Biomedical subjects

Anne J Ridley

Publications and source records attributed to Anne J Ridley.

At least 19 recordsLinked to original sources

PubMed2006

Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin-radixin-moesin (ERM) proteins.

Deletion of apoptotic cells from tissues involves their phagocytosis by macrophages, dendritic cells, and tissue cells. Although much attention has been focused on the participating ligands, receptors, and mechanisms of uptake, little is known of the disposition of the ingested cell within the phagosome. Here we show that uptake of apoptotic cells by macrophages or fibroblasts results in rapid phagosome maturation, whereas macrophage phagosomes containing Ig-opsonized target cells mature at a slower rate. The early maturation was shown to depend on activation of Rho acting through Rho kinase on ezrin-radixin-moesin proteins. Blockade of Rho signaling or inhibition of moesin both delayed maturation rates to those seen with opsonized targets. By contrast, phagosome maturation in dendritic cells was slower, similar between apoptotic and opsonized target cells, and unaffected by Rho inhibition. These observations have direct implications for the clearance of dying cells and the roles played by different phagocytes in antigen digestion and presentation.

Acids↗

PubMed2006

beta1-Integrins determine the dendritic morphology which enhances DC-SIGN-mediated particle capture by dendritic cells.

The morphology of antigen-presenting dendritic cells (DCs) is characterized by the presence of numerous long dendrites. The formation of these processes is shown to require the interaction between the beta1-integrin (CD29) on the surface of the DCs and fibronectin in the extracellular matrix. This interaction occurs at focal contacts formed at the tips of dendrites, which contain high concentrations of the beta1-integrins, actin and the cytoskeletal proteins vinculin, paxillin and talin. Dendrites contain an extensive microtubule (MT) network, and are retracted in the presence of the MT inhibitor colchicine, suggesting that MTs are essential for dendrite stability. The dendritic morphology is shown to contribute directly to an enhanced ability to capture dendritic cell specific ICAM-3 grabbing nonintegrin (DC-SIGN)-coated beads. Time-lapse photography demonstrates that dendrites are highly dynamic structures, with cells extending and retracting multiple dendrites in different directions over a 3-h period. This motility increases the area scanned by an individual DC by over 2-fold. The unusual combination of a dendritic morphology and high motility is likely to play a major role in the efficient function of DCs as sentinels of the immune system.

Actins↗

PubMed2006

Phosphoinositide 3-Kinase C2beta regulates cytoskeletal organization and cell migration via Rac-dependent mechanisms.

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Rac1 and Rac2 regulate macrophage morphology but are not essential for migration.

Rac GTPases are believed to contribute to migration in leukocytes by transducing signals from cell surface receptors to the actin and microtubule cytoskeletons. Mammals have three closely related Rac isoforms, Rac1, Rac2 and Rac3, and it is widely assumed that cell migration requires the activity of these Rac GTPases. We have previously shown that Rac1-null mouse macrophages have altered cell shape and reduced membrane ruffling but normal migration speed. Here we investigate the behaviour of macrophages lacking Rac2 (Rac2(-/-)) or Rac1 and Rac2 (Rac1/2(-/-)). Rac2(-/-) macrophages have reduced F-actin levels and lack podosomes, which are integrin-based adhesion sites, and their migration speed is similar to or slightly slower than wild-type macrophages, depending on the substrate. Unexpectedly, Rac1/2(-/-) macrophages, which do not express Rac1, Rac2 or Rac3, migrate at a similar speed to wild-type macrophages on a variety of substrates and perform chemotaxis normally, although their morphology and mode of migration is altered. However, Rac1(-/-) and Rac1/2(-/-) but not Rac2(-/-) macrophages are impaired in their ability to invade through Matrigel. Together, these data show that Rac1 and Rac2 have distinct roles in regulating cell morphology, migration and invasion, but are not essential for macrophage migration or chemotaxis.

Animals↗

PubMed2006

Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin.

Occludin is an integral-membrane protein that contributes to tight junction function. We have identified casein kinase I epsilon (CKI epsilon) as a binding partner for the C-terminal cytoplasmic domain of occludin by yeast two-hybrid screening. CKI epsilon phosphorylated occludin and co-localised and co-immunoprecipitated with occludin from human endothelial cells. Amino acids 265-318 of occludin were sufficient for CKI epsilon binding and phosphorylation. Deletion of the C-terminal 48 amino acids of occludin increased CKI epsilon binding and phosphorylation, suggesting that this region inhibits CKI epsilon binding. These data identify CKI epsilon as a novel occludin kinase that may be important for the regulation of occludin.

Casein Kinase 1 epsilon↗

PubMed2006

Rho GTPases and leukocyte adhesion receptor expression and function in endothelial cells.

Rho family GTPases are key signal transducers that regulate cell adhesion and migration and a variety of other cellular responses, including changes in gene expression. In this review, we discuss how Rho GTPases regulate signaling by endothelial cell receptors involved in leukocyte extravasation. First, Rho GTPases affect the expression of some leukocyte adhesion molecules on endothelial cells, such as intracellular adhesion molecule-1 and E-selectin, that can be induced by proinflammatory mediators, hypoxia, or shear stress. Second, Rho GTPases are activated by engagement of several leukocyte adhesion receptors and contribute to both early morphological changes and subsequent alterations in gene expression. Rho GTPases are therefore candidate targets for inhibiting leukocyte transendothelial migration in heart disease and chronic inflammatory disorders.

Animals↗

PubMed2006

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains.

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.

Actins↗

PubMed2006

Prostate-derived sterile 20-like kinase 2 (PSK2) regulates apoptotic morphology via C-Jun N-terminal kinase and Rho kinase-1.

We have reported previously that human prostate-derived sterile 20-like kinase (PSK) 1 alters actin cytoskeletal organization and binds to microtubules, regulating their organization and stability. We have shown a structurally related protein kinase PSK2, which lacks a microtubule-binding site, activated c-Jun N-terminal kinase (JNK), and induced apoptotic morphological changes that include cell contraction, membrane blebbing, and apoptotic body formation. Apoptotic stimuli increased the catalytic activity of endogenous PSK2 and JNK, and dominant negative JNK or a physiological inhibitor of JNK blocked these apoptotic morphological responses to PSK2, demonstrating a requirement for JNK. PSK2 also stimulated the cleavage of Rho kinase-1 (ROCK-I), and the activity of ROCK-I was required for PSK2 to induce cell contraction and membrane blebbing. The activation of caspases was also needed for the induction of membrane blebbing by PSK2, which was itself a substrate for caspase 3. PSK2 therefore regulates apoptotic morphology associated with the execution phase of apoptosis, which involves dynamic reorganization of the actin cytoskeleton, via downstream targets that include JNK and ROCK-I. Our findings suggest that PSKs form a subgroup of sterile 20 (STE20)-like kinases that regulate different cytoskeletal processes.

Actin Depolymerizing Factors↗

PubMed2006

CSF-1 and PI 3-kinase regulate podosome distribution and assembly in macrophages.

Podosomes are actin-rich adhesive foci found in several cell types, including macrophages. They have a core containing actin and actin-binding proteins and a peripheral ring of integrins and associated proteins. We show that podosomes are abundant in polarized mouse bone marrow-derived macrophages (BMM) and are found primarily in lamellae. We investigated the effects of CSF-1, which induces membrane ruffling, cell spreading, and subsequent polarization and migration, on podosome formation. CSF-1 induces a transient increase in podosome number and enhances the formation of circular arrays of podosomes. Conversely, CSF-1 withdrawal leads to a reduction in podosomes and a decrease in polarized cells. The PI 3-kinase inhibitor LY294002 induces loss of podosomes together with rapid retraction of lamellae and loss of polarity. Our results indicate that CSF-1 acts via PI 3-kinase to enhance podosome assembly and that this is linked to macrophage polarization.

Animals↗

PubMed2006

Inhibition of ROCK by RhoE.

RhoE belongs to the Rnd subfamily of small Rho-related GTP-binding proteins. Similar to other Rho proteins, RhoE regulates actin cytoskeleton dynamics. Expression of RhoE induces loss of actin stress fibers, and it also increases cell migration speed. In part, this is due to RhoE interaction with the RhoA effector ROCK I, a serine/threonine kinase that regulates the formation and contractility of stress fibers. Interestingly, RhoE does not interact with the highly homologous kinase ROCK II. RhoE binding inhibits ROCK I from phosphorylating its downstream target myosin light chain phosphatase, thus increasing the activity of the phosphatase to dephosphorylate myosin II, which results in reduced actomyosin contractility. RhoE itself is phosphorylated by ROCK I, and this may enhance RhoE regulation of ROCK I function. This chapter describes the method used for studying ROCK inhibition by RhoE.

Animals↗

PubMed2006

An in vitro model to study the role of endothelial rho GTPases during leukocyte transendothelial migration.

Tissue injury induces release of cytokines that stimulate the expression of adhesion receptors in the endothelial wall of neighboring vessels. These endothelial receptors recruit leukocytes from the bloodstream and facilitate their transendothelial migration (TEM) toward the inflamed area. The molecules involved in leukocyte-endothelium interaction and TEM have been studied in vivo and in vitro over the past 20 years. Human umbilical vein endothelial cells (HUVECs) are a popular in vitro model to analyze TEM, and have been used to investigate the role of Rho GTPases in this process. Here we describe methods to activate HUVECs, to investigate Rho GTPase-activation by the endothelial adhesion receptor ICAM-1, and to inhibit or activate Rho GTPases using C3 transferase or adenoviruses coding for dominant negative or constitutive active Rho GTPases. Finally, we describe how to image and quantitate leukocyte TEM by digital time-lapse microscopy.

ADP Ribose Transferases↗

PubMed2006

Mammalian cell microinjection assay to study the function of Rho family guanosine triphosphatases.

Microinjection is an excellent technique for studying the acute responses of cells to proteins and can be used to investigate the effects of mutations in proteins on their activity. It has been used widely to study the responses to Rho family guanosine triphosphatases and is particularly useful for cell types that are difficult to transfect. Here, we describe the procedure for microinjecting cells with purified recombinant proteins or with expression vectors encoding proteins, and for analyzing the cells after injection.

3T3 Cells↗

PubMed2005

Vav1 and Vav2 play different roles in macrophage migration and cytoskeletal organization.

Vav family proteins act as guanine nucleotide exchange factors for Rho family proteins, which are known to orchestrate cytoskeletal changes and cell migration in response to extracellular stimuli. Using mice deficient for Vav1, Vav2 and/or Vav3, overlapping and isoform-specific functions of the three Vav proteins have been described in various hematopoietic cell types, but their roles in regulating cell morphology and migration have not been studied in detail. To investigate whether Vav isoforms have redundant or unique functions in regulating adhesion and migration, we investigated the properties of Vav1-deficient and Vav2-deficient macrophages. Both Vav1-deficient and Vav2-deficient cells have a smaller adhesive area; yet, only Vav1-deficient cells have a reduced migration speed, which coincides with a lower level of microtubules. Vav2-deficient macrophages display a high level of constitutive membrane ruffling, but neither Vav1 nor Vav2 is required for colony stimulating factor-1-induced membrane ruffling and cell spreading. Our results suggest that the migration speed of macrophages is regulated independently of spread area or membrane ruffling and that Vav1 is selectively required to maintain a normal migration speed.

Animals↗

PubMed2005

Endothelial intercellular adhesion molecule (ICAM)-2 regulates angiogenesis.

Endothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as vascular endothelial (VE)-cadherin and CD31/platelet endothelial cell adhesion molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule intercellular adhesion molecule 2 (ICAM-2) is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2-deficient mice and ICAM-2-deficient endothelial cells, we show that the lack of ICAM-2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2-deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine. ICAM-2 signaling in human umbilical vein endothelial cells (HUVECs) was found to activate the small guanosine triphosphatase (GTPase) Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration, and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.

Animals↗

PubMed2005

RhoE function is regulated by ROCK I-mediated phosphorylation.

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.

Animals↗

PubMed2005

Rho GTPases and leucocyte-induced endothelial remodelling.

Leucocytes in the bloodstream respond rapidly to inflammatory signals by crossing the blood vessel wall and entering the tissues. This process involves adhesion to, and subsequent transmigration across, the endothelium, mediated by a cascade of interactions between adhesion molecules and stimulation of intracellular signalling pathways in both leucocytes and endothelial cells. This leads to changes in endothelial cell morphology that assist leucocyte extravasation, including endothelial cell contraction, intercellular junction disruption, increased permeability, remodelling of the endothelial apical surface and alterations in vesicle trafficking. Rho GTPases play a central role in many of the endothelial responses to leucocyte interaction. In this review, we discuss recent findings on leucocyte-induced alterations to endothelial cells, and the roles of Rho GTPases in these responses.

Animals↗

PubMed2005

Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy.

The directed migration of cells (chemotaxis) occurs not only during wound healing and inflammatory responses but also during embryonic development. However, the intracellular signaling pathways that enable a cell to detect a chemoattractant and subsequently migrate toward the source are not clearly defined. The Dunn chemotaxis chamber in conjunction with time-lapse microscopy is a powerful tool that enables the user to observe directly the morphological response of cells to a chemoattractant in real time. Here, we describe using the Dunn chemotaxis chamber to study the response of murine bone marrow-derived macrophages to colony stimulating factor-1. This is a particularly useful protocol as it can be adapted to study bone marrow-derived macrophages isolated from genetically modified mice and thus study the requirement for a specific protein in cell migration and chemotaxis.

Animals↗

PubMed2004

Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility.

Higher vertebrates have 3 Rho GTPases, RhoA, RhoB, and RhoC, which share 85% amino acid sequence identity. Here, we compare and contrast the roles of RhoA, B, and C in the regulation of the cytoskeleton and cell motility. Despite their similarity, some regulators and effectors show preferential interaction with RhoA, B, or C, and the three proteins show differences in function in cells. RhoA plays a key role in the regulation of actomyosin contractility. RhoB, which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival, while RhoC may be more important in cell locomotion. In cancer cells, the expression and activity of RhoA, B, and C is altered in different ways. Together, this evidence suggests that although the 3 isoforms of Rho are structurally highly homologous, they have different cellular functions.

Actomyosin↗