Instruct: Given a scientific claim, retrieve passages that support or refute the claim.
Query: Guanine nucleotide exchange factors (GEFs) mediate RhoA activation in response to tensional forces on fibronectin-binding integrins.
How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.
Sec7-related guanine nucleotide exchange factors (GEFs) initiate vesicle budding from the Golgi membrane surface by converting the GTPase ARF to a GTP-bound, membrane-associated form. Here we report the crystal structure of the catalytic Sec7 homology domain of Arno, a human GEF for ARF1, determined at 2.2 angstroms resolution. The Sec7 domain is an elongated, all-helical protein with a distinctive hydrophobic groove that is phylogenetically conserved. Structure-based mutagenesis identifies the groove and an adjacent conserved loop as the ARF-interacting surface. The sites of Sec7 domain interaction on ARF1 have subsequently been mapped, by protein footprinting experiments, to the switch 1 and switch 2 GTPase regions, leading to a model for the interaction between ARF GTPases and Sec7 domain exchange factors.
Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These 'focal complexes' were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.
To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.
Synapses are specialized cell-cell contacts that mediate communication between neurons. Most excitatory synapses in the brain are housed on dendritic spines, small actin-rich protrusions extending from dendrites. During development and in response to environmental stimuli, spines undergo marked changes in shape and number thought to underlie processes like learning and memory. Improper spine development, in contrast, likely impedes information processing in the brain, since spine abnormalities are associated with numerous brain disorders. Elucidating the mechanisms that regulate the formation and plasticity of spines and their resident synapses is therefore crucial to our understanding of cognition and disease. Rho-family GTPases, key regulators of the actin cytoskeleton, play essential roles in orchestrating the development and remodeling of spines and synapses. Precise spatio-temporal regulation of Rho GTPase activity is critical for their function, since aberrant Rho GTPase signaling can cause spine and synapse defects as well as cognitive impairments. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and inhibited by GTPase-activating proteins (GAPs). We propose that Rho-family GEFs and GAPs provide the spatiotemporal regulation and signaling specificity necessary for proper Rho GTPase function based on the following features they possess: (i) existence of multiple GEFs and GAPs per Rho GTPase, (ii) developmentally regulated expression, (iii) discrete localization, (iv) ability to bind to and organize specific signaling networks, and (v) tightly regulated activity, perhaps involving GEF/GAP interactions. Recent studies describe several Rho-family GEFs and GAPs that uniquely contribute to spinogenesis and synaptogenesis. Here, we highlight several of these proteins and discuss how they occupy distinct biochemical niches critical for synaptic development.
The integrin family of heterodimeric cell-surface receptors are fundamental in cell-cell and cell-matrix adhesion. Changes to either integrin-ligand affinity or integrin gene expression are central to a variety of disease processes, including inflammation, cardiovascular disease and cancer. In screening for novel activators of integrin-ligand affinity we identified the previously uncharacterised multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER). siRNA knockdown of Fam38A in epithelial cells inactivates endogenous beta1 integrin, reducing cell adhesion. Fam38A mediates integrin activation by recruiting the small GTPase R-Ras to the ER, which activates the calcium-activated protease calpain by increasing Ca(2+) release from cytoplasmic stores. Fam38A-induced integrin activation is blocked by inhibition of either R-Ras or calpain activity, or by siRNA knockdown of talin, a well-described calpain substrate. This highlights a novel mechanism for integrin activation by Fam38A, utilising calpain and R-Ras signalling from the ER. These data represent the first description of a novel spatial regulator of R-Ras, of an alternative integrin activation-suppression pathway based on direct relocalisation of R-Ras to the ER, and of a mechanism linking R-Ras and calpain signalling from the ER with modulation of integrin-ligand affinity.
Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.
SIGNIFICANCE The extracellular matrix (ECM) fulfills essential functions in multicellular organisms. It provides the mechanical scaffold and environmental cues to cells. Upon cell attachment, the ECM signals into the cells. In this process, reactive oxygen species (ROS) are physiologically used as signalizing molecules. RECENT ADVANCES ECM attachment influences the ROS-production of cells. In turn, ROS affect the production, assembly and turnover of the ECM during wound healing and matrix remodeling. Pathological changes of ROS levels lead to excess ECM production and increased tissue contraction in fibrotic disorders and desmoplastic tumors. Integrins are cell adhesion molecules which mediate cell adhesion and force transmission between cells and the ECM. They have been identified as a target of redox-regulation by ROS. Cysteine-based redox-modifications, together with structural data, highlighted particular regions within integrin heterodimers that may be subject to redox-dependent conformational changes along with an alteration of integrin binding activity. CRITICAL ISSUES In a molecular model, a long-range disulfide-bridge within the integrin β-subunit and disulfide bridges within the genu and calf-2 domains of the integrin α-subunit may control the transition between the bent/inactive and upright/active conformation of the integrin ectodomain. These thiol-based intramolecular cross-linkages occur in the stalk domain of both integrin subunits, whereas the ligand-binding integrin headpiece is apparently unaffected by redox-regulation. FUTURE DIRECTIONS Redox-regulation of the integrin activation state may explain the effect of ROS in physiological processes. A deeper understanding of the underlying mechanism may open new prospects for the treatment of fibrotic disorders.
The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.
Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits β1, β5, α1 as well as the expression of β-catenin and E-cadherin were detected by Western blot. We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin β1 in Piezo1-knockdown cells. Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.
The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here, we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.
Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.
Adherens junctions (AJs), which are organized by adhesion proteins and the underlying actin cytoskeleton, probably sense pulling forces from adjacent cells and modulate opposing forces to maintain tissue integrity, but the regulatory mechanism remains unknown at the molecular level. Although the possibility that α-catenin acts as a direct linker between the membrane and the actin cytoskeleton for AJ formation and function has been minimized, here we show that α-catenin recruits vinculin, another main actin-binding protein of AJs, through force-dependent changes in α-catenin conformation. We identified regions in the α-catenin molecule that are required for its force-dependent binding of vinculin by introducing mutant α-catenin into cells and using in vitro binding assays. Fluorescence recovery after photobleaching analysis for α-catenin mobility and the existence of an antibody recognizing α-catenin in a force-dependent manner further supported the notion that α-catenin is a tension transducer that translates mechanical stimuli into a chemical response, resulting in AJ development.
Cells migrate in multiple different ways depending on their environment, which includes the extracellular matrix composition, interactions with other cells, and chemical stimuli. For all types of cell migration, Rho GTPases play a central role, although the relative contribution of each Rho GTPase depends on the environment and cell type. Here, I review recent advances in our understanding of how Rho GTPases contribute to different types of migration, comparing lamellipodium-driven versus bleb-driven migration modes. I also describe how cells migrate across the endothelium. In addition to Rho, Rac and Cdc42, which are well known to regulate migration, I discuss the roles of other less-well characterized members of the Rho family.
Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovascularization to proceed. Endothelial cells associate with the extracellular matrix through specific interactions with a variety of cell adhesion receptors known as integrins. Peptides containing the tripeptide sequence RGD are known to bind with high affinity to the alphavbeta3 and alphavbeta5 integrins associated with angiogenesis. We present herein the synthesis and in vitro binding affinity of the RGD-containing peptide NC-100717 and a range of molecular probes derived from this intermediate.
A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.
Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.
Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle [4], and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts (our unpublished observations). We investigated how increased concentrations of RanBP1 disturb mitosis. We found that spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. We present evidence that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics.
Angiopoietin-1 (Ang-1), a ligand of the endothelium-specific receptor Tie-2, inhibits permeability in the mature vasculature, but the mechanism remains unknown. Here we show that Ang-1 signals Rho family GTPases to organize the cytoskeleton into a junction-fortifying arrangement that enhances the permeability barrier function of the endothelium. Ang-1 phosphorylates Tie-2 and its downstream effector phosphatidylinositol 3-kinase. This induces activation of one endogenous GTPase, Rac1, and inhibition of another, RhoA. Loss of either part of this dual effect abrogates the cytoskeletal and anti-permeability actions of Ang-1, suggesting that coordinated GTPase regulation is necessary for the vessel-sealing effects of Ang-1. p190 RhoGAP, a GTPase regulatory protein, provides this coordinating function as it is phosphorylated by Ang-1 treatment, requires Rac1 activation, and is necessary for RhoA inhibition. Ang-1 prevents the cytoskeletal and pro-permeability effects of endotoxin but requires p190 RhoGAP to do so. Treatment with p190 RhoGAP small interfering RNA completely abolishes the ability of Ang-1 to rescue endotoxemia-induced pulmonary vascular leak and inflammation in mice. We conclude that Ang-1 prevents vascular permeability by regulating the endothelial cytoskeleton through coordinated and opposite effects on the Rho GTPases Rac1 and RhoA. By linking Rac1 activation and RhoA inhibition, p190 RhoGAP is critical to the protective effects of Ang-1 against endotoxin. These results provide mechanistic evidence that targeting the endothelium through Tie-2 may offer specific therapeutic strategies in life-threatening endotoxemic conditions such as sepsis and acute respiratory distress syndrome.
To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4 degrees C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.
Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development [1-6]. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues [1-6]. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. Here we quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. We found that mechanical tension is approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632 [7], an inhibitor of Rho-kinase whose main effector is Myosin II [8]. Simulations using a vertex model [9] demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. Our results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries.
Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.
Follicular T helper (TFH) cells orchestrate the germinal center (GC) reaction locally. Local mechanisms regulating their dynamics and helper functions are not well defined. Here we found that GC-expressed ephrin B1 (EFNB1) repulsively inhibited T cell to B cell adhesion and GC TFH retention by signaling through TFH-expressed EPHB6 receptor. At the same time, EFNB1 promoted interleukin-21 production from GC TFH cells by signaling predominantly through EPHB4. Consequently, EFNB1-null GCs were associated with defective production of plasma cells despite harboring excessive TFH cells. In a competitive GC reaction, EFNB1-deficient B cells more efficiently interacted with TFH cells and produced more bone-marrow plasma cells, likely as a result of gaining more contact-dependent help. Our results reveal a contact-dependent repulsive guidance system that controls GC TFH dynamics and effector functions locally.
During development, mechanical forces cause changes in size, shape, number, position, and gene expression of cells. They are therefore integral to any morphogenetic processes. Force generation by actin-myosin networks and force transmission through adhesive complexes are two self-organizing phenomena driving tissue morphogenesis. Coordination and integration of forces by long-range force transmission and mechanosensing of cells within tissues produce large-scale tissue shape changes. Extrinsic mechanical forces also control tissue patterning by modulating cell fate specification and differentiation. Thus, the interplay between tissue mechanics and biochemical signaling orchestrates tissue morphogenesis and patterning in development.
Although GBP1 (guanylate binding protein 1) was among the first interferon-inducible proteins identified, its function is still largely unknown. Epidermal growth factor receptor (EGFR) activation by amplification or mutation is one of the most frequent genetic lesions in a variety of human tumors. These include glioblastoma multiforme (GBM), which is characterized by independent but interrelated features of extensive invasion into normal brain parenchyma, rapid growth, necrosis, and angiogenesis. In this study, we show that EGFR activation promoted GBP1 expression in GBM cell lines through a signaling pathway involving Src and p38 mitogen-activated protein kinase. Moreover, we identified YY1 (Yin Yang 1) as the downstream transcriptional regulator regulating EGFR-driven GBP1 expression. GBP1 was required for EGFR-mediated MMP1 (matrix metalloproteinase 1) expression and glioma cell invasion in vitro. Although deregulation of GBP1 expression did not affect glioma cell proliferation, overexpression of GBP1 enhanced glioma cell invasion through MMP1 induction, which required its C-terminal helical domain and was independent of its GTPase activity. Reducing GBP1 levels by RNA interference in invasive GBM cells also markedly inhibited their ability to infiltrate the brain parenchyma of mice. GBP1 expression was high and positively correlated with EGFR expression in human GBM tumors and cell lines, particularly those of the neural subtype. Together, these findings establish GBP1 as a previously unknown link between EGFR activity and MMP1 expression and nominate it as a novel potential therapeutic target for inhibiting GBM invasion.