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BACKGROUND: A major issue in Chronic Myeloid Leukemia (CML) is the persistence of quiescent leukemia stem cells (LSCs) in the hematopoietic niche under tyrosine kinase inhibitor (TKI) treatment. RESULTS: Here, using CFSE sorting, we show that low-proliferating CD34+ cells from CML patients in 3D co-culture hide under HS27A stromal cells during TKI treatment-a behavior less observed in untreated cells. Under the same conditions, Ba/F3p210 cells lose their spontaneous motility. In CML CD34+ and Ba/F3p210 cells, while Rac1 is completely inhibited by TKI, RhoA remains activated but is unable to signal to ROCK. Co-incubation of Ba/F3p210 cells with TKI, SKF-96365 (a calcium channel inhibitor), and EGF restores myosin II activation and amoeboid motility to levels comparable to untreated cells, sustaining the activation of ROCK. In CFSE+ CD34+ cells containing quiescent leukemic stem cells, co-incubation of TKI with SKF-96365 induced the expulsion of these cells from the HS27A niche. CONCLUSIONS: This study underscores the role of RhoA in LSC behavior under TKI treatment and suggests that SKF-96365 could remobilize quiescent CML LSCs through reactivation of the RhoA/ROCK pathway.

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Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.

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The overproduction of the toxic peptide amyloid-beta (Aß) generated from the cleavage of amyloid precursor protein (APP) is proposed to be a critical event in the development of Alzheimer's disease. Evidence suggests that the cleavage of APP occurs after its internalization from the cell surface. Previously, we identified a novel pathway for APP internalization, which trafficks cell surface APP directly to lysosomes by macropinocytosis, leading to its processing into Aß. We also demonstrated that ADP-ribosylation factor 6 (Arf6) is required for the macropinocytosis of APP. Here, we characterized the roles of Arf6's downstream effectors Rac1, Cdc42 and RhoA. Both pharmacological inhibition and siRNA knockdown of these proteins reduced the amount of APP colocalized with LAMP1-labeled lysosomes without affecting APP transport to early endosomes. Decreases in the production of both Aß40 and Aß42 were also observed by ELISA in response to inhibitor treatment. These findings together demonstrate that Rac1, Cdc42 and RhoA are components of the mechanism regulating the macropinocytosis of APP and targeting these components can reduce the production of Aß.

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Microglia, the largest population of brain immune cells, continuously interact with synapses to maintain brain homeostasis. In this study, we use conditional cell-specific gene targeting in mice with multi-omics approaches and demonstrate that the RhoGTPase Rac1 is an essential requirement for microglia to sense and interpret the brain microenvironment. This is crucial for microglia-synapse crosstalk that drives experience-dependent plasticity, a fundamental brain property impaired in several neuropsychiatric disorders. Phosphoproteomics profiling detects a large modulation of RhoGTPase signaling, predominantly of Rac1, in microglia of mice exposed to an environmental enrichment protocol known to induce experience-dependent brain plasticity and cognitive performance. Ablation of microglial Rac1 affects pathways involved in microglia-synapse communication, disrupts experience-dependent synaptic remodeling, and blocks the gains in learning, memory, and sociability induced by environmental enrichment. Our results reveal microglial Rac1 as a central regulator of pathways involved in the microglia-synapse crosstalk required for experience-dependent synaptic plasticity and cognitive performance.

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Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. The statoacoustic ganglion (SAG) develops into several lobes that are spatially arranged to connect appropriately with hair cells of the inner ear. To investigate the cellular behaviours involved in the 3D organization of the SAG, we use high-resolution confocal imaging of single-cell, labelled zebrafish neuroblasts (NBs), photoconversion, photoablation, and genetic perturbations. We show that otic NBs delaminate out of the otic epithelium in an epithelial-mesenchymal transition-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NBs require RhoGTPases in order to perform active migration. Furthermore, tracking of recently delaminated NBs revealed their directed migration and coalescence around a small population of pioneer SAG neurons. These pioneer SAG neurons, not from otic placode origin, populate the coalescence region before otic neurogenesis begins and their ablation disrupts delaminated NB migratory pathways, consequentially affecting SAG shape. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development.

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Objective: Activation of Rho-GTPases in macrophages causes inflammation and severe arthritis in mice. In this study, we explore if Rho-GTPases define the joint destination of pathogenic leukocytes, the mechanism by which they perpetuate rheumatoid arthritis (RA), and how JAK inhibition mitigates these effects. Methods: CD14+ cells of 136 RA patients were characterized by RNA sequencing and cytokine measurement to identify biological processes and transcriptional regulators specific for CDC42 hiCD14+ cells, which were summarized in a metabolic signature (MetSig). The effect of hypoxia and IFN-γ signaling on the metabolic signature of CD14+ cells was assessed experimentally. To investigate its connection with joint inflammation, the signature was translated into the single-cell characteristics of CDC42 hi synovial tissue macrophages. The sensitivity of MetSig to the RA disease activity and the treatment effect were assessed experimentally and clinically. Results: CDC42 hiCD14+ cells carried MetSig of genes functional in the oxidative phosphorylation and proteasome-dependent cell remodeling, which correlated with the cytokine-rich migratory phenotype and antigen-presenting capacity of these cells. Integration of CDC42 hiCD14+ and synovial macrophages marked with MetSig revealed the important role of the interferon-rich environment and immunoproteasome expression in the homeostasis of these pathogenic macrophages. The CDC42 hiCD14+ cells were targeted by JAK inhibitors and responded with the downregulation of immunoproteasome and MHC-II molecules, which disintegrated the immunological synapse, reduced cytokine production, and alleviated arthritis. Conclusion: This study shows that the CDC42-related MetSig identifies the antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. The accumulation of CDC42 hiCD14+ cells discloses patients perceptive to the JAKi treatment.

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Thyroid cancer (TC) is one of the most common endocrine system cancers, and its incidence is elevating. There is an urgent need to develop a deeper understanding of TC pathogenesis and explore new therapeutic target for its treatment. This study aimed to investigate the effects of pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) on the progression of TC. Herein, 29 pairs of TC and adjacent tissues were used to assess the expression of PLEKHG4. A xenograft model of mouse was established by subcutaneously injected with TC cells. Lung metastasis model was established through left ventricular injection. The results revealed that PLEKHG4 was up-regulated in human TC tissues. PLEKHG4 level was correlated with clinicopathological parameters of TC patients. In vitro assays revealed that PLEKHG4 promoted TC cell proliferation, migration, invasion, and epithelial-mesenchymal transformation. Knockdown of PLEKHG4 led to the opposite effects, and the loss of PLEKHG4 enhanced the apoptosis ability and inhibited the stemness properties of TC cells. These findings were further confirmed by the in vivo growth and lung metastasis of TC tumor. Mechanistically, PLEKHG4 promoted the activation of RhoGTPases RhoA, Cdc42, and Rac1. The inhibitors of these RhoGTPases reversed the PLEKHG4-induced malignant phenotypes. Additionally, ubiquitin-conjugating enzyme E2O (UBE2O), a large E2 ubiquitin-conjugating enzyme acted as an ubiquitin enzyme of PLEKHG4, facilitated its ubiquitination and degradation. In conclusion, PLEKHG4, regulated by UBE2O, promoted the thyroid cancer progression via activating the RhoGTPases pathway. UBE2O/PLEKHG4/RhoGTPases axis is expected to be a novel a therapeutic target for TC treatment.

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The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding-proteins and nucleation-promoting factors to perform fundamental cellular functions. We have previously shown that ERK3, an atypical MAPK, controls IL-8 production and chemotaxis (Bogueka et al., 2020). Here, we show in human cells that ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent RAC1 and CDC42 activation, maintenance of F-actin content, filopodia formation, and epithelial cell migration. Further, ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. ERK3 kinase activity was required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.

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Cell proliferation is central to epithelial tissue development, repair, and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodeling to faithfully segregate the genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPase spatiotemporal regulation during epithelial cell division, we generated a transgenic fluorescently tagged library for the 48 Drosophila Rho guanine exchange factors (RhoGEFs) and GTPase-activating proteins (GAPs), and we systematically characterized their endogenous distributions by time-lapse microscopy. Therefore, we unveiled candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division. Building on these findings, we established that the conserved RhoGEF Cysts and RhoGEF4 play sequential and distinct roles to couple cytokinesis with de novo junction formation. During ring contraction, Cysts via Rho1 participates in the neighbor mechanosensing response, promoting daughter-daughter cell membrane juxtaposition in preparation to de novo junction formation. Subsequently and upon midbody formation, RhoGEF4 via Rac acts in the dividing cell to ensure the withdrawal of the neighboring cell membranes, thus controlling de novo junction length and cell-cell arrangements upon cytokinesis. Altogether, our findings delineate how the RhoGTPases Rho and Rac are locally and temporally activated during epithelial cytokinesis, highlighting the RhoGEF/GAP library as a key resource to understand the broad range of biological processes regulated by RhoGTPases.

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Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell-cell contacts. Consistent with this, endothelial junctions are defective in CD93-/- mice, and the blood-brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

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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 guanine exchange factors (GEFs), GTPase-activating proteins and guanosine dissociation inhibitors (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.

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Collective cell movements drive normal development and metastasis. Drosophila border cells move as a cluster of 6-10 cells, where the role of the Rac GTPase in migration was first established. In border cells, as in most migratory cells, Rac stimulates leading-edge protrusion. Upstream Rac regulators in leaders have been identified; however, the regulation and function of Rac in follower border cells is unknown. Here, we show that all border cells require Rac, which promotes follower-cell motility and is important for cluster compactness and movement. We identify a Rac guanine nucleotide exchange factor, Cdep, which also regulates follower-cell movement and cluster cohesion. Scribble, Discs large, and Lethal giant larvae localize Cdep basolaterally and share phenotypes with Cdep. Relocalization of Cdep::GFP partially rescues Scribble knockdown, suggesting that Cdep is a major downstream effector of basolateral proteins. Thus, a Scrib/Cdep/Rac pathway promotes cell crawling and coordinated, collective migration in vivo.

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The C57BL/6.NOD-Aec1Aec2 mouse has been extensively studied to define the underlying cellular and molecular basis for the onset and development of Sjögren's syndrome (SS), a human systemic autoimmune disease characterized clinically as the loss of normal lacrimal and salivary gland functions leading respectively to dry eye and dry mouth pathologies. While an overwhelming majority of SS studies in both humans and rodent models have long focused primarily on pathophysiological events and the potential role of T lymphocytes in these events, recent studies in our murine models have indicated that marginal zone B (MZB) lymphocytes are critical for both development and onset of SS disease. Although migration and function of MZB cells are difficult to study in vivo and in vitro, we have carried out ex vivo investigations that use temporal global RNA transcriptomic analyses to track early cellular and molecular events in these exocrine glands of C57BL/6.NOD-Aec1Aec2 mice. In the present report, genome-wide transcriptome analyses of lacrimal glands indicate that genes and gene-sets temporally upregulated during early onset of disease define the Notch2/NF-kß14 and Type1 interferon signal transduction pathways, as well as identify chemokines, especially Cxcl13, and Rho-GTPases, including DOCK molecules, in the cellular migration of immune cells to the lacrimal glands. We discuss how the current results compare with our recently published salivary gland data obtained from similar studies carried out in our C57BL/6.NOD-Aec1Aec2 mice, pointing out both similarities and differences in the etiopathogeneses underlying the autoimmune response within the two glands. Overall, this study uses the power of transcriptomic analyses to identify temporal molecular bioprocesses activated during the preclinical covert pathogenic stage(s) of SS disease and how these findings may impact future intervention therapies as the disease within the two exocrine glands may not be identical.

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Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

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Leukocyte trafficking is an essential process of immunity, occurring as leukocytes travel within the bloodstream and as leukocyte migration within tissues. While it is now established that leukocytes can utilize the mesenchymal migration mode or amoeboid migration mode, differences in the migratory behavior of leukocyte subclasses and how these are realized on a molecular level in each subclass is not fully understood. To outline these differences, first migration modes and their dependence on parameters of the extracellular environments will be explained, as well as the intracellular molecular machinery that powers migration in general. Extracellular parameters are detected by adhesion receptors such as integrins. ß2-integrins are surface receptors exclusively expressed on leukocytes and are essential for leukocytes exiting the bloodstream, as well as in mesenchymal migration modes, however, integrins are dispensable for the amoeboid migration mode. Additionally, the balance of different RhoGTPases - which are downstream of surface receptor signaling, including integrins - mediate formation of membrane structures as well as actin dynamics. Individual leukocyte subpopulations have been shown to express distinct RhoGTPase profiles along with their differences in migration behavior, which will be outlined. Emerging aspects of leukocyte migration include signal transduction from integrins via actin to the nucleus that regulates DNA status, gene expression profiles and ultimately leukocyte migratory phenotypes, as well as altered leukocyte migration in tumors, which will be touched upon.

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Breast cancer affects one in eight women during their lifetime. Although diagnostic and therapeutic options have improved, recurrence, metastasis, and therapeutic resistance remain clinical challenges, which affect life quality and prognosis. The mevalonate pathway is an essential part of cellular homeostasis by providing a number of essential isoprenoid products including cholesterol. However, the disturbance of this pathway paralleled by increased bioavailability of its products and their direct involvement in several steps of tumorigenesis has highlighted the mevalonate pathway as a promising hub in cancer treatment. In this review, we will specifically discuss how the mevalonate pathway affects breast cancer biology in terms of supporting and modulating soluble and cellular factors and distinct steps of tumorigenesis. We will further summarize antitumor effects of the mevalonate pathway-inhibiting drugs, statins and amino-bisphosphonates, in breast cancer and discuss how they are used for future precision therapy.

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YAP/TAZ are transcription co-factors recently described responsive to pro-inflammatory cytokines and involved in inflammatory-related disorders. However, the role of tumor necrosis factor (TNF), a major pro-inflammatory cytokine, on YAP signaling is not well understood and controversial. Here, we observe in vitro, using wild type and YAP knockout HEK293 cells, that TNF triggers YAP nuclear translocation and transcriptional activity, thus being dependent on Rho family of GTPases. In response to TNF, YAP transcriptional activity orientates cell fate toward survival. Transcriptional analysis with Nanostring technology reveals that YAP modulates TNF-induced increase in fibro-inflammatory pathways such as NF-κB, inflammasomes, cytokines or chemokines signaling and pro-fibrotic pathways involving TGF-ß and extracellular matrix remodeling. Therefore, in response to TNF, YAP acts as a sustainer of the inflammatory response and as a molecular link between inflammation and fibrotic processes. This work identifies that YAP is critical to drive several biological effects of TNF which are involved in cancer and inflammatory disorders.

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GTPase activating proteins (GAPs) were initially considered as the inhibitors of cell signaling pathways because of their nature to activate the intrinsic GTPase activity of the RhoGTPases. But recent studies of dysregulated GAPs in many cancers such as glioblastoma, colorectal cancer, breast cancer, and renal cancer have elucidated the important roles of GAPs in carcinogenesis and GAPs have been shown to perform multiple nonconventional functions in different contexts. We have discussed the recent developments in the roles played by different types of srGAPs (SLIT-ROBO Rho GTPase-activating proteins) in cancer.

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The cytotoxic necrotizing factors (CNFs) are a family of Rho GTPase-activating single-chain exotoxins that are produced by several Gram-negative pathogenic bacteria. Due to the pleiotropic activities of the targeted Rho GTPases, the CNFs trigger multiple signaling pathways and host cell processes with diverse functional consequences. They influence cytokinesis, tissue integrity, cell barriers, and cell death, as well as the induction of inflammatory and immune cell responses. This has an enormous influence on host-pathogen interactions and the severity of the infection. The present review provides a comprehensive insight into our current knowledge of the modular structure, cell entry mechanisms, and the mode of action of this class of toxins, and describes their influence on the cell, tissue/organ, and systems levels. In addition to their toxic functions, possibilities for their use as drug delivery tool and for therapeutic applications against important illnesses, including nervous system diseases and cancer, have also been identified and are discussed.

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The hyaluronan-based extracellular matrix is expressed throughout nervous system development and is well-known for the formation of perineuronal nets around inhibitory interneurons. Since perineuronal nets form postnatally, the role of hyaluronan in the initial formation of neural circuits remains unclear. Neural circuits emerge from the coordinated electrochemical signaling of excitatory and inhibitory synapses. Hyaluronan localizes to the synaptic cleft of developing excitatory synapses in both human cortical spheroids and the neonatal mouse brain and is diminished in the adult mouse brain. Given this developmental-specific synaptic localization, we sought to determine the mechanisms that regulate hyaluronan synthesis and signaling during synapse formation. We demonstrate that hyaluronan synthase-2, HAS2, is sufficient to increase hyaluronan levels in developing neural circuits of human cortical spheroids. This increased hyaluronan production reduces excitatory synaptogenesis, promotes inhibitory synaptogenesis, and suppresses action potential formation. The hyaluronan receptor, CD44, promotes hyaluronan retention and suppresses excitatory synaptogenesis through regulation of RhoGTPase signaling. Our results reveal mechanisms of hyaluronan synthesis, retention, and signaling in developing neural circuits, shedding light on how disease-associated hyaluronan alterations can contribute to synaptic defects.

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