in macrophages indicate that they are not always essential for migration, although they do affect cell shape and adhesion.

Rho: theme and variations.

In addition to their roles in organizing the actin cytoskeleton, members of the Rho family of GTP-binding proteins have recently been implicated in a plethora of other functions, including the activation of kinase cascades and transcription factors, and the control of endocytosis and secretion. Alongside this expansion is proposed functions has been the identification of multiple target proteins that interact directly with Rho, Rac or Cdc42. Molecular connections are now being made along the signalling pathways activated by members of the Rho family.

Intracellular regulation. Rac and Bcr regulate phagocytic phoxes.

The NADPH oxidase of phagocytes is essential for defence against invading microorganisms. The small GTpase Rac seems to be critical for the activation and deactivation of the phox proteins that make up this enzyme.

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages.

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.

The small GTPases Rac and Rho as regulators of secretion in mast cells.

BACKGROUND: Regulated secretion by mast cells is known to be controlled by GTP-binding proteins, but the proteins involved have not been identified. Rac and Rho, two small GTPases related to the oncoprotein Ras, mediate transmission of signals from cell-surface receptors to the actin cytoskeleton. In rat mast cells, both Rac and Rho participate in effecting the centripetal redistribution of filamentous actin that is observed after stimulation of the cells. Rho is responsible for polymerization of actin filaments in the cell interior, whereas Rac is required for the entrapment in the interior of filaments released from the cortex. Such cytoskeletal changes could be important in control of the exocytotic process, so we examined whether Rac and Rho also play a role in regulated secretion by mast cells. RESULTS: We show that the constitutively active mutant proteins, V14RhoA and V12Rac1, enhance regulated secretion from permeabilized mast cells by increasing the proportion of cells that are competent to respond to stimulation. In addition, inhibition of endogenous Rac and Rho activity using inhibitors, N17Rac1 and C3 transferase, respectively, reduces the secretory response of mast cells to stimuli. CONCLUSION: These results provide direct evidence that, in mast cells, both Rac and Rho are components of the signalling pathway that leads to secretion.

Stimulation of actin stress fibre formation mediated by activation of phospholipase D.

BACKGROUND: Agonist-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine, generating the putative messenger phosphatidate (PA). Proposed functions for PA, and hence for PLD, include kinase activation, the regulation of small molecular weight GTP-binding proteins, actin polymerization and secretion. It has not been possible to define a physiological function for PLD activation as it is generally stimulated together with other signalling pathways, such as those involving phospholipases A2 and C, phosphatidylinositide (PI) 3-kinase and the p21(ras)/mitogen-activated protein (MAP) kinase cascade. RESULTS: We report that, in porcine aortic endothelial (PAE) cells, lysophosphatidic acid (LPA) stimulated PLD activity and rapidly generated PA in the absence of other phospholipase, PI 3-kinase or MAP kinase activities. PLD activation was controlled by a tyrosine kinase-regulated pathway. LPA also stimulated actin stress fibre formation, but was inhibited by butan-1-ol; the alcohol also reduced the accumulation of PA. The addition of PA to cells did not stimulate PLD activity, but did cause stress fibre formation in a manner that was insensitive to butan-1-ol. Stimulation of stress fibre formation by LPA and PA was sensitive to genistein, and was inhibited by micro-injection of the Rho-inhibiting C3 exotoxin into PAE cells. CONCLUSIONS: This study provides the first clear demonstration of a physiological role for PLD activity. In PAE cells, the stimulation of actin stress fibre formation was a consequence of PA generation and, therefore, PLD activation. The results suggest that PA generation is upstream of Rho activation, and imply a role for PLD in the regulation of Rho-mediated pathways.

Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation.

BACKGROUND: Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells. RESULTS: We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis. CONCLUSIONS: Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.

Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells.

Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.