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Hall, A. & Nobes, C. D. Rho GTPases: Molecular switches that control the organization and dynamics of the actin cytoskeleton. Phil. Trans. R. Soc. Lond. B 355, 965-970
Philosophical Transactions B
Abstract
The actin cytoskeleton plays a fundamental role in all eukaryotic cells it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. Work in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.
... We reasoned that if the Panx1 ablation promotes the formation of F-actin, then regulatory proteins that control actin dynamics and organization could mediate this effect. Thus, we evaluated the expression levels of actin-binding proteins (ABPs) and members of the Rho GTPase family, which are master regulators of the actin cytoskeleton [69]. Western blot analysis revealed an increased expression of Arp3, Drebrin, Cortactin 1, Rac1, Cdc42, and RhoA in hippocampal homogenates from Panx1-KO compared to WT samples ( Figure 8D,E; WT versus KO *** p < 0.001; two-way ANOVA test), indicating that the Panx1 deficiency affects actin remodeling by altering the expression of ABPs and Rho GTPases. ...
... Rho GTPases are important molecular "switches" that transduce extracellular signals to the actin cytoskeleton [68,69]. RhoA, Rac1, and Cdc42 are members of the Rho GTPases family, implicated in the maintenance and reorganization of dendritic structures [70,71]. ...
... One of the critical aspects of establishing and maintaining the neuronal structure is the well-controlled turnover of the cytoskeletal elements [104]. At this point, Rho GTPases are the main regulators that control the organization and dynamics of the actin/tubulin cytoskeleton [69]. Among them, Rac1, Cdc42, and RhoA play a major role in dendritic spine dynamics, connecting signals from the postsynaptic receptors to changes in ABPs and hence, actin polymerization/depolymerization [70,71]. ...
Enhanced activity and overexpression of Pannexin 1 (Panx1) channels contribute to neuronal pathologies such as epilepsy and Alzheimer’s disease (AD). The Panx1 channel ablation alters the hippocampus’s glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, Panx1-knockout (Panx1-KO) mice still retain the ability to learn, suggesting that compensatory mechanisms stabilize their neuronal activity. Here, we show that the absence of Panx1 in the adult brain promotes a series of structural and functional modifications in the Panx1-KO hippocampal synapses, preserving spontaneous activity. Compared to the wild-type (WT) condition, the adult hippocampal neurons of Panx1-KO mice exhibit enhanced excitability, a more complex dendritic branching, enhanced spine maturation, and an increased proportion of multiple synaptic contacts. These modifications seem to rely on the actin–cytoskeleton dynamics as an increase in the actin polymerization and an imbalance between the Rac1 and the RhoA GTPase activities were observed in Panx1-KO brain tissues. Our findings highlight a novel interaction between Panx1 channels, actin, and Rho GTPases, which appear to be relevant for synapse stability.
... We reasoned that if PANX1 ablation promotes the formation of F-actin, then regulatory proteins that control actin dynamics and organization could mediate this effect. Thus, we evaluated the levels of actin-binding proteins (ABPs) and the expression of the Rho-small-GTPase family, which are master regulators of the actin cytoskeleton [63]. Western blot analysis revealed an increased expression of Arp3, Drebrin, Cortactin 1, Rac1, Cdc42, and RhoA in hippocampal homogenates from PANX1-KO compared to WT samples (Fig. 6, D and E), indicating that the PANX1 de ciency affects actin remodeling by altering the expression of ABPs and small Rho GTPases. ...
... Western blot analysis revealed an increased expression of Arp3, Drebrin, Cortactin 1, Rac1, Cdc42, and RhoA in hippocampal homogenates from PANX1-KO compared to WT samples (Fig. 6, D and E), indicating that the PANX1 de ciency affects actin remodeling by altering the expression of ABPs and small Rho GTPases. Rho GTPases are important molecular "switches" that transduce extracellular signals to the actin cytoskeleton [63,61]. RhoA, Rac1, and Cdc42 are members of the Rho GTPases family, implicated in the maintenance and reorganization of dendritic structures [64,65]. ...
... The mammalian Rho family of small GTPases are key regulators that control the organization and dynamics of the actin cytoskeleton [63]. Among them, Rac1, Cdc42, and RhoA play a major role in dendritic spine dynamics, connecting signals from the postsynaptic receptors to changes in ABPs and hence, actin polymerization/depolymerization [64,65]. ...
Enhanced activity and overexpression of Pannexin 1 (PANX1) channels contribute to neuronal pathologies, such as epilepsy and Alzheimer’s disease (AD). In the hippocampus, the PANX1 channel ablation alters glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, PANX1-knockout (PANX1-KO) mice still preserve the ability to learn, suggesting that compensatory mechanisms work to stabilize neuronal activity. Here, we show that the absence of PANX1 in the adult brain promotes a series of structural and functional modifications in PANX1-KO CA1 hippocampal synapses, preserving spontaneous activity. Adult CA1 neurons of PANX1-KO mice exhibit enhanced excitability, a more complex dendritic branching, enhanced spine maturation, and multiple synaptic contacts compared to the WT condition. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in actin polymerization and an imbalance between Rac1 and RhoA GTPase activity is observed in the absence of PANX1. Our findings highlight a novel interaction between PANX1, actin, and small Rho GTPases, which appear to be relevant for synapse stability.
... We reasoned that if PANX1 ablation promotes the formation of F-actin, then regulatory proteins that control actin dynamics and organization could mediate this effect. Thus, we evaluated the levels of actin-binding proteins (ABPs) and the expression of the Rho-small-GTPase family, which are master regulators of the actin cytoskeleton [63]. Western blot analysis revealed an increased expression of Arp3, Drebrin, Cortactin 1, Rac1, Cdc42, and RhoA in hippocampal homogenates from PANX1-KO compared to WT samples (Fig. 6, D and E), indicating that the PANX1 de ciency affects actin remodeling by altering the expression of ABPs and small Rho GTPases. ...
... Western blot analysis revealed an increased expression of Arp3, Drebrin, Cortactin 1, Rac1, Cdc42, and RhoA in hippocampal homogenates from PANX1-KO compared to WT samples (Fig. 6, D and E), indicating that the PANX1 de ciency affects actin remodeling by altering the expression of ABPs and small Rho GTPases. Rho GTPases are important molecular "switches" that transduce extracellular signals to the actin cytoskeleton [63,61]. RhoA, Rac1, and Cdc42 are members of the Rho GTPases family, implicated in the maintenance and reorganization of dendritic structures [64,65]. ...
... The mammalian Rho family of small GTPases are key regulators that control the organization and dynamics of the actin cytoskeleton [63]. Among them, Rac1, Cdc42, and RhoA play a major role in dendritic spine dynamics, connecting signals from the postsynaptic receptors to changes in ABPs and hence, actin polymerization/depolymerization [64,65]. ...
Enhanced activity and overexpression of Pannexin 1 (PANX1) channels contribute to neuronal pathologies, such as epilepsy and Alzheimers disease (AD). In the hippocampus, the PANX1 channels ablation alters glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, PANX1-knockout (KO) mice still preserve the ability to learn, suggesting that compensatory mechanisms work to stabilize neuronal activity. Here, we show that the absence of PANX1 in the adult brain promotes a series of structural and functional modifications in KO hippocampal synapses, preserving spontaneous activity. Adult CA1 neurons of KO mice exhibit enhanced excitability, complex dendritic branching, spine maturation, and multiple synaptic contacts compared to the WT condition. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in actin polymerization and an imbalance between Rac1 and RhoA GTPase activity is observed in the absence of PANX1. Our findings highlight a novel interaction between PANX1, actin, and small Rho GTPases that appear to be relevant for synapse maintenance as a long-term compensatory mechanism for PANX1 deficiency.
... In the final part, I summarize a number of experimental strategies and methods that I 13 How an organism is created from a single cell is a challenging question. As complex as organisms are, their development is extremely reproducible and robust process. ...
... Most importantly, key proteins controlling AM network behaviour affect both the actin polymerization dynamics and MyoII contractility (Fig. 2B) 11 . Over the decades, evidence implicated Rho family GTPases as the central regulators of AM networks [11][12][13] . RhoA, Rac and Cdc42 are both activating and repressing various AM components including actin nucleators such N-WASP and motors like MyoII 11 . ...
... To clarify the roles of actin and MTs during zipping, the exact morphology of interacting LE cells needs correlation with cytoskeleton architecture as the process proceeds. Recently, electron tomography enabled a comprehensive three-dimensional (3D) description of an entire eukaryotic cell at the required resolution 13 . Here, we present a similar 3D reconstruction of an entire developmental process. ...
... Das Zytoskelett durchzieht als dreidimensionales Netzwerk das Zytosol und erfüllt maßgebliche Aufgaben bei der Stabilisierung der Zellen, bei Zellbewegungen, sowie bei intrazellulären Transportvorgängen (Vasioukhin et al., 2000;Bär et al., 2014;Moujaber und Stochaj, 2019). Es besteht aus drei Anteilen mit spezifischen Funktionen, dem Aktin-Zytoskelett (Gliem et al., 2010;Tsang et al., 2012a;Hall und Nobes, 2000;Vasioukhin et al., 2000), den Intermediärfilamenten (Bär et al., 2014;Seltmann et al., 2013a;Vielmuth et al., 2018c) und den Mikrotubuli (Moujaber und Stochaj, 2019). ...
... Das Aktinnetzwerk hat mehrere essentielle Funktionen in allen eukaryotischen Zellen, so auch in Keratinozyten (Hall und Nobes, 2000). Es sorgt für die mechanische Stabilität der Zellen und eine robuste Zelladhäsion (Tsang et al., 2012a;Vasioukhin et al., 2000). ...
... Es sorgt für die mechanische Stabilität der Zellen und eine robuste Zelladhäsion (Tsang et al., 2012a;Vasioukhin et al., 2000). Durch den schnellen und flexiblen Auf-und Abbau der Aktinfilamente werden aber auch dynamische Prozesse wie Zellform und -motilität (Hall und Nobes, 2000;Parsons et al., 2010), Phagozytose (Hall und Nobes, 2000), Proliferation und Differenzierung (Tsang et al., 2012a), Zellteilung und die Morphogenese während der embryonalen Entwicklung (Lee und Dominguez, 2010) ermöglicht. (Parsons et al., 2010;Vasioukhin et al., 2000) und Desmosomen Pasdar und Li, 1993;Pasdar et al., 1991). ...
Bei der Autoimmunerkrankung Pemphigus vulgaris führen Antikörper zur charakteristischen suprabasalen Akantholyse und Blasenbildung der Epidermis, indem sie an spezifische Antigene, Dsg3 (Desmoglein 3) und Dsg1 (Desmoglein 1), auf der Zelloberfläche der Keratinozyten binden. Die Art und Weise, wie die multiplen zellulären Pathomechanismen zusammenwirken und das potenziell tödliche Krankheitsbild hervorrufen, ist jedoch bislang noch weitgehend unklar. In der vorliegenden Arbeit wurden entscheidende, durch die Autoantikörper hervorgerufene, pathologische intrazelluläre Prozesse genauer untersucht und deren Stellenwert beleuchtet.
... Eps8 regulates tumour cell invasion and metastasis. Cell migration is a complex process involving reorganization of the actin cytoskeleton (76)(77)(78). Rho GTPase along with other cell processes regulates the organization of the actin cytoskeleton to promote coordinated changes in cell behaviour, in which members of Rho GTPases, including Rac, Cdc42 and Rho, affect different aspects of tumour cell motility (76)(77)(78). Rac promotes the formation of actin-rich membrane ruffle at the leading edge of migrating cells, called Lamellipodia (77). ...
... Cell migration is a complex process involving reorganization of the actin cytoskeleton (76)(77)(78). Rho GTPase along with other cell processes regulates the organization of the actin cytoskeleton to promote coordinated changes in cell behaviour, in which members of Rho GTPases, including Rac, Cdc42 and Rho, affect different aspects of tumour cell motility (76)(77)(78). Rac promotes the formation of actin-rich membrane ruffle at the leading edge of migrating cells, called Lamellipodia (77). Cdc42 modulates cell polarity and the formation of filopodia, thus controlling the direction of cell movement, and Rho facilitates stress fibre formation and maintains focal adhesion at the rear of the cells (78). ...
... Rac promotes the formation of actin-rich membrane ruffle at the leading edge of migrating cells, called Lamellipodia (77). Cdc42 modulates cell polarity and the formation of filopodia, thus controlling the direction of cell movement, and Rho facilitates stress fibre formation and maintains focal adhesion at the rear of the cells (78). In addition, Ras has also been revealed to participate in cell motility and function downstream of G i to mediate ovarian cancer cell migration (79). ...
Epidermal growth factor receptor pathway substrate 8 (Eps8) was initially identified as the substrate for the kinase activity of EGFR, improving the responsiveness of EGF, which is involved in cell mitosis, differentiation and other physiological functions. Numerous studies over the last decade have demonstrated that Eps8 is overexpressed in most ubiquitous malignant tumours and subsequently binds with its receptor to activate multiple signalling pathways. Eps8 not only participates in the regulation of malignant phenotypes, such as tumour proliferation, invasion, metastasis and drug resistance, but is also related to the clinicopathological characteristics and prognosis of patients. Therefore, Eps8 is a potential tumour diagnosis and prognostic biomarker and even a therapeutic target. This review aimed to describe the structural characteristics, role and related molecular mechanism of Eps8 in malignant tumours. In addition, the prospect of Eps8 as a target for cancer therapy is examined.
... The first Rho GTPase was identified in 1985, and over the subsequent decade, it became clear that these proteins are master organizers of actin cytoskeletal dynamics [1][2][3][4][5][6]. Due largely to this role, signaling from Rho GTPases is critical for a wide range of cellular processes, including cell motility, polarization, cell-cell adhesion, cell-matrix adhesion, membrane trafficking and gene expression [7][8][9]. Consequently, Rho GTPases play major roles in various physiological processes, including innate and adaptive immunity, neuronal development, vasculogenesis, and angiogenesis; they have also been implicated in several diseases, including cancer, autism spectrum disorders, metabolic disorders, and vascular disease [10], raising the potential for therapeutic interventions [11,12]. Exploration of Rho GTPase signaling over the past ~ 4 decades has revealed extensive signaling pathways and networks by which these proteins exert their actions [8,9,[12][13][14]. ...
... Consequently, Rho GTPases play major roles in various physiological processes, including innate and adaptive immunity, neuronal development, vasculogenesis, and angiogenesis; they have also been implicated in several diseases, including cancer, autism spectrum disorders, metabolic disorders, and vascular disease [10], raising the potential for therapeutic interventions [11,12]. Exploration of Rho GTPase signaling over the past ~ 4 decades has revealed extensive signaling pathways and networks by which these proteins exert their actions [8,9,[12][13][14]. In this review, we discuss the roles and controls of signaling downstream of Rho GTPases via the PAK group, with a particular focus on the type II PAKs: PAK4, PAK5 and PAK6. ...
Signaling from the Rho family small GTPases controls a wide range of signaling outcomes. Key among the downstream effectors for many of the Rho GTPases are the p21-activated kinases, or PAK group. The PAK family comprises two types, the type I PAKs (PAK1, 2 and 3) and the type II PAKs (PAK4, 5 and 6), which have distinct structures and mechanisms of regulation. In this review, we discuss signal transduction from Rho GTPases with a focus on the type II PAKs. We discuss the role of PAKs in signal transduction pathways and selectivity of Rho GTPases for PAK family members. We consider the less well studied of the Rho GTPases and their PAK-related signaling. We then discuss the molecular basis for kinase domain recognition of substrates and for regulation of signaling. We conclude with a discussion of the role of PAKs in cross talk between Rho family small GTPases and the roles of PAKs in disease.
... Nobes, 2000) and cytoskeleton remodeling(Chiu et al., 1999)(Mertens et al., 2003) , which are important for cell migration and metastasis. Similarly, TIAM2 was associated with tumor progression and unfavorable prognoses(Jiang et al., 2021)(Woroniuk et al., 2018). ...
Background: EPS8L1, an analog of epidermal growth factor receptor pathway substrate 8 (Eps8), was screened out in our previous work from clinical samples of patients with ovarian cancer. We also found that EPS8L1 was involved in various biological activities. In this study, the participation and mechanisms of EPS8L1 ion the migration and metastasis of ovarian cancer were investigated.
Methods: In vitro scratch-healing, transwell assay, and actin-staining studies were performed in SKOV-3 ovarian cancer cells with EPS8L1 overexpression or knockdown. An ovarian cancer mouse model with lung colonization was established to evaluate the in vivo colonization and migration. To identify correlated proteins, a bioinformatics assay was conducted and verified via qRT-PCR and Western blot.
Results: EPS8L1 knockdown inhibited cellular migration in vitro and reduced tumor colonization in vivo. The actin-staining and ELISA experiments suggested that EPS8L1 regulated actin formation as well as cytoskeleton remodeling. Furthermore, mRNA and protein expression confirmed that EPS8L1 regulated the downstream T-cell lymphoma invasion and metastasis 2 (TIAM2) molecule and stimulated the activation of Rac1. Additionally, the phosphorylation levels of P38, Erk, and Jnk in the MAPK pathway decreased after EPS8L1 knockdown.
Conclusions: The upregulation of EPS8L1 could promote the migration and metastasis of ovarian cancer cells by regulating cytoskeleton remodeling. The mechanism underlying this might be that EPS8L1 regulates TIAM2 to induce the transformation of Rac-GDP into Rac-GTP and then activates the downstream MAPK pathway. As a regulatory factor in cell migration and metastasis, EPS8L1 could be a new prognostic biomarker and a promising therapeutic target for ovarian cancer patients.
... Under certain conditions, the dissociated actin and polymerized actin are in equilibrium. When stimulated by external chemical or physical signals, the free G-actin monomer in the cell will combine to form F-actin in the polymerized state and then form actin filaments through its own spiral, which is called cytoskeletal remodeling [30,31]. Phalloidin specifically binds with polymerized actin but not with free actin monomers. ...
Background
Airway remodeling due to increased airway smooth muscle cell (ASMC) mass, likely due to enhanced proliferation, hypertrophy, and migration, has been proven to be highly correlated with decreased lung function in asthma patients. Vascular endothelial growth factor (VEGF) mediates vascular and extravascular remodeling and inflammation and has been proven to be involved in the progression of asthma. Previous studies have focused on the effects of VEGF on ASMC proliferation, but few researchers have focused on the effects of VEGF on human ASMC migration. The purpose of this study was to explore the effect of VEGF on the migration of ASMCs and its related signaling pathway mechanism to provide evidence for the treatment of airway remodeling.
Methods
We examined the effects of VEGF induction on ASMC migration and explored the mechanisms involved in ASMC migration.
Results
We found by wound healing and Transwell assays that VEGF promoted ASMC migration. Through the Cell Counting Kit-8 (CCK-8) experiment, we found that VEGF had no significant effect on the proliferation of ASMCs, which excluded the involvement of cell proliferation in the process of wound healing. Moreover, a cellular immunofluorescence assay showed that VEGF promoted F-actin reorganization, and Western blotting showed that VEGF improved RhoA activation and myosin phosphatase targeting subunit-1 (MYPT1) and myosin light chain (MLC) phosphorylation in ASMCs. Treatment with the ROCK inhibitor Y27632 significantly attenuated the effects of VEGF on MYPT1/MLC activation and cell migration.
Conclusion
In conclusion, the results suggest that the promigratory function of VEGF activates the RhoA/ROCK pathway, induces F-actin reorganization, improves the migration of ASMCs, and provides a better rationale for targeting the RhoA/ROCK pathway for therapeutic approaches in airway remodeling.
... Of interest, cytoskeletal architecture defects within EHT cells were observed followed by blood flow stagnation [6]. Recent studies in zebrafish have showed that Rho-GTPases, established molecular switches that control cytoskeleton dynamics [9], mediated blood flow-induced HSPC development via activation of Yes-activated protein (YAP) [10]. Moreover, defects in actin cytoskeleton remodeling also impaired lymphocyte development and activation [11]. ...
Background
In vertebrates, hematopoietic stem and progenitor cells (HSPCs) emerge from hemogenic endothelium in the floor of the dorsal aorta and subsequently migrate to secondary niches where they expand and differentiate into committed lineages. Glia maturation factor γ (gmfg) is a key regulator of actin dynamics that was shown to be highly expressed in hematopoietic tissue. Our goal is to investigate the role and mechanism of gmfg in embryonic HSPC development.
Methods
In-depth bioinformatics analysis of our published RNA-seq data identified gmfg as a cogent candidate gene implicated in HSPC development. Loss and gain-of-function strategies were applied to study the biological function of gmfg. Whole-mount in situ hybridization, confocal microscopy, flow cytometry, and western blotting were used to evaluate changes in the number of various hematopoietic cells and expression levels of cell proliferation, cell apoptosis and hematopoietic-related markers. RNA-seq was performed to screen signaling pathways responsible for gmfg deficiency-induced defects in HSPC initiation. The effect of gmfg on YAP sublocalization was assessed in vitro by utilizing HUVEC cell line.
Results
We took advantage of zebrafish embryos to illustrate that loss of gmfg impaired HSPC initiation and maintenance. In gmfg-deficient embryos, the number of hemogenic endothelium and HSPCs was significantly reduced, with the accompanying decreased number of erythrocytes, myelocytes and lymphocytes. We found that blood flow modulates gmfg expression and gmfg overexpression could partially rescue the reduction of HSPCs in the absence of blood flow. Assays in zebrafish and HUVEC showed that gmfg deficiency suppressed the activity of YAP, a well-established blood flow mediator, by preventing its shuttling from cytoplasm to nucleus. During HSPC initiation, loss of gmfg resulted in Notch inactivation and the induction of Notch intracellular domain could partially restore the HSPC loss in gmfg-deficient embryos.
Conclusions
We conclude that gmfg mediates blood flow-induced HSPC maintenance via regulation of YAP, and contributes to HSPC initiation through the modulation of Notch signaling. Our findings reveal a brand-new aspect of gmfg function and highlight a novel mechanism for embryonic HSPC development.
... In several stages of plant growth and development, such as root growth, leaf morphogenesis, sexual reproduction, and immunity, Rho GTPases play functional switches [3,4,7,46]. Rho GTPase regulators GAPs, GEFs, and GDIs control the cycling between the active GTP-bound and inactive GDP-bound forms [9,10,12]. ...
The primary regulators of Rho GTPases are GTPase-activating protein (GAP), guanine nucleotide exchange factor (GEF), and GDP dissociation inhibitor (GDI), which function as signaling switches in several physiological processes involved in plant growth and development. This study compared how the Rho GTPase regulators functioned in seven Rosaceae species. Seven Rosaceae species, divided into three subgroups, had a total of 177 regulators of Rho GTPases. According to duplication analysis, the expansion of GEF, GAP, and GDI families was facilitated by whole genome duplication or a dispersed duplication event. The balance of cellulose deposition to control the growth of the pear pollen tube, as demonstrated by the expression profile and antisense oligonucleotide approach. Moreover, protein-protein interactions indicated that PbrGDI1 and PbrROP1 could directly interact, suggesting that PbrGDI1 regulated the growth of the pear pollen tube through PbrROP1 signaling downstream. These results lay the foundations for future functional characterization of the GAP, GEF, and GDI gene families in Pyrus bretschneideri.
... The Eph receptor which belongs to the tyrosine kinase family also plays a role in cell-cell contact by binding the ephrin ligand on the neighboring cell and coupling the them [27]. The Rho family of GTPases controls cytoskeleton arrangement and dynamics [28] which is crucial in inhibiting protrusion of the leading edge upon cell-cell contact [29]. The Rac1 activity at the leading edge promotes actin polymerization which drives the protrusion [30]. ...
Tumor growth is a spatiotemporal birth-and-death process with loss of heterotypic contact-inhibition of locomotion (CIL) of tumor cells promoting invasion and metastasis. Therefore, representing tumor cells as two-dimensional points, we can expect the tumor tissues in histology slides to reflect realizations of spatial birth-and-death process which can be mathematically modeled to reveal molecular mechanisms of CIL, provided the mathematics models the inhibitory interactions. Gibbs process as an inhibitory point process is a natural choice since it is an equilibrium process of the spatial birth-and-death process. That is if the tumor cells maintain homotypic contact inhibition, the spatial distributions of tumor cells will result in Gibbs hard core process over long time scales. In order to verify if this is the case, we applied the Gibbs process to 411 TCGA Glioblastoma multiforme patient images. Our imaging dataset included all cases for which diagnostic slide images were available. The model revealed two groups of patients, one of which - the "Gibbs group," showed the convergence of the Gibbs process with significant survival difference. Further smoothing the discretized (and noisy) inhibition metric, for both increasing and randomized survival time, we found a significant association of the patients in the Gibbs group with increasing survival time. The mean inhibition metric also revealed the point at which the homotypic CIL establishes in tumor cells. Besides, RNAseq analysis between patients with loss of heterotypic CIL and intact homotypic CIL in the Gibbs group unveiled cell movement gene signatures and differences in Actin cytoskeleton and RhoA signaling pathways as key molecular alterations. These genes and pathways have established roles in CIL. Taken together, our integrated analysis of patient images and RNAseq data provides for the first time a mathematical basis for CIL in tumors, explains survival as well as uncovers the underlying molecular landscape for this key tumor invasion and metastatic phenomenon.
... Therefore, it seemed plausible to search for more specific regulators of cell migration. The small GTPases of the Rho family -Cdc42, Rac, and Rho -are essential regulators of the actin cytoskeleton rearrangements and cell motility [4,5]. Each of these small GTPases has a strictly defined role; they also mutually regulate each other. ...
Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression that leads to the acquisition by cancer cells the capacity for migration using the mesenchymal motility mode regulated by the Rac→WAVE→Arp2/3 signaling pathway. Earlier it was shown that proteins interacting with Rac can regulate mesenchymal migration and thus determine the metastatic potential of the cells. The search for new regulators of cell migration is an important theoretical and practical task. The adaptor protein Anks1a is one of the proteins interacting with Rac, whose expression is altered in many types of tumors. The aim of this study was to find whether Anks1a affects the migration of cancer cells and to identify the mechanism underlying this effect. It was suggested that Anks1a can influence cancer cell migration either as a Rac1 effector or by activating human epidermal growth factor receptor 2 (HER2) exchange. We investigated how upregulation and inhibition of Anks1a expression affected migration of breast cancer cells with different HER2 status. Anks1a was shown to interact with the activated form of Rac1. In the MDA-MB-231 cells (triple negative cancer), which lack HER2, Anks1a accumulated at the active cell edge, which is characterized by enrichment with active Rac1, whereas no such accumulation was observed in the HER2-overexpressing SK-BR-3 cells. Downregulation of the ANKS1a expression with esiRNA had almost no effect on the cancer cell motility, except a slight increase in the average migration rate of MDA-MB-231 cells. Among three cell lines tested, overexpression of Anks1a increased the migration rate of HER2-overexpressng SK-BR-3 cells only. We showed that Anks1a is an effector of activated Rac1, but its influence on the cell migration in this capacity was minimal, at least in the studied breast cancer cells. Anks1a affected the motility of breast cancer cells due to its involvement in the EGF receptor exchange.
... The involvement of Rho GTPases and their activating/ inactivating regulators in this process is supported by the fact that RHOA, CDC42, and RAC1 participate in most intracellular events that link extracellular signaling with cytoskeletal reorganization (Hall and Lalli, 2010). Actin cytoskeleton remodeling is essential for cell migration and neurite elongation, two cellular processes that share ultrastructural features and molecular regulations (Hall and Nobes, 2000;Burridge and Wennerberg, 2004;Hua et al., 2005;Govek et al., 2011). During migration, neurons form F-Actin-rich filopodial and lamellipodial membrane protrusions at the peripheral region of the growth cone toward the direction of movement (Cooper, 2013). ...
GTPases of the Rho family are components of signaling pathways linking extracellular signals to the control of cytoskeleton dynamics. Among these, RAC1 plays key roles during brain development, ranging from neuronal migration to neuritogenesis, synaptogenesis, and plasticity. RAC1 activity is positively and negatively controlled by guanine nucleotide exchange factors (GEFs), guanosine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), but the specific role of each regulator in vivo is poorly known. ARHGAP15 is a RAC1-specific GAP expressed during development in a fraction of migrating cortical interneurons (CINs) and in the majority of adult CINs. During development, loss of ARHGAP15 causes altered directionality of the leading process of tangentially migrating CINs, along with altered morphology in vitro. Likewise, time-lapse imaging of embryonic CINs revealed a poorly coordinated directional control during radial migration, possibly due to a hyper-exploratory behavior. In the adult cortex, the observed defects lead to subtle alteration in the distribution of CALB2-, SST-, and VIP-positive interneurons. Adult Arhgap15-knock-out mice also show reduced CINs intrinsic excitability, spontaneous subclinical seizures, and increased susceptibility to the pro-epileptic drug pilocarpine. These results indicate that ARHGAP15 imposes a fine negative regulation on RAC1 that is required for morphological maturation and directional control during CIN migration, with consequences on their laminar distribution and inhibitory function.
... Those genes are promising candidates to explain ASD neuropathology. Rho GTPase signaling is essential for cellular signaling and cytoskeleton dynamics [32,33]. One interesting example is the p21-activated kinase 3 (PAK3), which is a downstream component of the Rho-GTPase cascade. ...
With a prevalence of 2-4% of the worldwide population, neurodevelopmental disorders (NDDs) comprise a heterogeneous group of disorders associated with neurodevelopmental dysfunction, including intellectual disability (ID), autism spectrum disorder (ASD), Down syndrome (DS) and attention-deficit/hyperactivity disorder (ADHD) among others. However, due to their heterogeneity and overlapping clinical features, NDDs such as ASD are often misdiagnosed, while for others with more distinct symptoms, such as Rett syndrome or DS, the mechanisms underlying their pathogenesis remain elusive. Last year, important steps in the mechanistic understanding of several NDDs have been achieved. New preclinical models demonstrated causality between PAK3 mutations and disorders associated with social deficiencies. ARID1B mutations have been linked to neuroectoderm specification in Coffin-Siris syndrome and DNA damage was established as an important pathologic mechanism in Aicardi-Goutières syndrome. Moreover, alterations in basic molecular processes including translation and histone acet-ylation have been established as major traits in the pathology of X-linked ID and Rett syndrome, revealing new pathogenetic mechanisms. Last year, advances in bioinformatics have begun to shed light on the human re-peatome, a largely unexplored part of our genome, and how alterations in these sequences have a central role in ASD. The role of mitochondria in neuropathology was clarified last year with the discovery of previously unknown vesicles derived from mitochondria with a putative role in DS. An interesting discovery in the field of basic neurodevelopment showed that during postnatal brain development, changes in genome architecture and tran-scriptional dynamics progress independently of sensory experience. Finally, our neurocentric views of NDDs are changing as new players such as astrocytes are revealed to be crucial in neuropathology. The role of astrocytes has been clarified for some pathologies such as ASD and DS, linking well-known genetic mutations to impaired astrocyte function. Abbreviations ADAR1-double-stranded RNA-specific adeno-sine deaminase, AGS-Aicardi-Goutières syndrome, ASD-autism spectrum disorder, ATP-adenosine tri-phosphate, cGAS-GMP-AMP synthase, cKO-conditional knockout, Dip-C-diploid chromatin confor-mation capture, DNA-deoxyribonucleic acid, DS-Down syndrome, EV-extracellular vesicles, GABA-gamma-aminobutyric acid, γH2AX: H2A-histone family member X, hiPSC-human induced pluripo-tent stem cells, Iba1-ionized calcium-binding adaptor molecule 1, ID-intellectual disability, IFN-inter-feron, IP3R2-2 inositol 1,4,5-trisphosphate receptors , LC-MS-liquid chromatography-mass spec-trometry, MeCP2-methyl CpG binding protein 2, NDD-neurodevelopmental disorder, RNA-ribonu-cleic acid, RNASEH2-ribonuclease H2, NMDA-N-methyl-d-aspartic acid or N-methyl-d-aspartate, NOVA1-neuro-oncological ventral antigen 1, PSD95-postsynaptic density 95, RTT-Rett syndrome , RPS4Y1-40S ribosomal protein S4, TADs-topologically associated domains, TRs-tandem-repeat sequence, TREX1-three prime repair exonu-clease 1, tRNA-transfer RNAs, XLID-X-linked intellectual disability.
... Another major downstream effector of PI3K is the small GTPase Rac [45,46], a wellknown regulator of cytoskeletal dynamics [47,48], which is also directly implicated in the regulation of fibrosis [49][50][51]. Post-cataract surgery explant cultures were co-immunolabeled at D1 and D2 post-wounding for active Rac ( Figure 4A,C,D,F) and vimentin ( Figure 4B,C,E,F), a cytoskeletal protein enriched in the protrusions that mesenchymal leader cells extend at the leading edge [35]. ...
The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as resident immune cells to produce and organize a fibronectin-EDA rich provisional matrix and transition to collagen I-producing αSMA+ myofibroblasts. While the PI3K effector Akt was alone insufficient to regulate myofibroblast differentiation, our studies revealed a role for Rac, another potential PI3K effector, in this process. Our studies further uncovered a critical role for PI3K p110α in signaling the proliferation of CD44+ leader cells, which is important to the emergence and expansion of myofibroblasts. Thus, these studies identify activation of PI3K p110α as a critical signaling input following wounding to the development and progression of fibrotic disease.
... A previous study indicated that Rho-associated coiled-coil containing protein kinase (ROCK) signaling regulates actin filament assembly and is probably a key factor in the regulation of these keratocyte transitions 12 . It is well known that ROCK is involved in a variety of physiological functions, including chemotaxis, neural growth, smooth muscle contraction [13][14][15] in addition to the assembly and organization of actomyosin filaments [16][17][18] . In fact, the ROCK subspecies, ROCK1 and 2 are widely distributed in tissues including ocular tissues 19 , and are also involved in the development of several types of disease including glaucoma, cataracts, retinopathy, and corneal dysfunction [20][21][22][23] . ...
The objective of the current study was to examine the roles of ROCK1 and 2 on the spatial architecture of human corneal stroma. We examined the effects of a pan-ROCK inhibitor (pan-ROCK-i), ripasudil, and a ROCK2 inhibitor (ROCK2-i), KD025 on the expression of genes that encode for ECM proteins including collagen (COL) 1, 4, 6, and fibronectin (FN), their regulators, a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14, and ER stress-related factors of two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs), and the physical properties of 3D HCSF spheroids. A gene expression analysis using ROCK-is indicated that KD025 (ROCK2 selective ROCK inhibitor) induced more significant changes than Rip (ripasudil, pan-ROCK inhibitor), suggesting that ROCK2 might be more extensively involved in the metabolism of ECM proteins and cell architectures of the 2D cultured HCSFs than ROCK1. In terms of the physical properties, size and stiffness of the 3D HCSFs spheroids, Rip caused a significant enlargement and this enhancement was concentration-dependent while KD025 also exerted a similar but less pronounced effect. In contrast, Rip and KD025 modulated physical stiffness differently, in that Rip caused a substantial decrease and KD025 caused an increase. Such diverse effects between Rip and KD025 were also observed for the gene expressions of ECM proteins, their regulators, and ER-stress related factors. The findings presented herein suggest that the ROCK1 and 2 influence the spatial architecture of 3D HCFS spheroids in different manners.
... In a breast cancer setting, the activity of Rac1 can be co-opted and has been demonstrated to be upregulated in many cancers (Feng et al., 2014;Vega and Ridley, 2008;Yamaguchi et al., 2020). This has been partially linked to the deregulation of specific Rac1 GEFs (guanine nucleotide exchange factors) such as Tiam1 (Adams et al., 2010;Bourguignon et al., 2000aBourguignon et al., , 2000bLi et al., 2016) or P-Rex1 (Barrio-Real et al., 2014, 2016Liu et al., 2016;Srijakotre et al., 2020;Zhong et al., 2019) and can also be attributed to alterations of GEF/GAP (GTPase activating proteins) ratios or GDI (guanine nucleotide disassociation inhibitor) de-regulation (Hall and Nobes, 2000;Machesky and Sansom, 2012;Ridley, 2015). In some cases, this upregulation in activity can also be due to specific point mutations in Rac1 (Kawazu et al., 2013) or the upregulation of the alternative splice isoform of Rac1b (Schnelzer et al., 2000). ...
Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.
... Generally, two major types of endocytosis can be distinguished in the cells: phagocytosis and pinocytosis (1). Phagocytosis is carried out against particles larger than >0.5 mm by specialized cells to protect the organism from microbes and eliminate damaged cells (2). ...
Invertebrates, including earthworms, are applied to study the evolutionarily conserved cellular immune processes. Earthworm immunocytes (so-called coelomocytes) are functionally similar to vertebrate myeloid cells and form the first line of defense against invading pathogens. Hereby, we compared the engulfment mechanisms of THP-1 human monocytic cells, differentiated THP-1 (macrophage-like) cells, and Eisenia andrei coelomocytes towards Escherichia coli and Staphylococcus aureus bacteria applying various endocytosis inhibitors [amantadine, 5-( N -ethyl- N -isopropyl) amiloride, colchicine, cytochalasin B, cytochalasin D, methyl-ß-cyclodextrin, and nystatin]. Subsequently, we investigated the messenger RNA (mRNA) expressions of immune receptor-related molecules ( TLR , MyD88 , BPI ) and the colocalization of lysosomes with engulfed bacteria following uptake inhibition in every cell type. Actin depolymerization by cytochalasin B and D has strongly inhibited the endocytosis of both bacterial strains in the studied cell types, suggesting the conserved role of actin-dependent phagocytosis. Decreased numbers of colocalized lysosomes/bacteria supported these findings. In THP-1 cells TLR expression was increased upon cytochalasin D pretreatment, while this inhibitor caused a dropped LBP/BPI expression in differentiated THP-1 cells and coelomocytes. The obtained data reveal further insights into the evolution of phagocytes in eukaryotes. Earthworm and human phagocytes possess analogous mechanisms for bacterial internalization.
... RhoA activity was furthermore linked to possible apoptotic signalling in pemphigus (251). The effects of RhoA inactivation include reduced incorporation of DP into the desmosomal plaque (190), reduced anchorage of the desmosomes to the keratin cytoskeleton (227) and depletion of Dsg3 and Dsg1 finally leading to loss of cell adhesion (250,(252)(253)(254). All these effects can be explained by the fact that RhoA via Rho kinase regulates the actin binding protein adducin (192). ...
Pemphigus is a severe autoimmune disease impairing barrier functions of epidermis and mucosa. Autoantibodies primarily target the desmosomal adhesion molecules desmoglein (Dsg) 1 and Dsg 3 and induce loss of desmosomal adhesion. Strikingly, autoantibody profiles in pemphigus correlate with clinical phenotypes. Mucosal-dominant pemphigus vulgaris (PV) is characterised by autoantibodies (PV-IgG) against Dsg3 whereas epidermal blistering in PV and pemphigus foliaceus (PF) is associated with autoantibodies against Dsg1. Therapy in pemphigus is evolving towards specific suppression of autoantibody formation and autoantibody depletion. Nevertheless, during the acute phase and relapses of the disease additional treatment options to stabilise desmosomes and thereby rescue keratinocyte adhesion would be beneficial. Therefore, the mechanisms by which autoantibodies interfere with adhesion of desmosomes need to be characterised in detail. Besides direct inhibition of Dsg adhesion, autoantibodies engage signalling pathways interfering with different steps of desmosome turn-over. With this respect, recent data indicate that autoantibodies induce separate signalling responses in keratinocytes via specific signalling complexes organised by Dsg1 and Dsg3 which transfer the signal of autoantibody binding into the cell. This hypothesis may also explain the different clinical pemphigus phenotypes.
... Transforming protein RhoA, Ras-related C3 botulinum toxin substrate 1 and Rho modulator Rab GDP dissociation inhibitor beta, Rho guanine nucleotide exchange factor 2 were found up-regulated in early stage of KCS along with many Rasrelated Rab-proteins (Fig. 1). DLC1 is a tumour suppressor and negatively regulator of RhoA, B and C. Transdifferentiation of native keratocytes and its organisation of actomyosin network are regulated by Rho GTPases (Hall and Nobes, 2000). Rho signalling regulates the assembly of actomyosin filaments and focal adhesions, influences gene expression, cell proliferation, endocytic and exocytic pathways, and cellular transformation (Schmandke et al., 2007) and activation of RhoA was found necessary for TGF-β mediated expression of α-SMA in keratocytes; a marker of smooth muscle transformation of keratocytes (Harvey et al., 2007). ...
Rho GTPases activity in tear fluid as well as ocular surface epithelium (OSEC) during progression of Keratoconjunctivitis sicca (KCS) and their modulation with Rosuvastatin in dogs was studied. Based on Schirmer’s tear test, KCS cases were staged as early (n= 21) and late KCS (N=14). Tear and ocular surface epithelial cell samples were collected from all cases for protein and gene expression studies. Rho GTPase activity in different stages of KCS was evaluated by tear fluid 1D-SDS-PAGE, MALDI-TOF/MS, western blotting and corneal tissue immuno-histochemistry (IHC). Protein expression studies showed increased expression of Rho GTPases in early and late KCS. Effect of Rosuvastatin on progression of KCS was evaluated on 12 client owned dogs divided into two groups A (control group) and B (treatment group) having six dogs in each. Group A dogs were treated with topical eye drops alone whereas, group B animals were treated with topical eye drops and Rosuvastatin orally @ 0.2 mg/kg B.W. 14 days. Tear proteomic study revealed significant down-regulation of Rho GTPase-activating protein 7 (DLC1) and up-regulation Rho modulators and Ras-related Rab proteins in KCS cases. Group B dogs showed better response in terms of corneal clarity both clinically and photographically compared to group A. Expression of Rho GTPase proteins was increased in group B compared A post treatment. On Real-Time PCR assay, mRNA expression of RhoA and GDI2 was contained post Rosuvastatin treatment in OSCEs. Rosuvastatin treatment reduced the gene expression of Rho GTPases and blocked the progression of KCS in dogs.
... These cells are able to rearrange actin cytoskeleton which could lead to internalization of molecules and pathogens, such as bacteria (Conner and Schmid, 2003, Aderem and Underhill, 1999, Dramsi and Cossart, 1998. Among the proteins that are involved in this process, Rho GTPases play an important role (Hall and Nobes, 2000). ...
Tunneling nanotubes (TNTs) are F-actin rich structures that connect distant cells, allowing the transport of many cellular components, including vesicles, organelles and different kind of molecules. TNTs are implicated in key cellular processes, such as development, immunity and tissue regeneration, but also in the transmission of various pathogens. Several molecular factors have been identified to participate in the regulation of TNT formation. One of the early molecular factors that is implicated in TNT formation is the exocyst complex. This complex is also involved in the tethering of secretory vesicles during secretion, which suggest that proteins that regulate vesicle trafficking could have a role in TNT formation. We have hypothesized that the formation of TNTs is modulated by proteins that participate in both, the regulation of vesicle trafficking and the remodelling of the actin cytoskeleton, and that these two processes are key for the formation of these structures. Since Rab GTPases are the major regulators of vesicle trafficking and also participate in actin cytoskeleton regulation, we examined the role of this protein family in TNT formation. First, we performed a screening of several different Rab proteins for its effect on TNT-dependent vesicle transfer. We found that Rab8a, Rab11a and Rab35 have a positive effect on vesicle transfer. Additional studies demonstrated that Rab8a and Rab11a overexpression also increase the number of TNT connected cells. Upon overexpression of VAMP3 (another protein involved in vesicle trafficking), we also observed an increase in the number of TNT connected cells. Further analysis showed that all three proteins, i.e. Rab11a, Rab8a and VAMP3, show an effect on TNT formation in a cascade dependent manner. To establish a relationship between Rab11a and Rab8a, we checked the role of Rabin8 (a protein that interacts with Rab11 and activates Rab8), on TNT formation and we found that it has no role in TNT formation. Additionally, we checked another protein whose function is similar to Rabin8, i.e. GRAB (guanine nucleotide exchange factor for Rab3A) and its role in TNT formation. The results show that GRAB overexpression increases TNT formation, but it acts in a pathway independent of Rab11 and Rab8a to regulate TNT formation. The analysis of Rab35, a protein involved in endocytic recycling, cytokinesis and neurite outgrowth, showed that the GTP-Rab35 bound form also increases TNT formation. Neurite outgrowth is an essential process in order to establish neural connectivity and vesicle recycling plays a crucial role in this process. Rab35 interacts with several proteins, that are involved in vesicle trafficking such as such as ACAP2 (acts as GAP of ARF6), MICAL-L1 (molecule interacting with CasL-like 1, which plays a role in vesicle recycling) EHD1 (a molecular scissor that has a role in vesicle scission). At the ARF6 positive endosomes, Rab35 recruits ACAP2 and MICAL-L1, and forms a complex that binds to EHD1 to regulate neurite outgrowth.Our data strongly suggest that these effectors may also be involved in the formation of TNTs. Individually, ACAP2, EHD1 and ARF6-GDP regulate TNT formation in a positive manner. But MICAL-L1 overexpression in cells shows no effect on TNTs. Also, preliminary data, indicates that Rab35 and EHD1 acts in a cascade mechanism to regulate TNT formation. This indicates that TNT formation and neurite outgrowth may act in a similar, but not exact pathway. The molecules identified here that have a role in TNT formation, constitute potential molecular targets for therapies aiming to block the spreading of pathogens that transfer through TNTs.This study proves that proteins that have a role in vesicle trafficking and neurite outgrowth, such as Rab proteins, are also involved in TNT formation.
... The wide-ranging exposure to QC showed a sharp upregulation in cell cytoskeletal features at 24 h with insignificant changes at 48 h. The altered expression of these genes could be correlated with the Cancer progression is characterized by cell motility, shape, and invasiveness, and the cytoskeleton plays a crucial role in the cell cycle, morphogenesis, and migration [3,18]. Our results have shown that the motility and invasiveness of MCF7 cells is decreased in the presence of QC, which were documented through wound healing and invasion assays. ...
Invasive ductal carcinoma (IDC) is the most recurrent cancer, accounting for 80% of all breast cancers worldwide. Originating from the milk duct, it eventually invades the fibrous tissue of the breast outside the duct, proliferation takes 1–2 months for each division. Quinacrine (QC), an FDA-approved small molecule, has been shown to have anti-cancer activity in numerous cancerous cell lines through diverse pathways; ultimately leading to cell death. Here, we have investigated the mode of action of QC in MCF7 cells. This study demonstrated the modulation of cellular cytoskeleton, such as the formation of distinct filopodial and lamellipodial structures and spikes, through the regulation of small-GTPases. We also observed that QC induces a signaling cascade by inducing apoptotic cell death by increasing ROS generation and altering HSP70 expression; which presumably involves ERK regulation. Our findings show that QC could be an attractive chemotherapeutic agent having a “shotgun” nature with potential of inducing different signaling pathways leading to apoptotic cell death. This opens new avenues for research on developing QC as an effective therapeutic agent for the treatment of invasive ductal carcinomas.
... The activation of Rho small GTPase family members is a trademark for increased motility and tissue invasiveness of tumor cells [10][11][12]. Rho small GTPases are essential molecular regulators of dynamic actin filament remodeling [13,14], and of the eight members of this family, RhoA, Rac1, and Cdc42 are the most studied with respect to their roles in cytoskeletal actin filament regulators of cell shape and movement [15,16]. The regulation and function of Rho small GTPase family members depend on GTP-to-GDP hydrolysis, and the induced protein conformational changes are regulated by the coordinated actions of guanine nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs), and guanine nucleotide dissociation inhibitors (Rho GDIs) [17,18]. ...
CTRP8 is the least studied member of the C1Q-TNF related peptide family. We identified CTRP8 as a ligand of the G protein coupled receptor RXFP1 in glioblastoma multiforme (GBM). The CTRP8-RXFP1 ligand-receptor system protects human GBM cells against the DNA alkylating damage inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1-STAT3 signaling cascade and targeted the mono-functional glycosylase N-methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ induced DNA damaged sites. CTRP8 also improved survival of GBM cells by up-regulating anti-apoptotic BCl-2 and BCL-XL. Here, we have identified JAK3 as a novel member of a novel CTRP8-RXFP1-JAK3-STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F-actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N-WASP/Arp3/4 and actin elongation factor profilin-1. Activation of the RXFP1-JAK3-STAT3-Cdc42 axis by both RXFP1 agonists, CTRP8 and RLN2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F-actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The Cdc42 mediated F-actin remodeling and pro-migratory action of the novel RXFP1-JAK3-STAT3-Cdc42 axis was blocked by JAK3 inhibitor Tofacitinib and STAT3 inhibitor S3I-201 and provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM.
... The Rho family of small GTP-binding proteins are major regulators of microfilaments (Hall and Nobes, 2000). In early work, Speck et al. (2003) found that defects in fly moesin could be counteracted by antagonizing Rho activity, suggesting that ERM proteins might be able to regulate contractility in some manner. ...
Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II–containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.
... However, the intracellular effectors involved in these responses have been very poorly characterized. The RhoGTPases are key players in the control of cytoskeleton dynamics during neuron migration and neurite extension in development (Hall & Lalli, 2010;Hall & Nobes, 2000;Ueyama, 2019). Rnd3 (or RhoE) is an atypical RhoGTPase with no GTPase activity and is therefore considered to be a constitutive-active enzyme (Chardin, 2006;Foster et al. 1996;Riou et al. 2010). ...
The forebrain includes the cerebral cortex, the thalamus, and the striatum and globus pallidus (GP) in the subpallium. The formation of these structures and their interconnections by specific axonal tracts take place in a precise and orchestrated time and spatial‐dependent manner during development. However, the knowledge of the molecular and cellular mechanisms that are involved is rather limited. Moreover, while many extracellular cues and specific receptors have been shown to play a role in different aspects of nervous system development, including neuron migration and axon guidance, examples of intracellular signaling effectors involved in these processes are sparse. In the present work, we have shown that the atypical RhoGTPase, Rnd3, is expressed very early during brain development and keeps a dynamic expression in several brain regions including the cortex, the thalamus, and the subpallium. By using a gene‐trap allele (Rnd3gt) and immunological techniques, we have shown that Rnd3gt/gt embryos display severe defects in striatal and thalamocortical axonal projections (SAs and TCAs, respectively) and defects in GP formation already at early stages. Surprisingly, the corridor, an important intermediate target for TCAs is still present in these mutants. Mechanistically, a conditional genetic deletion approach revealed that Rnd3 is primarily required for the normal development of Medial Ganglionic Eminence‐derived structures, such as the GP, and therefore acts non‐cell autonomously in SAs and TCAs. In conclusion, we have demonstrated the important role of Rnd3 as an early regulator of subpallium development in vivo and revealed new insights about SAs and TCAs development.
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... Transforming protein RhoA, Ras-related C3 botulinum toxin substrate 1 and Rho modulator Rab GDP dissociation inhibitor beta, Rho guanine nucleotide exchange factor 2 were found up-regulated in early stage of KCS along with many Rasrelated Rab-proteins (Fig. 1). DLC1 is a tumour suppressor and negatively regulator of RhoA, B and C. Transdifferentiation of native keratocytes and its organisation of actomyosin network are regulated by Rho GTPases (Hall and Nobes, 2000). Rho signalling regulates the assembly of actomyosin filaments and focal adhesions, influences gene expression, cell proliferation, endocytic and exocytic pathways, and cellular transformation (Schmandke et al., 2007) and activation of RhoA was found necessary for TGF-β mediated expression of α-SMA in keratocytes; a marker of smooth muscle transformation of keratocytes (Harvey et al., 2007). ...
... The Rho GTPases, a subfamily of hydrolases of the Ras superfamily, are known for their essential roles in controlling actin cytoskeleton organization and dynamics in cells; therefore, Rho GTPases are able to regulate cell growth, migration, morphogenesis, survival and membrane trafficking [155][156][157][158]. Rho GTPases are activated by binding to guanosine triphosphate (GTP) and deactivated by binding to guanosine diphosphate (GDP), the process regulated by positive regulatory proteins: guanine nucleotide exchange factors (GEFs), and negative regulatory proteins: GTPase-activating proteins (GAPs) and guanine-nucleotide-dissociation inhibitors (GDIs) [159,160]. ...
Long-term potentiation (LTP) and long-term depression (LTD) are important cellular mechanisms underlying learning and memory processes. N -Methyl- d -aspartate receptor (NMDAR)-dependent LTP and LTD play especially crucial roles in these functions, and their expression depends on changes in the number and single channel conductance of the major ionotropic glutamate receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) located on the postsynaptic membrane. Structural changes in dendritic spines comprise the morphological platform and support for molecular changes in the execution of synaptic plasticity and memory storage. At the molecular level, spine morphology is directly determined by actin cytoskeleton organization within the spine and indirectly stabilized and consolidated by scaffold proteins at the spine head. Palmitoylation, as a uniquely reversible lipid modification with the ability to regulate protein membrane localization and trafficking, plays significant roles in the structural and functional regulation of LTP and LTD. Altered structural plasticity of dendritic spines is also considered a hallmark of neurodevelopmental disorders, while genetic evidence strongly links abnormal brain function to impaired palmitoylation. Numerous studies have indicated that palmitoylation contributes to morphological spine modifications. In this review, we have gathered data showing that the regulatory proteins that modulate the actin network and scaffold proteins related to AMPAR-mediated neurotransmission also undergo palmitoylation and play roles in modifying spine architecture during structural plasticity.
... Il s'agit d'un mécanisme actif impliquant des récepteurs de surface spécifiques (Aderem et Underhill 1999) ainsi que des cascades de signalisation faisant intervenir des membres de la famille des Rho-GTPases (Wolpert L. et al. 2000). Il existe trois différents modes de phagocytose caractérisés par la nature de la particule internalisée ainsi que par la nature des récepteurs reconnaissant cette particule ( Figure 12). ...
Le récepteur à la transferrine (TfR1) est un récepteur très bien conservé au cours de l’évolution qui permet l’entrée du fer (Fe) lié à la transferrine (Fe-Tf) dans les cellules. Une fois internalisé, le fer se détache de la Tf et est exporté dans le cytoplasme tandis que le complexe Tf/TfR1 est recyclé à la membrane plasmique. Le TfR1 est internalisé par un mécanisme clathrine dépendant et peut être soit recyclé à la membrane plasmique par la voie rapide, soit être dirigé vers le compartiment endosomal de recyclage (ERC). Une fois dans l’ERC, le TfR1 peut être soit recyclé à la membrane plasmique par la voie lente, soit être dirigé vers les lysosomes. De nombreuses protéines régulent le routage des protéines internalisées comme les protéines Rab. Il s’agit de petites GTPases impliquées dans les échanges moléculaires entre les différents compartiments cellulaires.Etant la principale voie d’entrée du fer dans la cellule,le TfR1 est exprimé par la plupart des cellules et est surexprimé par les cellules hautement prolifératives dont certaines cellules cancéreuses. Le TfR1 est très étudié comme cible thérapeutique dans le développement de nouvelles stratégies anti-cancéreuses. A24 est un anticorps murin anti-TfR1 dont l’action anti-proliférative et pro-apoptotique ont été démontrées dans plusieurs hémopathies malignes.Nous nous sommes demandés comment la fixation de A24 sur le TfR1 peut produire des effets différents de la fixation de la Fe-Tf. En générant un fragment Fab, nous avons d’abord démontré que le routage du TfR1 dépend de la valence de son ligand. Nous avons ensuite démontré que la fixation monovalente du TfR1 par la Fe-Tf et le Fab induisent son recyclage sans passer par l’ERC. Nous avons également démontré que la fixation divalente du TfR1 par A24 induit son routage depuis l’ERC vers les lysosomes par une voie dépendante de Rab12.
... The roles of Rho family GTPases (Cdc42, Rac, and Rho) in regulating actin assembly and myosin contraction are well-known [44,45]. These switch-like proteins are central regulators of cell migration, funneling signals from the cell's environment to downstream components ...
Mathematical and computational models can assist in gaining an understanding of cell behavior at many levels of organization. Here, we review models in the literature that focus on eukaryotic cell motility at 3 size scales: intracellular signaling that regulates cell shape and movement, single cell motility, and collective cell behavior from a few cells to tissues. We survey recent literature to summarize distinct computational methods (phase-field, polygonal, Cellular Potts, and spherical cells). We discuss models that bridge between levels of organization, and describe levels of detail, both biochemical and geometric, included in the models. We also highlight links between models and experiments. We find that models that span the 3 levels are still in the minority.
... In mammalian cells, RhoA is also located at the front of migrating cells. These different localizations of RhoA have been explained by the intracellular location where myosin-actin contraction occurs in different types of migrating cells (Etienne-Manneville and Hall, 2002;Hall and Nobes, 2000;Wong et al., 2007). Our current data suggest that rear located GTP-RacE suppresses mTORC2-AKT signaling and allows polymerization of actin only at the leading edge of migrating cells ( Figure 6H). ...
The activation of G-protein-coupled receptors (GPCRs) leads to the activation of mTORC2 in cell migration and metabolism. However, the mechanism that links GPCRs to mTORC2 remains unknown. Here, using Dictyostelium cells, we show that GPCR-mediated chemotactic stimulation induces hetero-oligomerization of phosphorylated GDP-bound Rho GTPase and GTP-bound Ras GTPase in directed cell migration. The Rho-Ras hetero-oligomers directly and specifically stimulate mTORC2 activity toward AKT in cells and after biochemical reconstitution using purified proteins in vitro. The Rho-Ras hetero-oligomers do not activate ERK/MAPK, another kinase that functions downstream of GPCRs and Ras. Human KRas4B functionally replace Dictyostelium Ras in mTORC2 activation. In contrast to GDP-Rho, GTP-Rho antagonizes mTORC2-AKT signaling by inhibiting the oligomerization of GDP-Rho with GTP-Ras. These data reveal that GPCR-stimulated hetero-oligomerization of Rho and Ras provides a critical regulatory step that controls mTORC2-AKT signaling.
... The roles of Rho family GTPases (Cdc42, Rac, and Rho) in regulating actin assembly and myosin contraction are well-known [44,45]. These switch-like proteins are central regulators of cell migration, funneling signals from the cell's environment to downstream components that shape its motility. ...
Mathematical and computational models can assist in gaining an understanding of cell behavior at many levels of organization. Here, we review models in the literature that focus on eukaryotic cell motility at 3 size scales: intracellular signaling that regulates cell shape and movement, single cell motility, and collective cell behavior from a few cells to tissues. We survey recent literature to summarize distinct computational methods (phase-field, polygonal, Cellular Potts, and spherical cells). We discuss models that bridge between levels of organization, and describe levels of detail, both biochemical and geometric, included in the models. We also highlight links between models and experiments. We find that models that span the 3 levels are still in the minority.
... As master regulators of the cytoskeleton (Hall and Nobes, 2000;Abreu-Blanco et al., 2014), it is therefore conceivable that RHO GTPases should play a major role in the regulation of nuclear shape. The findings reported herein support a model in which RAC1, among other RHO GTPases, acts as a key orchestrator of nuclear morphology by a process dependent on the tubulin cytoskeleton and LINC complex integrity to promote an invasive phenotype in melanoma cells. ...
RHO GTPases are key regulators of the cytoskeletal architecture, which impacts on a broad range of biological processes in malignant cells including motility, invasion and metastasis, thereby affecting tumor progression. One of the constrains during cell migration, is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus poses a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the LINC complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.
[Media: see text]
... Rho GTPases transmit signals from the membrane receptors to the cytoskeleton and cell adhesions. Small GTPase Rac1 promotes actin assembly at the cell periph ery to produce lamellipodia and membrane ruffles [30]. The inhibitor of Rac1 GDP/GTP exchange activity NSC23766 suppressed protrusions, induced the loss of lamella and lamellipodia, disturbed the gamma CYA net work, and enhanced formation of beta and gamma CYA stress fibers in the interphase cells [2]. ...
We investigated distribution and functions of beta- and gamma-cytoplasmic actins (CYAs) at different stages of non-neoplastic epithelial cell division using laser scanning microscopy (LSM). Here, we demonstrated that beta- and gamma-CYAs are spatially segregated in the early prophase, anaphase, telophase, and cytokinesis. Small interfering RNA (siRNA) experiments revealed that in both beta-CYA- and gamma-CYA-depleted cells, the number of cells was significantly reduced compared with the siRNA controls. Beta-CYA depletion resulted in an enlargement of the cell area in metaphase and high percentage of polynuclear cells compared with the siRNA control, indicating a potential failure of cytokinesis. Gamma-CYA depletion resulted in a reduced percentage of mitotic cells. We also observed the interdependence between the actin isoforms and the microtubule system in mitosis: (i) a decrease in the gamma-CYA led to impaired mitotic spindle organization; (ii) suppression of tubulin polymerization caused impaired beta-CYA reorganization, as incubation with colcemid blocked the transfer of short beta-actin polymers from the basal to the cortical compartment. We conclude that both actin isoforms are essential for proper cell division, but each isoform has its own specific functional role in this process.
... In our study, we observed a decreased in expression of K-Ras, N-Ras, and H-Ras proteins as well as reduced levels of activated pan-Ras, K-Ras, p-p44/42, and Akt by co-treatment with DHA and diclofenac. Members of the Ras family of GTPases have been implicated in lung carcinogenesis and are known to mediate signaling pathways that regulate proliferation, survival, and metastasis in cancer cells [38,[48][49][50]. KRAS is the most frequently mutated isoform in lung cancer representing 19% of the cases, followed by NRAS (1%) and HRAS (< 1%). ...
Simple Summary
Polyunsaturated fatty acids (PUFAs) and non-steroidal anti-inflammatory drugs (NSAIDs) have limited anticancer capacities when used alone. We examined whether combining NSAIDs with docosahexaenoic (DHA) would increase their anticancer activity on lung cancer cell lines. Our results indicate that combining DHA and NSAIDs increased their anticancer activities by altering the expression of critical proteins in the RAS/MEK/ERK and PI3K/Akt pathways. The data suggest that DHA combined with low dose diclofenac provides more significant anticancer potential, which can be further developed for chemoprevention and adjunct therapy in lung cancer.
Abstract
Polyunsaturated fatty acids (PUFAs) and non-steroidal anti-inflammatory drugs (NSAIDs) show anticancer activities through diverse molecular mechanisms. However, the anticancer capacities of either PUFAs or NSAIDs alone is limited. We examined whether combining NSAIDs with docosahexaenoic (DHA), commonly derived from fish oils, would possibly synergize their anticancer activity. We determined the viability of lung cancer cell lines (NCI-H1573, A549, NCI-H1299, and NCI-H1975) after exposure to DHA and various NSAIDs. We further conducted cell apoptosis assays and analyzed apoptosis-associated proteins and some key proteins in the RAS/MEK/ERK and PI3K/Akt pathways using western blot analysis. We also determined the impact of the treatment on the expression of inducible cancer-related genes using nCounter PanCancer Pathways gene expression analysis. The results showed that the combination of DHA and NSAIDs increased suppression of cell viability in all the lung cancer cell lines tested compared to each of the compounds used alone, with diclofenac being the most potent NSAID tested. This synergistic effect is especially significant in A549 and NCI-H1573 cells. The combination treatment was more effective at inhibiting clonogenic cell growth and anchorage-independent growth in soft agar, inducing caspase-dependent apoptosis, and altering expression of critical proteins in the RAS/MEK/ERK and PI3K/Akt pathways. The data from this study demonstrate that DHA combined with low dose diclofenac provides greater anticancer potential, which can be further developed for chemoprevention and adjunct therapy in lung cancer.
... SMO, a functional G protein-coupled receptor (GPCR), demonstrates selectivity toward heterotrimeric G i proteins [90]. GPCRs are known to activate small GTPases which play key roles in cytoskeletal reorganization [91]. These small GTPases are monomeric G proteins that act as molecular switches to quickly regulate cellular processes [92]. ...
The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform.
... In order for this to occur, a delicate balance must exist between intrinsic and extrinsic repair processes, allowing migration and proliferation of cells to the site of injury to allow effective tendon repair [7][8][9][10]. The actin cytoskeleton is important in facilitating the cellular migratory and proliferative processes and proteins that regulate the actin cytoskeleton are integral to the repair process [11][12][13]. ...
Background:
Surgical repair of tendons is common, but function is often limited due to the formation of flexor tendon adhesions which reduce the mobility and use of the affected digit and hand. The severity of adhesion formation is dependent on numerous cellular processes many of which involve the actin cytoskeleton. Flightless I (Flii) is a highly conserved cytoskeletal protein, which has previously been identified as a potential target for improved healing of tendon injuries. Using human in vitro cell studies in conjunction with a murine model of partial laceration of the digital flexor tendon, we investigated the effect of modulating Flii levels on tenocyte function and formation of adhesions.
Methods:
Human tenocyte proliferation and migration was determined using WST-1 and scratch wound assays following Flii knockdown by siRNA in vitro. Additionally, mice with normal and increased levels of Flii were subjected to a partial laceration of the digital flexor tendon in conjunction with a full tenotomy to immobilise the paw. Resulting adhesions were assessed using histology and immunohistochemistry for collagen I, III, TGF-β1and -β3 RESULTS: Flii knockdown significantly reduced human tenocyte proliferation and migration in vitro. Increasing the expression of Flii significantly reduced digital tendon adhesion formation in vivo which was confirmed through significantly smaller adhesion scores based on collagen fibre orientation, thickness, proximity to other fibres and crimping. Reduced adhesion formation was accompanied with significantly decreased deposition of type I collagen and increased expression of TGF-β1 in vivo.
Conclusions:
These findings suggest that increasing the level of Flii in an injured tendon may be beneficial for decreasing tendon adhesion formation.
... Another relevant interference in host cell functions emerging from host nucleus-parasite transcriptional cross talk is indicated by a large cluster formed by generic GO terms of response to antibiotics, response to toxic substances, which were connected to clusters referring to membrane trafficking, positive regulation of phosphorylation, and Rho GTPase activation. The latter clusters are known as molecular switches (70). The findings can be interpreted as an orchestrated adaptation of host cells to intracellular parasitism linked to signaling pathways and cytoskeleton-dependent membrane trafficking. ...
Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro ) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis , we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus.
... Despite the knowledge of the above mentioned gephyrin interactions, thus far, none of these proteins was proven to be directly responsible for the postsynaptic clustering of GABAaR s. (Hall, 1998;Raftopoulou and Hall, 2004). Some of the known target proteins are tyrosine kinases, serine/threonine kinases, lipid kinases, phospholipases and scaffold proteins (Hall and Nobes, 2000). Over the years, more than half of the known receptor tyrosine kinases have been linked to at least one Rho family member, highlighting the possibility of receptor tyrosine kinases being involved in an array of signalling cascades, hence affecting different cellular processes (Schiller, 2006). ...
γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory receptors in the central nervous system. These pentameric receptors contain different subunits, with the most common subtypes consisting of two α, two β and a single γ2 subunit. Changes in GABAAR expression, cellular distribution and function lead to neurological disorders including epilepsy, anxiety and depression. Postsynaptic clustering of GABAARs is thought to be important for their correct function, but the exact mechanisms involved remain unclear. A key molecule involved in the synaptic localisation of GABAARs is the tubulin linker protein gephyrin, as demonstrated by the loss of most synaptic GABAARs in gephyrin knockout mice. Recently, a direct interaction between gephyrin and the GABAARs α2 subunit was shown. However, other studies have implicated the involvement of an alternatively spliced isoform of the γ2 subunit (γ2L) in GABAAR clustering. In addition, knockout mice for the GDP/GTP exchange factor collybistin, which facilitates the correct targeting of gephyrin to postsynaptic membranes, also resulted in the loss of GABAAR clustering in several brain regions. I have examined the potential role of GABAAR α-, β- and γ-subunits in postsynaptic receptor clustering using a range of multidisciplinary techniques. Using the yeast-two hybrid system, I have demonstrated a direct interaction between selected GABAAR α subunit intracellular M3-M4 loops with subdomains of gephyrin and collybistin. Interestingly, I observed an enhanced interaction between gephyrin and the GABAAR α2 subunit in the presence of collybistin in a yeast tri-hybrid system, suggesting the formation of a complex containing these three proteins. However, I have also found that the M3-M4 loop of the GABAAR γ2L subunit localises to the submembrane compartment in mammalian cells, where it co-localises with gephyrin/collybistin aggregates. In conclusion, my data suggest a complex regulation of GABAAR clustering involving multiple GABAAR subunits and additional membrane-associated proteins.
Skin wounds are prone to fungal infections and may threaten patients’ lives in severe cases. However, conventional treatment protocols rarely promote skin wound repair by modulating microRNA (miRNA). A novel binuclear cobalt(ii) complex with the chemical formula [Co2(MBBA)2(HPT)2(H2O)2]·2H2O (1) was synthesized through the reaction of 2-(4-methylbenzoyl) benzoic acid and 3-(pyridin-2-yl)-1H-1,2,4-triazole in the presence of 3,4-pyridine dicarboxylic acid as the template reagent. With natural polysaccharide hyaluronic acid and carboxymethyl chitosan as raw material, based on the chemical synthesis of epidermal cell growth factor for the load of metal gel particles, the microstructure and skin wound healing effect were studied. Molecular docking simulation showed that neither the carboxyl nor triazole group formed binding interactions with the active sites on proteins. Instead, the carbonyl group interacted with the active sites through hydrogen bonding. This study not only provides an effective solution to promote skin wound repair by modulating miRNAs but also provides useful ideas for synthesizing organometallic ligand-based hydrogels and their applications in biomedical fields.
RhoGTPases are well known for being controllers of cell cytoskeleton and share common features in the way they act and are controlled. These include their switch from GDP to GTP states, their regulations by different GEFs, GAPs and GDIs, and their similar structure of active sites/membrane anchors. These very similar features often lead to the common consideration that the differences in their biological effects mainly arise from the different types of regulators and specific effectors associated with each GTPase. Focusing on data obtained through biosensors, live cell microscopy, and recent optogenetic approaches, we highlight in this review that the regulation of RhoA appears to depart from Cdc42 and Rac1 modes of regulation through its enhanced lability at the plasma membrane. RhoA presents a high dynamic turnover at the membrane that is regulated not only by GDIs but also by GEFs, effectors, and a possible soluble conformational state. This peculiarity of RhoA regulation may be important for the specificities of its functions, such as the existence of activity waves or its putative dual role in the initiation of protrusions and contractions. RhoA is a key regulator of cell cytoskeleton dynamics, usually described as a simple switch residing at the plasma membrane. In this review, we show that the highly diverse patterns of RhoA activity call for additional layers of regulation: RhoA residence time at the plasma membrane, localization in space and time, and positive/negative feedback loops.
This book provides up-to-date information on the crucial interaction of pathogenic bacteria and professional phagocytes, the host cells whose purpose is to ingest, kill, and digest bacteria in defense against infection. The introductory chapters focus on the receptors used by professional phagocytes to recognize and phagocytose bacteria, and the signal transduction events that are essential for phagocytosis of bacteria. Subsequent chapters discuss specific bacterial pathogens and the strategies they use in confronting professional phagocytes. Examples include Helicobacter pylori, Streptococcus pneumoniae, and Yersinae, each of which uses distinct mechanisms to avoid being phagocytosed and killed. Contrasting examples include Listeria monocytogenes and Mycobacterium tuberculosis, which survive and replicate intracellularly, and actually cooperate with phagocytes to promote their entry into these cells. Together, the contributions in this book provide an outstanding review of current knowledge regarding the mechanisms of phagocytosis and how specific pathogenic bacteria avoid or exploit these mechanisms.
Nanoplastic particles (NPs) can be produced or derived from the degradation of several daily used products and can therefore be found in the air, water, and food. Every day, these microscopic particles are confronted by different routes of exposure. Recent investigations have shown the internalization of these particles, differing in size and modification, in vivo in aquatic organisms and terrestrial organisms, as well as in vitro in different human cell lines. During the last years, the number of studies investigating the effects of NPs using widely different model systems and experimental approaches is exponentially growing, thus providing information about NPs, especially about polystyrene particle toxicity on health. To facilitate the grasping of the most relevant information, an overview is provided on the toxic effects of NPs coming from studies in cellular systems and in vivo in model organisms and on aspects which can be of particular relevance for particle toxicity (e.g., particle internalization mechanisms and structural modifications). Major achievements and gaps in the field as well as the point of view on how more systematic studies and exploitation of in vivo model organisms may improve the knowledge on important aspects of NPs are also pointed out. Nanoplastic particles (NPs) are everywhere in the environment raising the concern about their possible risks for humans. This review provides a comprehensive overview of the described toxic effects of NPs in mammalian cells and model organisms. Major achievements and gaps are discussed and studies in model organisms, e.g., C. elegans, are suggested to unravel important aspects of NPs in vivo.
Whilst mutations in genes encoding RHO GTPase proteins are rare in different
type of cancers, altered expression of several RHO GTPases has been reported in a variety of human
malignancies. As key regulators of gene expression, lncRNAs coordinate a wide range of molecular
processes, including post-translational regulation through miRNA sponging. The purpose of the
present study was to address the current state of knowledge about the lncRNAs involved in the
regulation of the expression of the RHO GTPases including RHOA, RHOB, RHOC, RAC1, and
CDC42, with a specific focus on the regulatory mechanism of lncRNAs as the molecular sponges
of miRNAs. Considering the critical roles of lncRNAs in malignancies, lncRNA-based therapeutics
are representing promising approaches in cancer treatment through novel technologies. In this
regard, well-characterized examples of lncRNAs associated with tumorigenicity present experimental
frameworks for future studies in this rapidly evolving field.
Pemphigus is a severe autoimmune disease impairing barrier functions of epidermis and mucosa. Autoantibodies primarily target the desmosomal adhesion molecules desmoglein (Dsg) 1 and Dsg 3 and induce loss of desmosomal adhesion. Strikingly, autoantibody profiles in pemphigus correlate with clinical phenotypes. Mucosal-dominant pemphigus vulgaris (PV) is characterized by autoantibodies (PV-IgG) against Dsg3 whereas epidermal blistering in PV and pemphigus foliaceus (PF) is associated with autoantibodies against Dsg1. Therapy in pemphigus is evolving towards specific suppression of autoantibody formation and autoantibody depletion. Nevertheless, during the acute phase and relapses of the disease additional treatment options to stabilize desmosomes and thereby rescue keratinocyte adhesion would be beneficial. Therefore, the mechanisms by which autoantibodies interfere with adhesion of desmosomes need to be characterized in detail. Besides direct inhibition of Dsg adhesion, autoantibodies engage signalling pathways interfering with different steps of desmosome turnover. With this respect, recent data indicate that autoantibodies induce separate signalling responses in keratinocytes via specific signalling complexes organized by Dsg1 and Dsg3 which transfer the signal of autoantibody binding into the cell. This hypothesis may also explain the different clinical pemphigus phenotypes.
This work strongly suggests that acute or chronic latent Toxoplasma gondii infections play a key role in development of Raynaaud's phenomenon, and explains several so far unknown molecular pathophysiological mechanisms involved in this clinical entity.
The immune response of the host towards a decellularized scaffold is complex. Not only can a number of immune cells influence this process, but also the characteristics, preparation and modification of the decellularized scaffold can significantly impact this reaction. Such factors can, together or alone, trigger immune cells to polarize towards either a pro-healing or pro-inflammatory direction. In this article, we have comprehensively reviewed factors which may influence the immune response of the host towards a decellularized scaffold, including the source of the biomaterial, biophysical properties or modifications of the scaffolds with bioactive peptides, drugs and cytokines. Furthermore, the underlying mechanism has also been recapitulated.
Exposure to di-(2-ethylhexyl) phthalate (DEHP), a widely used kind of plasticizer, can result in neurodevelopment impairments and learning and memory disorders. We studied the effects and possible mechanisms of maternal DEHP treatment on hippocampal synaptic plasticity in offspring. Pregnant Wistar rats were randomly divided into four groups and received 0, 30, 300, 750 (mg/kg)/d DEHP by gavage from gestational day (GD) 0 to postnatal day (PN) 21. Our data showed that DEHP exposure impaired hippocampal synaptic plasticity, damaged synaptic ultrastructure, and decreased synaptic protein levels in male pups. Furthermore, DEHP decreased the density of dendritic spines, affected F-actin polymerization, and downregulated the Rac1/PAK/LIMK1/cofilin signaling pathway in male offspring. However, the alterations in the hippocampi of female offspring were not observed. These results illustrate that maternal DEHP exposure could impair hippocampal synaptic plasticity by affecting synaptic structure and dendritic spine development in male offspring, which may be attributed to altered cytoskeleton construction induced by downregulation of the Rac1/PAK/LIMK1/cofilin signaling pathway.
ROP GTPases (Rho-related GTPases from plant), a unique subgroup of the Rho family in plants, is a group of key regulators of different signaling pathways controlling plant growth and development, cell polarity and differentiation, and plant response against biotic and abiotic stresses. The present study determined the potential regulatory mechanism of potato ROP GTPase (StRac1) against Phytophthora infestans (P. infestans) infection. Protein secondary structure analysis indicated that StRAC1 is a Rho GTPase. The expression level of StRac1 was variable in different tissues of potato, with the highest expression in young leaves of both Shepody and Hutou potato varieties. After challenging with P. infestans, the expression level of StRac1was higher in resistance varieties Zihuabai and Longshu 7 than in susceptible varieties Shepody and Desiree. StRAC1 fusion with GFP subcellularly localized at the plasma membrane (PM) in tobacco epidermal cells. The potato with transient or stable over-expression of CA-StRac1(constitutively active form of StRac1)exhibited a dramatic enhancement of its resistance against P. infestans infections. The increased resistance level in transgenic potato was accompanied with elevated H2O2 levels. Importantly, silencing StRac1 via virus-induced gene silencing (VIGS) in potato resulted in higher susceptibility to P. infestans infection than in control plants. In summary, our data reveal that StRac1 regulates potato resistance against P. infestans via positively modulating the accumulation of H2O2.
Professional phagocytes, such as neutrophils and monocytes, have an NADPH oxidase that generates superoxide and other reduced oxygen species important in killing microorganisms. Several components of the oxidase complex have been identified as targets of genetic defects causing chronic granulomatous disease. The complex consists of an electron transport chain that has as its substrate cytosolic NADPH and which discharges superoxide into the cavity of the intracellular phagocytic vacuole. The only electron transport component identified so far is a low-potential cytochrome b, apparently the only membrane component required. At least three cytosolic factors are also necessary, two of which, p67phOx and p47phOx, have been identified by their absence in patients with chronic granulomatous disease. A third component, sigma 1, is required for stimulation of oxidase activity in a cell-free system. The active components of purified sigma 1 are two proteins that associate as heterodimers, and here we report that these are the small GTP-binding protein p21rac1 and the GDP-dissociation inhibitor rhoGDI.
The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote
formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now
show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin
microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia,
as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection.
This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes
and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin
were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating
cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance
of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.
Rho and Rac, two members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins, regulate a variety of signal
transduction pathways in eukaryotic cells. Upon stimulation of phagocytic cells, Rac enhances the activity of the enzyme nicotinamide
adenine dinucleotide phosphate (reduced) (NADPH) oxidase, resulting in the production of superoxide radicals. Activation of
the NADPH oxidase requires the assembly of a multimolecular complex at the plasma membrane consisting of two integral membrane
proteins, gp91phox and p21phox, and two cytosolic proteins, p67phox and p47phox. Rac1 interacted directly with p67phox in
a GTP-dependent manner. Modified forms of Rac with mutations in the effector site did not stimulate oxidase activity or bind
to p67phox. Thus, p67phox appears to be the Rac effector protein in the NADPH oxidase complex.
Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho family of GTPases.
Genetic screening and biochemical studies during the past few years have led to the discovery of a family of cell growth regulatory proteins and oncogene products for which the Dbl oncoprotein is a prototype. These putative guanine nucleotide exchange factors for Rho family small GTP-binding proteins (G proteins) all contain a Dbl homology domain in tandem with a pleckstrin homology domain, and seem to activate specific members of the Rho family of proteins to elicit various biological functions in cells. The Dbl homology domain is directly responsible for binding and activating the small G proteins to mediate downstream signaling events, whereas the pleckstrin homology domain may serve to target these positive regulators of G proteins to specific cellular locations to carry out the signaling task. Despite the increasing interest in the Dbl family of proteins, there is still a good deal to learn regarding the biochemical mechanisms that underlie their diverse biological functions.
The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of
Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and
subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically
to the nucleotide-depleted state. A full-length cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited
sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or
Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation
of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate
that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.
Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell-cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell-cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell- cell contact formation and the remodelling of actin filaments in epithelial cells.
Dorsal closure in Drosophila embryos involves the migration of two lateral epithelia toward the dorsal midline to establish the dorsal ectoderm. Previous work showed that this morphogenetic movement depends on the activities of a Jun amino (N)-terminal kinase kinase (JNKK) encoded by the hemipterous (hep) gene, and of a JNK encoded by basket. Hep is required for cell determination in the leading edge of migrating epithelia, by controlling specific expression of the puckered (puc) gene in these cells. During dorsal closure, decapentaplegic (dpp), a member of the transforming growth factor-beta (TGF-beta) superfamily, is expressed in the row of cells making up the leading edge of the epithelia. Here, we show that the small GTPases Dcdc42, Drac1, and the Hep JNKK control dpp expression in this migratory process. Appropriate dpp and puc expression in the leading edge also depends on the inhibitory function of the puc gene. Further, our data suggest that the leading edge is the source of a JNK autocrine signal, and exclude a role of Dpp as such a ligand. Dorsal closure couples JNK and dpp signaling pathways, a situation that may be conserved in vertebrate development.
The mean trophic level of the species groups reported in Food and Agricultural Organization global fisheries statistics declined
from 1950 to 1994. This reflects a gradual transition in landings from long-lived, high trophic level, piscivorous bottom
fish toward short-lived, low trophic level invertebrates and planktivorous pelagic fish. This effect, also found to be occurring
in inland fisheries, is most pronounced in the Northern Hemisphere. Fishing down food webs (that is, at lower trophic levels)
leads at first to increasing catches, then to a phase transition associated with stagnating or declining catches. These results
indicate that present exploitation patterns are unsustainable.
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.
Cell movement is essential during embryogenesis to establish tissue patterns and to drive morphogenetic pathways and in the adult for tissue repair and to direct cells to sites of infection. Animal cells move by crawling and the driving force is derived primarily from the coordinated assembly and disassembly of actin filaments. The small GTPases, Rho, Rac, and Cdc42, regulate the organization of actin filaments and we have analyzed their contributions to the movement of primary embryo fibroblasts in an in vitro wound healing assay. Rac is essential for the protrusion of lamellipodia and for forward movement. Cdc42 is required to maintain cell polarity, which includes the localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of movement. Rho is required to maintain cell adhesion during movement, but stress fibers and focal adhesions are not required. Finally, Ras regulates focal adhesion and stress fiber turnover and this is essential for cell movement. We conclude that the signal transduction pathways controlled by the four small GTPases, Rho, Rac, Cdc42, and Ras, cooperate to promote cell movement.
Cdc42, a Rho-family GTPase, has been implicated in several signal transduction pathways, including organization of the actin cytoskeleton, activation of the c-Jun N-terminal MAP kinase (JNK) and stimulation of the nuclear transcription factor kappa B (NF(kappa)B). We report here that exposure of fibroblasts to the inflammatory cytokines tumor necrosis factor (alpha) (TNF(alpha)) and interleukin-1 (IL-1) triggers the activation of Cdc42 leading first to filopodia formation and subsequently to Rac and Rho activation. Inhibition of Cdc42 completely suppresses cytokine-induced actin polymerization, but not activation of JNK or NF(kappa)B. The latent membrane protein 1 of Epstein-Barr virus, LMP1, is thought to mimic constitutively activated TNF family receptors. When expressed in fibroblasts, LMP1 stimulates Cdc42-dependent filopodia formation as well as JNK and NF(kappa)B activation. Using LMP1 mutants, we show that activation of Cdc42 and JNK/NF(kappa)B occur through distinct pathways and that Cdc42 activation is independent of LMP1's interaction with TRADD and TRAF proteins.
Focal adhesions (FAs) are clustered integrins and associated proteins that mediate cell adhesion and signaling. A green fluorescent
protein–β1 integrin chimera was used to label FAs in living cells. In stationary cells, FAs were highly motile, moving linearly
for several plaque lengths toward the cell center. FA motility was independent of cell density and resulted from contraction
of associated actin fibers. In migrating cells, FAs were stationary and only moved in the tail. FA motility in stationary
cells suggests that cell movement may be regulated by a clutch-like mechanism by which the affinity of integrins to substrate
may be altered in response to migratory cues.
The small GTPase Rho induces the formation of actin stress fibres and mediates
the formation of diverse actin structures. However, it remains unclear how
Rho regulates its effectors to elicit such functions. Here we show that GTP-bound
Rho activates its effector mDia1 by disrupting mDia1's intramolecular
interactions. Active mDia1 induces the formation of thin actin stress fibres,
which are disorganized in the absence of activity of the Rho-associated kinase
ROCK. Moreover, active mDia1 transforms ROCK-induced condensed actin fibres
into structures reminiscent of Rho-induced stress fibres. Thus mDia1 and ROCK
work concurrently during Rho-induced stress-fibre formation. Intriguingly,
mDia1 and ROCK, depending on the balance of the two activities, induce actin
fibres of various thicknesses and densities. Thus Rho may induce the formation
of different actin structures affected by the balance between mDia1 and ROCK
signalling.
The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.
Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.
Rho and rac, two members of the ras-related superfamily of small GTPases, regulate the polymerization of actin to produce stress fibers and lamellipodia, respectively. We report here that cdc42, another member of the rho family, triggers the formation of a third type of actin-based structure found at the cell periphery, filopodia. In addition to stress fibers, rho controls the assembly of focal adhesion complexes. We now show that rac and cdc42 also stimulate the assembly of multimolecular focal complexes at the plasma membrane. These complexes, which are associated with lamellipodia and filopodia, contain vinculin, paxillin, and focal adhesion kinase, but are distinct from and formed independently of rho-induced focal adhesions. Activation of cdc42 in Swiss 3T3 cells leads to the sequential activation of rac and then rho, suggesting a molecular model for the coordinated control of cell motility by members of the rho family of GTPases.
The c-fos serum response element (SRE) forms a ternary complex with the transcription factors SRF (serum response factor) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38. Functional Rho is required for regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway.
The Rho subfamily of GTPases is involved in control of cell morphology in mammals and yeast. The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA did not exhibit this activity, and none of the proteins activated the ERK subgroup of MAPKs. JNK, but not ERK, activation was also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation.
c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.
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Rho-like GTPases have been implicated in the regulation of the actin cytoskeleton which controls the morphology, adhesion and motility of cells. Like Ras proteins, they become activated when bound GDP is exchanged for GTP, a process catalysed by GDP-dissociation stimulator (GDS) proteins. Several GDS proteins specific for Rho-like GTPases have been identified. Most of these contain a conserved catalytic domain, the DBL-homology (DH) domain, and activate Cdc42 or Rho but not Rac. We have isolated the invasion-inducing Tiam1 gene, which also encodes a protein with a DH domain. Here we show that Tiam1 is a GDS protein for Rho-like GTPases in vitro. In fibroblasts, Tiam1 induces a similar phenotype as constitutively activated (V12)Rac1, including membrane ruffling, and this is inhibited by dominant negative (N17)Rac1. Moreover, T-lymphoma cells expressing V12Rac1 become invasive, indicating that the Tiam1-Rac signalling pathway could be operating in the invasion and metastasis of tumour cells.
We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.
Directed endothelial cell (EC) movement is required for the development and repair of blood vessels and plays a critical role in angiogenic processes obligatory for large tumor formation. We now report that ras proteins have a critical role in regulation of movement of normal mammalian cells. Bovine aortic EC microinjected with oncogenic Ha-ras enter further into an artificial wound than uninjected cells. Treatment with oncogenic Ha-ras also converts the cell paths from nearly linear in control cells to apparent 'random-walk' trajectories in treated cells, suggesting that oncogenic ras alters the normal control processes regulating cell motility. Botulinum toxin C blocks ras-stimulated motility indicating that a member of the p21 rho family is a downstream participant in the motile pathway. In related experiments we have observed that microinjection of the neutralizing, ras-specific, Y13-259 monoclonal antibody completely blocks both basal and basic fibroblast growth factor-stimulated movement of aortic EC. Y13-259 blocks the initiation of EC movement, as well as the continued progress of cells already in motion, suggesting that ras activity is continuously required throughout the motile process. Together these data indicate that ras is an integral component of the signaling pathway regulating cell movement.
The authors are grateful for financial support from the National Institutes of Health (grants GM23244 and GM53905), and to very helpful comments on the manuscript from Elliot Elson, Vlodya Gelfand, Paul Matsudaira, Julie Theriot, and Sally Zigmond. D. A. L. and A. F. H. would also like to thank Alan Wells, and Anna Huttenlocher and Rebecca Sandborg, respectively, for stimulating conversations on this subject, and Sean Palecek for Figure 2Figure 2. Finally, we extend our apologies to all our colleagues in the field whose work we were unable to cite formally because of imposed reference limitations.
Dbl, a guanine nucleotide exchange factor (GEF) for members of the Rho family of small GTPases, is the prototype of a family of 15 related proteins. The majority of proteins that contain a DH (Dbl homology) domain were isolated as oncogenes in transfection assays, but two members of the DH family, FGD1 (the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus) and Vav, have been shown to be essential for normal embryonic development. Mutations to the FGD1 gene result in a human developmental disorder affecting specific skeletal structures, including elements of the face, cervical vertebrae and distal extremities. Homozygous Vav-/- knockout mice embryos are not viable past the blastocyst stage, indicating an essential role of Vav in embryonic implantation.
Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1.
We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.
Yeast cells respond to mating pheromones by activating a signal transduction pathway involving a seven transmembrane receptor/G protein complex linked to a mitogen-activated protein kinase module. Regulation of the G protein signal is controlled by the receptor and Sst2p; Sst2p may function as a GTPase-activating protein for the G protein alpha subunit. The Ste20 kinase acts in the linkage between the G protein and the MAP kinase module. Experiments suggest that binding of the Rho-like GTPase Cdc42p to Ste20p is not required for the mating response, yet is needed for the pseudohyphal growth response which involves many of the same kinases.
The Rho GTPases form a subgroup of the Ras superfamily of 20- to 30-kD GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions. These proteins are ubiquitously expressed across the species, from yeast to man. The mammalian Rho-like GTPases comprise at least 10 distinct proteins: RhoA, B, C, D, and E; Rac1 and 2; RacE; Cdc42Hs, and TC10. A comparison of the amino acid sequences of the Rho proteins from various species has revealed that they are conserved in primary structure and are 50%–55% homologous to each other. Like all members of the Ras superfamily, the Rho GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. Until recently, members of the Rho subfamily were believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors. However, research from a number of laboratories over the past few years has revealed that the Rho GTPases play crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development. Consequently, a major challenge has been to unravel the underlying molecular mechanisms by which the Rho GTPases mediate these various activities. Many targets of the Rho GTPases have now been identified and further characterization of some of them has provided major insights toward our understanding of Rho GTPase function at the molecular level. This review aims to summarize the general established principles about the Rho GTPases and some of the more recent exciting findings, hinting at novel, unanticipated functions of the Rho GTPases.
The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells. In addition to providing
a structural framework around which cell shape and polarity are defined, its dynamic properties provide the driving force
for cells to move and to divide. Understanding the biochemical mechanisms that control the organization of actin is thus a
major goal of contemporary cell biology, with implications for health and disease. Members of the Rho family of small guanosine
triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with
multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion.
Directed cell movement is an immensely complex process that depends on the co-operative interaction of numerous cellular components. Work over the past three decades has suggested that microtubules play an important role in the establishment and maintenance of the direction of cell migration. This chapter summarizes recent work from our laboratory designed to determine the roles of the microtubules and centrosome position relative to the direction of cell migration in a variety of cell types, and discusses these observations in the context of work from other laboratories. The results suggest that microtubules are required for stabilization of the direction of migration in many, but not all, cell types. For the centrosome to act as a stabilizer of cell migration requires that it is repositioned behind the leading edge. However, the process of repositioning does not precede the extension of a leading edge and the establishment of a new direction of cell migration. Rather, the centrosome follows the repositioning of the leading edge in response to other stimuli and, in doing so, stabilizes cell movement.
Focal adhesions (FAs) are clustered integrins and associated proteins that mediate cell adhesion and signaling. A green fluorescent protein-beta1 integrin chimera was used to label FAs in living cells. In stationary cells, FAs were highly motile, moving linearly for several plaque lengths toward the cell center. FA motility was independent of cell density and resulted from contraction of associated actin fibers. In migrating cells, FAs were stationary and only moved in the tail. FA motility in stationary cells suggests that cell movement may be regulated by a clutch-like mechanism by which the affinity of integrins to substrate may be altered in response to migratory cues.
Cooperation between mDia1 and ROCK in Rhoinduced actin reorganization
- N Watanabe
- T Kato
- A Fujita
- T Ishizaki
- S Naruyima
Watanabe, N., Kato, T., Fujita, A., Ishizaki, T. & Naruyima, S.
1999 Cooperation between mDia1 and ROCK in Rhoinduced actin reorganization. Nature Cell Biol. 1, 136^143.
Pheromone signalling and polarised morphogenesis in yeast
- Leberer E.
The small GTP-binding protein Rac regulates growth factor-induced membrane ru¥ing
- A J Ridley
- H F Paterson
- C L Johnston
- D Diekmann
- A Hall