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Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia

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Abstract

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.

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... These protrusions, called filopodia or lamellipodia, are bounded to extracellular matrix via focal adhesions, composed of several proteins such as Focal Adhesion Kinase (FAK), Paxilin or Src [3]. Focal adhesion are highly dynamic structures regulated by the RhoGTPase family proteins [4]. Activated RhoGTPases modulate notably actin cytoskeleton organization and migration processes [5,6]. ...
... Type 3 Inositol (1,4,5)-Trisphosphate Receptor: a migratory modulator with a unique calcium signature in breast cancer cells. Vautrin-Glabik A., XIV International Meeting of the European Calcium Society, Valladolid, Spain 09/2016 Impact du niveau d'expression du récepteur de l'inositol 1,4,5-trisphosphate de type 3 (IP 3 R3)sur la capacité migratoire et la signature calcique de cellules cancéreuses mammaires humaines. ...
... The Physiological Society, Cardiff, 07/2015 IP 3 R3 silencing induced cytoskeletal reorganization through RhoA / FAK / Actin pathway in breast cancer cell line. Vautrin-Glabik A., et al, Soumis à BBA Molecular Cell Research, 12/2017 Type 3 Inositol(1,4,5)-Trisphosphate Receptor regulates breast cancer cell migration through an oscillatory calcium signal.Mound A, Vautrin-Glabik A., et al. Oncotarget, 8(42):72324-41, 2017 The anti-tumor NC1 domain of collagen XIX inhibits the FAK/PI3K/Akt/mTOR signaling pathway through αvβ3 integrin interaction. ...
Thesis
Le cancer du sein est le cancer féminin le plus fréquent et le plus létal chez la femme dans le monde. Malgré l'amélioration du dépistage dans les phases précoces du développement tumoral, il demeure difficile de traiter les phases tardives lorsque les processus métastatiques sont engagés. Le développement métastatique dépend notamment de l'acquisition de capacités migratoires par les cellules épithéliales impliquant un remodelage du cytosquelette, hautement dépendant de la concentration calcique intracellulaire. Alors que les travaux se sont intéressés à l'implication des canaux ioniques membranaires dans les processus de migration, le rôle des récepteurs à l'inositol 1,4,5-trisphosphate (IP₃Rs) reste peu étudié et donc méconnu. Dans un premier temps nous avons montré une augmentation du niveau d'expression d'IP₃R3 avec le niveau du potentiel migratoire de trois lignées cancéreuses mammaires humaines : MCF-7 (les moins migrantes), MDA-MB-231 et MDA-MB-435s (les plus migrantes). D'autre part, nous montrons que la modulation de l'expression d'IP₃R3 module leurs capacités migratoires: elles sont diminuées par l'inhibition d'IP₃R3, alors qu'elles sont augmentées par la surexpression d'IP₃R3 dans les cellules mammaires peu migrantes. De plus, l'inhibition d'IP₃R3 révèle un signal calcique oscillant, alors que sa surexpression induit un signal calcique maintenu dans ces cellules. Dans un second temps, nous avons mis en évidence une corrélation inverse entre le niveau d'expression d'IP₃R3 et la morphologie arrondie de ces trois lignées cellulaires. En effet, plus la morphologie cellulaire est arrondie, plus l'expression d'IP₃R3 est importante. Par ailleurs, l'inhibition d'IP₃R3 induit un arrondissement des cellules migrantes et une diminution des protrusions membranaires accompagnés d’une diminution de leur adhésion. Ces résultats suggèrent fortement l'implication d'IP₃R3 dans la modulation des acteurs du cytosquelette. En effet, l'inhibition d'IP₃R3 induit une diminution de l'expression de l'ARHGAP18, de l'activité de RhoA et de l'expression de Cdc42. Ces Rho GTPases permettent à leur tour une diminution de la phosphorylation de FAK_⁸⁶¹ et la réorganisation du cytosquelette d'actine et de profiline. Enfin, dans un modèle migratoire, le profil oscillant s'établit dès réalisation d'une blessure et prédomine 3 h après dans les cellules du front de migration alors qu'il se met en place dans les cellules à l'arrière uniquement après 3 h. L'intervalle spatio-temporel de ce signal calcique oscillant reflète une dynamique calcique intracellulaire nécessaire aux processus migratoires et au remodelage du cytosquelette des cellules du cancer du sein. En conclusion, nos résultats révèlent un rôle clé de l'IP₃R3 dans les processus migratoires et dans le remodelage du cytosquelette de profilactine via la voie ARHGAP18/ RhoA/ FAK associé à une modulation du profil calcique intracellulaire
... Hippo signaling can be ON or OFF [50,141]. If Hippo are ON, this is corresponding to YAP/TAZ being inactivated. ...
... Ebata et al. reported the importance of Rho family activation in cancer metastasis [50]. Rho GTPases, such as Rho, Ras-related C3 botulinum toxin substance 1 (Rac), and cell division control protein 42 homolog (Cdc42) regulate the activation of actin regulators, contributing to the formation of filopodia, lamellipodia, and stress fibers, respectively [141]. The formation of actin-mediated structures, such as lamellipodia, filopodia, podosomes, and invadopodia, is associated with the invasion of cancer cells [140,141]. ...
... Rho GTPases, such as Rho, Ras-related C3 botulinum toxin substance 1 (Rac), and cell division control protein 42 homolog (Cdc42) regulate the activation of actin regulators, contributing to the formation of filopodia, lamellipodia, and stress fibers, respectively [141]. The formation of actin-mediated structures, such as lamellipodia, filopodia, podosomes, and invadopodia, is associated with the invasion of cancer cells [140,141]. Rho signaling is related to the ECM stiffness and the ECM proteins have an important role in the activation of several biochemical pathways. Gagné et al. recently demonstrated that integrin-linked kinase (ILK) specifically induced the initiation of fibronectin, fibrillogenesis, during cell spreading, promoting the Rho/ROCK-dependent cell contractility and maturation of the integrin-actin structures [151]. ...
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In this review, the mechanobiology of colorectal cancer (CRC) are discussed. Mechanotransduction of CRC is addressed considering the relationship of several biophysical cues and biochemical pathways. Mechanobiology is focused on considering how it may influence epithelial cells in terms of motility, morphometric changes, intravasation, circulation, extravasation, and metastization in CRC development. The roles of the tumor microenvironment, ECM, and stroma are also discussed, taking into account the influence of alterations and surface modifications on mechanical properties and their impact on epithelial cells and CRC progression. The role of cancer-associated fibroblasts and the impact of flow shear stress is addressed in terms of how it affects CRC metastization. Finally, some insights concerning how the knowledge of biophysical mechanisms may contribute to the development of new therapeutic strategies and targeting molecules and how mechanical changes of the microenvironment play a role in CRC disease are presented.
... The small GTPases Rho, Rac, and Cdc42, govern the formation of actin stress fibers, lamellipodia, and filopodia, respectively [87]. In humans, there are 20 members in the Rho family which are subdivided into subfamilies: Rho, Rac, Cdc42, RhoU/V, RhoD/F, Rnd, RhoH, and RhoBTB [88]. ...
... The small GTPases, RhoA and Rac1, are particularly well-studied as regulators of the endothelial barrier function, where RhoA and Rac1 activation leads to barrier disruption and stabilization, respectively [85,[92][93][94][95]. The small GTPases Rho, Rac, and Cdc42, govern the formation of actin stress fibers, lamellipodia, and filopodia, respectively [87]. In humans, there are 20 members in the Rho family which are subdivided into subfamilies: Rho, Rac, Cdc42, RhoU/V, RhoD/F, Rnd, RhoH, and RhoBTB [88]. ...
... ECs are known to form lamellipodia, which are protrusive actin-based structures [102]. Lamellipodia assembly is mainly governed by Rac1 [87,88]. Lamellipodia push the plasma membrane out from the cell interior, and this pressure can increase or decrease the energy required for the activation of any endothelial mechanosensory by SS. ...
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Chronic low-grade vascular inflammation and endothelial dysfunction significantly contribute to the pathogenesis of cardiovascular diseases. In endothelial cells (ECs), anti-inflammatory or pro-inflammatory signaling can be induced by different patterns of the fluid shear stress (SS) exerted by blood flow on ECs. Laminar blood flow with high magnitude is anti-inflammatory, while disturbed flow and laminar flow with low magnitude is pro-inflammatory. Endothelial mechanosensors are the key upstream signaling proteins in SS-induced pro- and anti-inflammatory responses. Being transmembrane proteins, mechanosensors, not only experience fluid SS but also become regulated by the biomechanical properties of the lipid bilayer and the cytoskeleton. We review the apparent effects of pro-inflammatory factors (hypoxia, oxidative stress, hypercholesterolemia, and cytokines) on the biomechanics of the lipid bilayer and the cytoskeleton. An analysis of the available data suggests that the formation of a vicious circle may occur, in which pro-inflammatory cytokines enhance and attenuate SS-induced pro-inflammatory and anti-inflammatory signaling, respectively.
... Maximal activation ranges from few seconds to several minutes, with a decline in the activity thereafter. Activation of Rac1, Cdc42 or RhoA results in characteristic changes in the actin cytoskeleton 25,43 . For instance, Rac1 activation induces the formation of actin-rich lamellipodia and membrane ruffles 43 . ...
... Activation of Rac1, Cdc42 or RhoA results in characteristic changes in the actin cytoskeleton 25,43 . For instance, Rac1 activation induces the formation of actin-rich lamellipodia and membrane ruffles 43 . When Cdc42 is active, prominent membrane extensions termed filopodia and smaller microspikes are formed, whereas RhoA activation results in increased formation of stress fibers 43 . ...
... For instance, Rac1 activation induces the formation of actin-rich lamellipodia and membrane ruffles 43 . When Cdc42 is active, prominent membrane extensions termed filopodia and smaller microspikes are formed, whereas RhoA activation results in increased formation of stress fibers 43 . Hence, we first optimized the timepoint in which we could observe the formation of actin-rich structures in response to the presence of either melanocores or melanosomes. ...
Article
In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred – either in a membrane‐bound melanosome or as a melanosome core, i.e., melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin‐dependent uptake mechanisms, namely Rac1, CtBP1/BARS, Cdc42 or RhoA, together with inhibition of Rac1‐dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct internalization routes of melanin internalization. Furthermore, we show that melanocore uptake induces Protease‐activated receptor‐2 (PAR‐2) internalization by keratinocytes to a higher extent than melanosomes. Since skin pigmentation was shown to be regulated by PAR‐2 activation, our results further support the melanocore‐based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.
... In 3D collagen gels, a "leader" glioblastoma cell reorganizes the collagen and creates a track for invasive glioblastoma cells to follow [33]. These cells generate leading pseudopodia, which are short-lived, actin-rich membrane protrusions, and filopodia, which are long-lived, finger-like protrusions [34]. Generation of these extensions that explore the environment is thought to be the mechanism in vivo to move along the white-matter tracts and blood vessels. ...
... Taken together, better understanding of In 3D collagen gels, a "leader" glioblastoma cell reorganizes the collagen and creates a track for invasive glioblastoma cells to follow [33]. These cells generate leading pseudopodia, which are short-lived, actin-rich membrane protrusions, and filopodia, which are long-lived, finger-like protrusions [34]. Generation of these extensions that explore the environment is thought to be the mechanism in vivo to move along the white-matter tracts and blood vessels. ...
... Especially, Ephrins and Ephrin receptors have an important role in bidirectional signaling in glioblastoma invasion. Ephrins mediate cell-to-cell signaling and control tissue organization, and the Ephrin pathway is overactivated in invasive glioblastoma cells [34]. Overactivation of Ephrin pathway elements, especially EphA2 and EphA3, has been associated with invasiveness and poor patient outcomes [34,68]. ...
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Glioblastoma is the most common and malignant primary brain tumor, defined by its highly aggressive nature. Despite the advances in diagnostic and surgical techniques, and the development of novel therapies in the last decade, the prognosis for glioblastoma is still extremely poor. One major factor for the failure of existing therapeutic approaches is the highly invasive nature of glioblastomas. The extreme infiltrating capacity of tumor cells into the brain parenchyma makes complete surgical removal difficult; glioblastomas almost inevitably recur in a more therapy-resistant state, sometimes at distant sites in the brain. Therefore, there are major efforts to understand the molecular mechanisms underpinning glioblastoma invasion; however, there is no approved therapy directed against the invasive phenotype as of now. Here, we review the major molecular mechanisms of glioblastoma cell invasion, including the routes followed by glioblastoma cells, the interaction of tumor cells within the brain environment and the extracellular matrix components, and the roles of tumor cell adhesion and extracellular matrix remodeling. We also include a perspective of high-throughput approaches utilized to discover novel players for invasion and clinical targeting of invasive glioblastoma cells.
... Activated RAC, RHO bind to and activate the Wiskott-Aldrich syndrome protein (WASp) fami activation, members of the WASp family bind to and activate the Arp2/3 c mately leading to actin polymerization. RAC, RHO, and CDC42 function as lators of actin stress fiber dynamics [38]. CDC42 can function ahead of RA ultimately leading to the sequential activation of RAC and RHO, respectively of the RHO family of GTPases using Clostridium difficile toxin B prevents act polymerization and endocytosis, highlighting the importance of these sma the regulation of actin dynamics and endocytic uptake [39]. ...
... Following activation, members of the WASp family bind to and activate the Arp2/3 complex, ultimately leading to actin polymerization. RAC, RHO, and CDC42 function as central regulators of actin stress fiber dynamics [38]. CDC42 can function ahead of RAC and RHO, ultimately leading to the sequential activation of RAC and RHO, respectively. ...
... Invasion was not restored by replenishing cholesterol [40] with this concentration of simvastatin, a lower concentration than that of early work showing apoptotic responses to simvastatin [93]. Multiple effects are rendered through small GTPases, including CDC42, a CaaX domain-containing host cell protein that relies on post-translational prenylation for membrane localization [38,94] (Figure 3). As prenylation decreases following treatment with simvastatin, CDC42 becomes sequestered in the cytoplasm, no longer anchored at the host cell membrane [40]. ...
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An emergent approach to bacterial infection is the use of host rather than bacterial-directed strategies. This approach has the potential to improve efficacy in especially challenging infection settings, including chronic, recurrent infection due to intracellular pathogens. For nearly two decades, the pleiotropic effects of statin drugs have been examined for therapeutic usefulness beyond the treatment of hypercholesterolemia. Interest originated after retrospective studies reported decreases in the risk of death due to bacteremia or sepsis for those on a statin regimen. Although subsequent clinical trials have yielded mixed results and earlier findings have been questioned for biased study design, in vitro and in vivo studies have provided clear evidence of protective mechanisms that include immunomodulatory effects and the inhibition of host cell invasion. Ultimately, the benefits of statins in an infection setting appear to require attention to the underlying host response and to the timing of the dosage. From this examination of statin efficacy, additional novel host-directed strategies may produce adjunctive therapeutic approaches for the treatment of infection where traditional antimicrobial therapy continues to yield poor outcomes. This review focuses on the opportunistic pathogen, Staphylococcus aureus, as a proof of principle in examining the promise and limitations of statins in recalcitrant infection.
... Rho GTPases can also be differentially lipidated, which would alter their interaction with membranes [Hodge and Ridley, 2016]. The classical view of the mode of action of the three canonical Rho GTPases (Rac1, Cdc42 and RhoA) was that they were regulating actin remodelling in the cell and they were each responsible for one type of structure -Rac1 for formation of lamellipodia [Michiels et al., 1997], Cdc42 for initiation of filopodia [Nobes and Hall, 1995] and RhoA for stress fibers [Chrzanowska- Wodnicka and Burridge, 1996]. However, once it was possible to spatiotemporally map Rho GTPase activation [Fritz and Pertz, 2016,Pertz, 2010a,Machacek et al., 2009,Pertz et al., 2006, it became clear that the "Rho GTPasecentric" view, where one Rho GTPase is sequentially regulated by GEFs, GAPs and GDIs and is responsible for singular functions, is too simplified. ...
... One of the first Cdc42 functional descriptions in migrating 3T3 fibroblasts was achieved by injecting cells with active Cdc42. Within 5 min cells started forming filopodia -long, finger-like actin protrusions [Nobes and Hall, 1995]. Cdc42 would signal the formation of filopodia through its effectors -formin families mDia and FMNL, and TOCA-1-N-WASP complex [Kühn and Geyer, 2014, Kühn et al., 2015, Watson et al., 2017, Bu et al., 2009. ...
Thesis
When cells migrate, they need to coordinate multiple signaling pathways in space and time to orchestrate the dynamics of their cytoskeleton and the distribution of their internal organelles. Yet, it remains unclear how the different signaling programs are coupled, and to what extent a proper cell polarity is required for migration. Such questions are difficult to answer because of the numerous feedbacks in the cell signaling circuitry. During my thesis, using dynamic micropatterning, optogenetics, pharmacological and trafficking assays, and live-cell imaging, I found that a feedback between RhoGTPases’ biochemical gradients -that dictate local membrane activity- and cell internal organization -that defines the global polarity axis of the cell- allows cell migration to be persistent. I first demonstrated that Golgi complex positioning is instructive for the selection of the direction of movement using a well-controlled dynamic micropatterning assay. Second, by pharmacologically dispersing Golgi complex and disrupting polarized secretion, I showed that persistent migration breaks down and becomes random. Third, I found evidence that the Golgi orientation biases protruding activity, most likely by directing the secretion of adhesive molecules. Fourth, by applying sustained optogenetic perturbations, I proved that a localized Cdc42 RhoGTPase gradient is able to orient the Golgi complex. Taken together, I propose a model of persistent cell migration based on the coupling between protrusion dynamics, driven by RhoGTPases’ activities, and polarized secretion, biased by organelle positioning.
... There are more than 20 Rho family members, of which RhoA, Rac1, and Cdc42 are the most studied ones. The classical view of Rho GTPase came from microinjections of the active mutants of RhoA, Rac1, or Cdc42, which induced formation of stress fiber, lamellipodia, or filopodia, respectively (Nobes and Hall, 1995;. ...
... Microinjection of an active mutant of Cdc42 induced filopodia formation (Nobes and Hall, 1995). To induce actin reorganization, active Cdc42 interacts with N-Wiskott-Aldrich syndrome protein (N-WASP), releasing C-terminal of N-WASP for interaction with Actin-Related Proteins (Arp) 2/3 complex. ...
Thesis
Most cellular membranes contain phospholipids (PL) with saturated and mono-unsaturated acyl chains. However, polyunsaturated PL (PUPL) are abundant membrane components in some specialized cells, notably neurons and endothelial cells. Previously, we showed that docosahexaenoic acid (DHA), a polyunsaturated fatty acid that belongs to the omega 3 family, reduced the energy cost for membrane tubulation, thereby facilitating vesicle formation in endocytosis processes. Here, we investigate the effects of DHA in PL on two distinct bacterial invasion processes in endothelial cells, which rely on either RhoA GTPase inactivation or the activation of Rho GTPases. The first process is bacterial-toxin induced formation of transendothelial cell macroapertures (TEMs) or tunnels, which represents a novel path for bacterial extravasation in tissues through the endothelium. The second process is extraintestinal pathogenic Escherichia coli (ExPEC) invasion of endothelial cells. We modified the lipidic profile of PL in terms of acyl chains in Human Umbilical Vein Endothelial Cells (HUVEC) by defined fatty acid diets. Under DHA-diet conditions where the level of DHA-PL in the plasma membrane increased, we observed that the frequency of TEM openings increased in cells treated with the RhoA inactivating C3 exoenzyme from Clostridium botulinum. The facilitating effect on TEM nucleation was attributed to the thinner cell thickness resulting from DHA-PL enrichment. DHA-PL-enriched environment also triggered an increase of the density of TEMs that display smaller apertures and shorter lifetimes. The product of TEMs maximal size by their number remained constant, indicating the total area occupied by TEMs in a cell remains similar. We therefore show a homeostatic regulation in the host cell in order to balance the membrane tension to prevent cell rupture induced by bacterial toxins. As a broader evidence of the importance of PULP-levels in membranes, we found that increase of DHA-PL at the plasma membrane reduced bacterial toxin-induced invasion of endothelial cells by uropathogenic E. coli. Altogether, our results shed light on the role of DHA-containing PUPL on actin-driven large-scale plasma membrane deformations triggered by two families of bacterial toxins, which jeopardize Rho GTPase signaling to allow cell or tissue invasion by pathogenic bacteria.
... Through these two domains, ExoS targets cytoskeletal components in different host cell types, including neutrophils, leukocytes, and epithelial cells [89]. The N-terminal Rho GTPases are critical for actin polymerization and cytoskeletal dynamics [90]. The N-terminal domain of ExoS is a mimic of eukaryotic RhoGAP domain so that it can prevent small GTPases Rho, Rac, and Cdc42 from activation by keeping them in inactivate GDPbound form [31,87,91]. Expression of the N-terminal RhoGAP domain in cultured cells stimulates reorganization of actin stress fibers, contributing to the collapse of the actin cytoskeleton and rounded cellular phenotype [31]. ...
... Through these two domains, ExoS targets cytoskeletal components in different host cell types, including neutrophils, leukocytes, and epithelial cells [89]. The N-terminal Rho GTPases are critical for actin polymerization and cytoskeletal dynamics [90]. The Nterminal domain of ExoS is a mimic of eukaryotic RhoGAP domain so that it can prevent small GTPases Rho, Rac, and Cdc42 from activation by keeping them in inactivate GDPbound form [31,87,91]. Expression of the N-terminal RhoGAP domain in cultured cells stimulates reorganization of actin stress fibers, contributing to the collapse of the actin cytoskeleton and rounded cellular phenotype [31]. ...
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Pseudomonas (P.) aeruginosa is an opportunistic pathogen that causes serious infections and hospital-acquired pneumonia in immunocompromised patients. P. aeruginosa accounts for up to 20% of all cases of hospital-acquired pneumonia, with an attributable mortality rate of ~30–40%. The poor clinical outcome of P. aeruginosa-induced pneumonia is ascribed to its ability to disrupt lung barrier integrity, leading to the development of lung edema and bacteremia. Airway epithelial and endothelial cells are important architecture blocks that protect the lung from invading pathogens. P. aeruginosa produces a number of virulence factors that can modulate barrier function, directly or indirectly, through exploiting cytoskeleton networks and intercellular junctional complexes in eukaryotic cells. This review summarizes the current knowledge on P. aeruginosa virulence factors, their effects on the regulation of the cytoskeletal network and associated components, and molecular mechanisms regulating barrier function in airway epithelial and endothelial cells. A better understanding of these processes will help to lay the foundation for new therapeutic approaches against P. aeruginosa-induced pneumonia.
... Our data demonstrate that mGBP-2 inhibits cell migration by altering the activity of members of the Rho family of GTPases, master regulators of the actin cytoskeleton [44]. The 67NR cells which express mGBP-2 were of a more mesenchymal appearance than the 4T1 cells, which grew in tightly associated colonies (data not shown). ...
... Interestingly, when mGBP-2 levels were significantly reduced in 67NR cells, the morphology became rounder ( Figure 5) and the presence of lamellipodia were more common ( Figure 5). These are features associated with activation of Rac1 [44]. Consistent with this, Rac1 activity was lost in the presence of mGBP-2 but when mGBP-2 was absent was robustly activated ( Figure 6). ...
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Breast cancer is the most common cancer in women. Despite advances in early detection and treatment, it is predicted that over 43,000 women will die of breast cancer in 2021. To lower this number, more information about the molecular players in breast cancer are needed. Guanylate-Binding Protein-2 has been correlated with better prognosis in breast cancer. In this study, we asked if the expression of GBP-2 in breast cancer merely provided a biomarker for improved prognosis or whether it actually contributed to improving outcome. To answer this, the 4T1 model of murine breast cancer was used. 4T1 cells themselves are highly aggressive and highly metastatic, while 67NR cells, isolated from the same tumor, do not leave the primary site. The expression of GBP-2 was examined in the two cell lines and found to be inversely correlated with aggressiveness/metastasis. Proliferation, migration, and invadosome formation were analyzed after altering the expression levels of GBP-2. Our experiments show that GBP-2 does not alter the proliferation of these cells but inhibits migration and invadosome formation downstream of regulation of Rho GTPases. Together these data demonstrate that GBP-2 is responsible for cell autonomous activities that make breast cancer cells less aggressive.
... Fingerprints of UPR activation were reported during the brain development in mouse, Caenorhabditis elegans and D. melanogaster models (reviewed previously 40 ). Interestingly, PERK expression also affects brain development at the level of neurogenesis and the generation of intermediate progenitors and projection neurons of the brain cortex 41 . Moreover, the alternative activity of IRE1α described here may be relevant in the context of other diseases. ...
... This underlines a mutual antagonistic role between RHO-family GTPases RAC1 and RHO/ROCK that display exclusive functions in mesenchymal and amoeboid-like migration respectively [38][39][40]. By modulating these RHO GTPases, several molecules as such FILGAPP and ARHGAP22 control the mesenchymal/amoeboid switch in cancer cells [34,40,41]. Importantly, the key marker of the amoeboid invasiveness is its independence from ECM proteolytic degradation [35,36]. ...
Thesis
Tumor cells are exposed to cell-intrinsic and extrinsic perturbations that alter the proper functioning of the endoplasmic reticulum (ER); a cellular condition known as ER stress. This condition engages an adaptive response termed as unfolded protein response (UPR). IRE1 is the most evolutionary conserved ER stress sensor of the UPR. Activation of the IRE1 RNase activity controls the expression of the transcription factor XBP1s and the degradation of mRNA through a process termed RIDD. IRE1 activity has been linked to cell migration and invasion in different types of tumors. However, no evidence regarding the role of IRE1 in melanoma cell migration/invasion has been published. Importantly in 2018 our group described a novel function of IRE1 independent of its catalytic activity, where IRE1 acted as a scaffold favoring cell migration through FLNA, an actin crosslinking protein involved in cell migration. This was demonstrated in non-tumoral cells. Therefore, in this thesis, we initially aimed to uncover the contribution of IRE1/FLNA signaling in cell migration and invasion in melanoma cells, its impact on the metastatic process. By using genetic and pharmacologic approaches, we found that deficiency of IRE1 expression or RNase activity inhibition enhances cell migration and invasion of human metastatic melanoma cell lines, indicating that the IRE1 branch could be acting as a suppressor of cell migration and invasion in metastatic melanoma cells. Notably, we were not able to corroborate the IRE1-dependent phosphorylation of FLNA. Importantly, processes that are known to be regulated by FLNA like actin cytoskeleton and cell adhesion were not affected by IRE1 depletion in human metastatic melanoma cells. These findings suggest that the regulation of cell migration/invasion by IRE1 is an FLNA-independent mechanism. Analyzing a gene expression database of melanoma tumors, we found that tumors identified with high RIDD activity presented a significant decrease in the expression of genes involved in melanoma metastasis. Our findings suggest that IRE1 through RIDD acts as a suppressor of metastatic melanoma cell migration and invasion. The results obtained in this thesis constitute the first approximation on the implication of IRE1 in metastasis-related processes in melanoma, such as cell migration and invasion.
... The alteration of cell phenotypes across cell cycle stages should be inexorable from a molecular-level perspective. Under such a condition, there must be certain molecules that connect the cell cycle to cell dynamics beyond Rho GTPases, the primary molecular switches for cytoskeletal remodeling 10 . These molecules must be associated with regulators of the cell cycle, the cyclin-dependent kinases (CDKs) 6,7 . ...
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Heterogeneity of cell phenotypes remains a barrier in progressing cell research and a challenge in conquering cancer-related drug resistance. Cell morphology, the most direct property of cell phenotype, evolves along the progression of the cell cycle; meanwhile, cell motility, the dynamic property of cell phenotype, also alters over the cell cycle. However, a quantifiable research understanding the relationship between the cell cycle and cell migration is missing. Herein, we coordinate the migratory behaviours of NIH 3T3 fibroblasts to their corresponding phases of the cell cycle, the G1, the S, and the G2 phases, and explain the relationship through the spatiotemporal arrangements between the Rho GTPases’ signals and cyclin-dependent kinase inhibitors, p21Cip1, and p27Kip1. Taken together, we demonstrate that both cell morphology and the dynamic subcellular behaviour are homogenous within each stage of the cell cycle phases but heterogenous between phases through quantitative cell analyses and an interactive molecular mechanism between the cell cycle and cell migration, posing potential implications in countering drug resistance.
... Upon activation of T cells, cytoskeletal actin reorganizes dramatically, resulting in protrusion of filopodia and lamellipodia [37]. Signaling through CD28 can induce filopodia formation [38]. ...
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Enhanced HSV-1 production is found in activated T-lymphocytes, but the mechanism is still unknown. In this paper, the HSV-1 entry step in CD3+CD4−CD8−Jurkat T lymphocytes was investigated. Observation under electron microscopy revealed the level of filopodia formation on the surface of activated Jurkat cells was significantly higher than that of non-activated Jurkat cells especially after adding HSV-1 for 15 min. A significant increase of actin protein was demonstrated in HSV-1 infected, activated Jurkat cells compared to HSV-1 infected, non-activated Jurkat cells. After the cells were treated with 2.5 and 5 µg/mL cytochalasin D, an inhibitor of actin polymerization that causes depolymerization of actin’s filamentous form, the actin protein was decreased significantly, resulting in an absence of filopodia formation. In summary, this is the first study revealing that HSV-1 induced filopodia formation through actin polymerization in activated T cells similar to epithelial, mucosal and neuronal cells. This phenomenon supported the virus entry resulting to increased yield of HSV-1 production.
... Three Rac1, Rac2, Rac3 share 89% sequence similarity, except the C-terminal region (3,6), whereas RhoG shares only 70%-72% sequence similarity with the other three Racs and may thus act differently within the subfamily. Typically, Rac-subfamily proteins stimulate the formation of membrane ruffles and lamellipodia by interacting with a panel of effector proteins, such as Wiskott-Aldrich syndrome protein (WASP) and p21activated kinases (PAK) (7,8). These interactions activate the Arp2/3 complex and subsequently induce actin polymerization (9)(10)(11)(12). ...
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The role of RhoG in T cell development is redundant with other Racs subfamily members, and this redundancy may be attributed to redundant signal transduction pathways. However, the absence of RhoG increases TCR signalling and proliferation, implying that RhoG activity is critical during late T cell activation following antigen-receptor interaction. Moreover, RhoG is required to halt signal transduction and prevent hyper-activated T cells. Despite increase in TCR signalling, cell proliferation is inhibited, implying that RhoG induces T cell anergy by promoting the activities of transcription factors, including nuclear factor of activated T cell (NFAT)/AP-1. The role of NFAT plays in T cell anergy is inducing the transcription of anergy-associated genes, such as IL-2, IL-5, and IFN-g. Although information about RhoG in T cell-related diseases is limited, mutant forms of RhoG, Ala151Ser and Glu171Lys have been observed in thymoma and hemophagocytic lymphohistiocytosis (HLH), respectively. Current information only focuses on these two diseases, and thus the role of RhoG in normal and pathological circumstances should be further investigated. This approach is necessary because RhoG and its associated proteins represent prospective targets for attack particularly in the therapy of cancer and immune-mediated illnesses.
... Il est connu que la réorganisation du cytosquelette est nécessaire à la formation des structures cytoplasmiques impliquées dans la migration cellulaire, et la famille Rho de petites GTPases constitue un facteur clé de ce processus (Burridge, 2017). Les lamellipodes fournissent la force qui propulse la cellule vers l'avant pendant la migration et l'activité de Rac 1 est nécessaire à la formation des lamellipodes via la polymérisation et l'extension de l'actine (Nobes and Hall, 1995). Il a été démontré que l'épuisement de Rac1 supprime la formation d'adhérences focales dans des cellules de carcinome (Guo et al., 2006). ...
Thesis
Malgré les diagnostics précoces et les avancées thérapeutiques, le taux de mortalité chez les patients diagnostiqués d’un CCR au stade métastatique reste très élevé. L’objectif de ce travail a été d’étudier le rôle de la chimiokine CXCL12 et ses deux récepteurs CXCR4 et CXCR7 dans processus métastatique. J’ai comparé l’expression de la chimiokine CXCL12 dans des tumeurs coliques humaines avec les tissus sains associés puis je me suis intéressée aux mécanismes de régulation de cette expression et plus particulièrement la régulation épigénétique. J’ai montré que le promoteur de CXCL12 est méthylé dans 35% des CCR et qu’un défaut d’acétylation des histones du promoteur entraîne la perte d’expression de CXCL12. Des enzymes impliquées dans la régulation des mécanismes épigénétiques, potentiellement liées à ce défaut d’acétylation ont été identifiées par l’analyse des tumeurs par PCR Array et parmi ces facteurs, j’ai identifié l’histone acétyl-transférase PCAF dont l’expression est diminuée dans les tumeurs. Enfin, pour comprendre le rôle respectif de CXCR4 et de CXCR7 dans la dissémination métastatique, j’ai invalidé l’expression du gène des récepteurs dans la lignée colique humaine SW480 par Crispr-Cas9, puis j’ai comparé la capacité migratoire des cellules in vitro et leur potentiel métastatique in vivo. L’induction d’une perte d’expression du récepteur CXCR7 n’a pas eu d’impact sur le développement des métastases pulmonaire et hépatique in vivo, mais a entraîné une baisse de la migration in vitro.
... Cdc42 is known to regulate the attachment of actin fibers to the sites of cell-cell adhesion that are stabilized by the junctional proteins. Thus, inhibition of Cdc42 could have reduced levels of junction proteins other than N-cadherin such as occludin 76 , which might have resulted in disruption of the spheroids. ...
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Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.
... Furthermore, ASC-CM strongly activates Rac1 and Cdc42 in microglia [22] (Figure 3A). As described on many occasions, Rac1 induces the formation of lamellipodia [52,53], the mesh-like polymerization of F-actin. These structures are easily detectable by contrast-phase microscopy as a rim along the cell edges. ...
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Neurological disorders, including neurodegenerative diseases, are often characterized by neuroinflammation, which is largely driven by microglia, the resident immune cells of the central nervous system (CNS). Under these conditions, microglia are able to secrete neurotoxic substances, provoking neuronal cell death. However, microglia in the healthy brain carry out CNS-supporting functions. This is due to the ability of microglia to acquire different phenotypes that can play a neuroprotective role under physiological conditions or a pro-inflammatory, damaging one during disease. Therefore, therapeutic strategies focus on the downregulation of these neuroinflammatory processes and try to re-activate the neuroprotective features of microglia. Mesenchymal stem cells (MSC) of different origins have been shown to exert such effects, due to their immunomodulatory properties. In recent years, MSC derived from adipose tissue have been made the center of attention because of their easy availability and extraction methods. These cells induce a neuroprotective phenotype in microglia and downregulate neuroinflammation, resulting in an improvement of clinical symptoms in a variety of animal models for neurological pathologies, e.g., Alzheimer’s disease, traumatic brain injury and ischemic stroke. In this review, we will discuss the application of adipose tissue-derived MSC and their conditioned medium, including extracellular vesicles, in neurological disorders, their beneficial effect on microglia and the signaling pathways involved.
... Rac1, one of mammalian Rho GTPases, is implicated in regulation of membrane ruffling, cytoskeletal rearrangement, migration, and apoptosis (Nobes and Hall, 1995;Mattila and Lappalainen, 2008). Before performing the proper function, Rac1 needs prenylation modification for anchoring to plasm membranes and interaction with downstream signaling effectors (Gao et al., 2009). ...
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Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a severe side effect of long-term administration of bisphosphonates such as zoledronic acid (ZA), but its pathogenesis remains unclear. Impairment of the clearance of apoptotic cells (termed “efferocytosis”) by ZA may be associated with the pathogenesis of BRONJ. The aim of this study was to investigate whether ZA might inhibit macrophage efferocytosis and promote osteocytic apoptosis, and the underlying mechanisms responsible for the disturbing balance between clean and generation of osteocytic apoptosis. We found that ZA significantly promoted the apoptosis of osteocyte and pre-osteoblast via BRONJ mouse models and in vitro MC3T3-E1 but also inhibited the efferocytosis of macrophage on apoptotic cells. Moreover, supplement with geranylgeraniol (GGOH), a substrate analog for geranylgeranylation of Rac1, could restore Rac1 homeostasis and rescue macrophage efferocytosis. GGOH partially inhibits MC3T3-E1 apoptosis induced by ZA via downregulation of Rac1/JNK pathway. We also examined the Rac1 distribution and activation conditions in bone marrow-derived macrophages (BMDMs) and MC3T3-E1 under ZA treatment, and we found that ZA impaired Rac1 migration to BMDM membrane, leading to round appearance with less pseudopodia and efferocytosis inhibition. Moreover, ZA simultaneously activated Rac1, causing overexpression of P-JNK and cleaved caspase 3 in MC3T3-E1. Finally, the systemic administration of GGOH decreased the osteocytic apoptosis and improved the bone healing of the extraction sockets in BRONJ mouse models. Taken together, our findings provided a new insight and experimental basis for the application of GGOH in the treatment of BRONJ.
... RhoA activity is also regulated by prenylation at its C-terminus (12). RhoA is well-known to play an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival (13)(14)(15)(16)(17). In T cells, RhoA modulates T cell polarization, thymocyte development and adhesion, and thymic egress (18)(19)(20)(21)(22)(23)(24)(25). ...
Article
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RhoA of the Rho GTPase family is prenylated at its C-terminus. Prenylation of RhoA has been shown to control T helper 17 (Th17) cell-mediated colitis. By characterizing T cell-specific RhoA conditional knockout mice, we have recently shown that RhoA is required for Th2 and Th17 cell differentiation and Th2/Th17 cell-mediated allergic airway inflammation. It remains unclear whether RhoA plays a cell-intrinsic role in regulatory T (Treg) cells that suppress effector T cells such as Th2/Th17 cells to maintain immune tolerance and to promote tumor immune evasion. Here we have generated Treg cell-specific RhoA-deficient mice. We found that homozygous RhoA deletion in Treg cells led to early, fatal systemic inflammatory disorders. The autoimmune responses came from an increase in activated CD4+ and CD8+ T cells and in effector T cells including Th17, Th1 and Th2 cells. The immune activation was due to impaired Treg cell homeostasis and increased Treg cell plasticity. Interestingly, heterozygous RhoA deletion in Treg cells did not affect Treg cell homeostasis nor cause systemic autoimmunity but induced Treg cell plasticity and an increase in effector T cells. Importantly, heterozygous RhoA deletion significantly inhibited tumor growth, which was associated with tumor-infiltrating Treg cell plasticity and increased tumor-infiltrating effector T cells. Collectively, our findings suggest that graded RhoA expression in Treg cells distinguishes tumor immunity from autoimmunity and that rational targeting of RhoA in Treg cells may trigger anti-tumor T cell immunity without causing autoimmune responses.
... Ras gene mutations correspond to a common feature of many tumor types, and preliminary studies suggest that the inhibitory activities of statins are primarily attributable to the inhibition of the mevalonate pathway downstream target Rho GTPase [17][18][19] . There are approximately 20 Rho GTPase superfamily members that have been described to date, including RhoA, Rac1, and Cdc42 20 . In addition to regulating normal cellular differentiation, apoptosis, and proliferation, these signaling molecules are closely associated with tumor development and metastatic progression 21,22 . ...
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Statins are first-line drugs used to control patient lipid levels, but there is recent evidence that statin treatment can lower colorectal cancer (CRC) incidence by 50% and prolong CRC patient survival through mechanisms that are poorly understood. In this study, we found that the treatment of APC min mice by the mevalonate pathway inhibitor lovastatin significantly reduced the number of colonic masses and improved hypersplenism and peripheral anemia. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) analysis of colonic mass tissues showed a potent inhibitory effect in both Wnt/β-catenin signaling and YAP/TAZ signaling in the lovastatin treatment group. The results of our transcriptomic analyses in RKO indicated that lovastatin regulated several proliferation-related signaling pathways. Moreover, lovastatin suppressed important genes and proteins related to the canonical Wnt/β-catenin and alternative Wnt-YAP/TAZ signaling pathways in RKO and SW480 cells, and these effects were rescued by mevalonic acid (MVA), as confirmed through a series of Western blotting, RT-PCR, and reporter assays. Given that statins suppress oncogenic processes primarily through the inhibition of Rho GTPase in the mevalonate pathway, we speculate that lovastatin can inhibit certain Rho GTPases to suppress both canonical Wnt/β-catenin signaling and alternative Wnt-YAP/TAZ signaling. In RKO cells, lovastatin showed similar inhibitory properties as the RhoA inhibitor CCG1423, being able to inhibit β-catenin, TAZ, and p-LATS1 protein activity. Our results revealed that lovastatin inhibited RhoA activity, thereby suppressing the downstream canonical Wnt/β-catenin and alternative Wnt-YAP/TAZ pathways in colon cancer cells. These inhibitory properties suggest the promise of statins as a treatment for CRC. Altogether, the present findings support the potential clinical use of statins in non-cardiovascular contexts and highlight novel targets for anticancer treatments.
... The Rho family GTPase have also emerged as regulators of endothelial junction proteins, mediating vascular barrier integrity via such interaction. Rac1/Cdc42 is thought to spatially influence the forming of filopodia and lamellipodia, which can promote vascular barrier development [33][34][35][36]. They also promoted endothelial cells migration and adhesion through the assembly and organization of the actin cytoskeleton [37]. ...
Article
Vascular barrier dysfunction is considered as the initial and critical event in atherosclerosis progression. Recent studies have revealed that treatment with piceatannol (PIC) alleviates both acute and chronic responses to vascular injury. We investigated whether PIC treatment would have beneficial effects on glucolipotoxicity-induced endothelial barrier dysfunction. Target proteins of PIC were identified from several online databases. Then, we confirmed the effect of PIC on endothelial barrier function. PIC treatment mitigated the impairment of endothelial cell motility, adhesion and migration ability associated with high glucose/lipid stimulation. PIC stabilized cytoskeletal reorganization and expression of cell cytoskeletal associated proteins GTPase. PIC reversed changes in critical vascular junction proteins and thus preserved endothelial barrier function and permeability. Finally, we confirmed that reducing of nuclear factor kappa B (NF-κB)/p65 activation and elimination of reactive oxygen species (ROS) were involved in the protective effect of PIC against glucolipotoxicity-induced vascular barrier injury. We identify PIC as a promising therapeutic strategy for glucolipotoxicity-induced endothelial barrier injury.
... The analysis of mean influorescence intensity of F-actin showed similar results (Figure 4c). Expression of CDC42 and p70 S6 kinase, which are the regulators of cytoskeleton (Ip et al., 2011;Nobes & Hall, 1995), was significantly upregulated at dox4d and at dox4d-4d. Notably, the expression was restored to the aging state at dox4d-8d, and was activated at dox4d-4d+4d (Supplementary Figure S4e, Figure 4f). ...
Article
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Rejuvenation of nucleus pulposus cells (NPCs) in degenerative discs can reverse intervertebral disc degeneration (IDD). Partial reprogramming is used to rejuvenate aging cells and ameliorate progression of aging tissue to avoiding formation of tumors by classical reprogramming. Understanding the effects and potential mechanisms of partial reprogramming in degenerative discs provides insights for development of new therapies for IDD treatment. The findings of the present study show that partial reprogramming through short-term cyclic expression of Oct-3/4, Sox2, Klf4, and c-Myc (OSKM) inhibits progression of IDD, and significantly reduces senescence related phenotypes in aging NPCs. Mechanistically, short-term induction of OSKM in aging NPCs activates energy metabolism as a "energy switch" by upregulating expression of Hexokinase 2 (HK2) ultimately promoting redistribution of cytoskeleton and restoring the aging state in aging NPCs. These findings indicate that partial reprogramming through short-term induction of OSKM has high therapeutic potential in the treatment of IDD.
... Cdc42 in turn can activate Rac1 (154,155,164). Not only does Cdc42 regulate the actin cytoskeleton, it also has an important function in regulating cell polarity, which is well conserved in many eukaryotic organisms (141). ...
Article
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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. β2-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.
... This complex was thereafter named the WAVE regulatory complex (WRC or WAVE complex) (Fig. 3). The identification of the WRC explained the previously observed links between Rac1 and WRC components and their importance in the formation of lamellipodia, thin sheet-like membrane ruffling often found at the leading edge of migrating cells (Kobayashi et al., 1998;Nobes and Hall, 1995;Ridley et al., 1992;Rogers et al., 2003;Scita et al., 1999). These studies also reconciled the original debates about WRC activity regulation, establishing that the WRC is basally inhibited in the cytosol and is recruited to and activated at the plasma membrane by various ligands to promote Arp2/3-mediated actin polymerization ( Fig. 3A) (Kurisu and Takenawa, 2009;Rottner et al., 2021). ...
Article
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Proteins of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in regulating actin cytoskeletal dynamics in a wide range of cellular processes. Genetic mutations or misregulation of these proteins are tightly associated with many diseases. The WASP-family proteins act by transmitting various upstream signals to their conserved WH2-Central-Acidic (WCA) peptide sequence at the C-terminus, which in turn binds to the Arp2/3 complex to stimulate the formation of branched actin networks at membranes. Despite this common feature, the regulatory mechanisms and cellular functions of distinct WASP-family proteins are very different. Here, we summarize and clarify our current understanding of WASP-family proteins and how disruption of their functions is related to human disease.
... Our previous studies have proven that the nanowave-like structure has an influence on cell mechanotransduction, possibly through focal adhesion, cytoskeletal tension, and YAP signaling pathways [36,37,58,59]. Rho GTPase activation is essential for focal adhesion assembly and disassembly, while focal adhesions anchor actin stress fibers and in turn facilitate intracellular vesicular trafficking, which may affect transfection efficiency [60]. Future endeavors may therefore focus on understanding the roles of specific mechanotransductions and endocytosis pathways, which would indicate that by means of physicochemical stimuli one can modulate these mechanisms; therefore, specific chemical modifications of the transfection complexes might not be necessary. ...
Article
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Gene delivery holds great promise for bioengineering, biomedical applications, biosensors, diagnoses, and gene therapy. In particular, the influence of topography on gene delivery is considered to be an attractive approach due to low toxicity and localized delivery properties. Even though many gene vectors and transfection systems have been developed to enhance transfection potential and combining it with other forms of stimulations could even further enhance it. Topography is an interesting surface property that has been shown to stimulate differentiation, migration, cell morphology, and cell mechanics. Therefore, it is envisioned that topography might also be able to stimulate transfection. In this study, we tested the hypothesis “topography is able to regulate transfection efficiency”, for which we used nano- and microwave-like topographical substrates with wavelengths ranging from 500 nm to 25 µm and assessed the transfectability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and myoblasts. For transfection, Lipofectamine 2000 and a gene encoding plasmid for red-fluorescent protein (m-Cherry) were used and topography-induced cell morphology and transfection efficiency was analyzed. As a result, topography directs cell spreading, elongation, and proliferation as well as the transfection efficiency, which were investigated but were found not to be correlated and dependent on the cell type. A 55% percent improvement of transfection efficiency was identified for hBM-MSCs grown on 2 µm wrinkles (24.3%) as compared to hBM-MSCs cultured on flat controls (15.7%). For myoblast cells, the highest gene-expression efficiency (46.1%) was observed on the 10 µm topography, which enhanced the transfection efficiency by 64% as compared to the flat control (28.1%). From a qualitative assessment, it was observed that the uptake capacity of cationic complexes of TAMRA-labeled oligodeoxynucleotides (ODNs) was not topography-dependent but that the intracellular release was faster, as indicated by the positively stained nuclei on 2 μm for hBM-MSCs and 10 μm for myoblasts. The presented results indicate that topography enhances the gene-delivery capacity and that the responses are dependent on cell type. This study demonstrates the important role of topography on cell stimulation for gene delivery as well as understanding the uptake capacity of lipoplexes and may be useful for developing advanced nonviral gene delivery strategies.
... Cdc42 is also exploited by C. jejuni and P. aeruginosa for host cell invasion [26]. Cdc42 along with the rac helps to stimulate the assembly of multimolecular focal complexes at plasma membrane and promotes filopodia formation [27]. ...
Article
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Helicobacter pullorum is a human zoonotic pathogen transmitted through poultry where it is associated with vibrionic hepatitis and colitis. Hemolysin co-regulated protein (Hcp) is an important structural as well as effector protein of type six secretory system; however, its role in H. pullorum invasion and pathogenesis has not been elucidated. In this study, we predicted the Helicobacter pullorum Hcp (HpuHcp) structure and identified Campylobacter jejuni Hcp (CjHcp) as its nearest homologue. Analysis of the predicted structure shows several common bacterial Hcp motifs like Protein kinase C phosphorylation site, Casein kinase II phosphorylation site, N-myristoylation site, cAMP-and cCGMP-dependent protein kinase phosphorylation site, N-glycosylation site. The presence of unique microbodies C-terminal targeting signal domain was present in HpuHcp which was seen for the first time in CjHcp. This could indicate that Hcp is a structural protein as well as a secretory protein. Moreover, the presence of a deamidase domain, similar to the tecA of Burkholderia cenocepacia an opportunistic pathogen, may help in bacterial internalization as it depolymerises the membranous actin by deamidation of the host cell Rho GTPases cdc42 and Rac1, which was supported by increased invasion of hepatocytes by Hcp-positive isolates.
... Increased filopodia elongation was observed similarly with both protein conditions (control: 3.4 µm ± 0.23 µm; WT DLGAP4: 6.71 µm ± 1.08 µm, p = 0.036; mutant DLGAP4: 6.61 µm ± 0.5 µm, p = 0.041) (Fig. 7c). Actin stress fibers are long bundles of filaments extending across the cell, making links to the extracellular matrix via integrins and focal adhesion complexes 44 . Organized stress fibers were not significantly affected (Fig. 7d). ...
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Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4 . The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.
... YAP/ TAZ plays an important role in focal adhesion signaling by modulating interaction of focal adhesions (FAs), mainly integrins, to the F-actin in the actin cytoskeleton and fibronectin expressed in the ECM by myofibroblasts (Winograd-Katz et al., 2014;Zent and Guo, 2018). The focal adhesions mediate force transmission between the ECM and actin cytoskeleton via the Rho/ROCK pathway, a pathway that is critical for EMT-induction in the lens, to inhibit phosphorylation of YAP thus facilitating its nuclear translocation (Nobes and Hall, 1995;Huveneers and Danen, 2009;Kim and Gumbiner, 2015;Burridge and Guilluy, 2016;Korol et al., 2016;Nardone et al., 2017). Recent work from our laboratory has shown that increased expression of YAP1 in lens sections from a mouse model of ASC provides further evidence of involvement of YAP/TAZ in lens fibrosis (Taiyab and coworkers, unpublished observation). ...
Article
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Injury to the ocular lens perturbs cell-cell and cell-capsule/basement membrane interactions leading to a myriad of interconnected signaling events. These events include cell-adhesion and growth factor-mediated signaling pathways that can ultimately result in the induction and progression of epithelial-mesenchymal transition (EMT) of lens epithelial cells and fibrosis. Since the lens is avascular, consisting of a single layer of epithelial cells on its anterior surface and encased in a matrix rich capsule, it is one of the most simple and desired systems to investigate injury-induced signaling pathways that contribute to EMT and fibrosis. In this review, we will discuss the role of key cell-adhesion and mechanotransduction related signaling pathways that regulate EMT and fibrosis in the lens.
... The small Rho GTPase Cdc42, has been shown to control the formation of actin based filopodia and dynamic cell adhesions called focal complexes to the extracellular matrix (Nobes and Hall, 1995). Thus, we next investigated a potential interplay of Myo1f and Cdc42 in filopodia formation, by using wild-type mCherry-Cdc42 and a constitutively active mutant thereof, mCherry-Cdc42-Q61L. ...
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The unique threonine protease Tasp1 impacts not only ordered development and cell proliferation but also pathologies. However, its substrates and the underlying molecular mechanisms remain poorly understood. We demonstrate that the unconventional Myo1f is a Tasp1 substrate and unravel the physiological relevance of this proteolysis. We classify Myo1f as a nucleo-cytoplasmic shuttle protein, allowing its unhindered processing by nuclear Tasp1 and an association with chromatin. Moreover, we show that Myo1f induces filopodia resulting in increased cellular adhesion and migration. Importantly, filopodia formation was antagonized by Tasp1-mediated proteolysis, supported by an inverse correlation between Myo1f concentration and Tasp1 expression level. The Tasp1/Myo1f-axis might be relevant in human hematopoiesis as reduced Tasp1 expression coincided with increased Myo1f concentrations and filopodia in macrophages compared to monocytes and vice versa. In sum, we discovered Tasp1-mediated proteolysis of Myo1f as a mechanism to fine-tune filopodia formation, inter alia relevant for cells of the immune system.
... Erhöhung der MLC-Phosphorylierung führt zu Ausbildung von kontraktilen Aktin-Myosin-Filamenten, den sogenannten Stressfasern (Bresnick, 1999 (Bamburg et al., 1999;Maekawa et al., 1999). (Kozma et al., 1995;Miki et al., 1998;Nobes & Hall, 1995;Rohatgi R. et al., 1999). (Hanna & El-Sibai, 2013;Ridley & Hall, 1992). ...
Thesis
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This study investigates toxin-host-interactions of the bacterial deamidating toxins Cytotoxic necrotizing factor S and Pasteurella multocida toxin. These two virulence factors belong to the dermonecrotic toxin family and act as intracellular toxins. By deamidation of a crucial glutamine residue in G-proteins they render them constitutively active. Through this covalent modification they’re able to impair various signaling pathways and thereby manipulate central functions of the host cell by posttranslational modification of GTP-binding proteins. The purpose of these secreted Toxins is the adaption of pathogens to their host environment. The goal of the first part of this study was the recombinant expression and characterization of the potential deamidating toxin Cytotoxic necrotizing factor S (CNFS). The sequence of the toxin was found in a probably human pathogenic Salmonella enterica strain. In vitro CNFS showed deamidase- as well as transglutaminase-activity on proteins of the Rho-family. Effector-protein pulldowns in eukaryotic cells revealed RhoA, Rac1 and Cdc42 as intracellular substrates of the toxin. The substrates were confirmed by observation of the characteristic morphological changes of the actin cytoskeleton. Linkedto the organization of the actin cytoskeleton is the regulation of tight junction structures and the related endothelial/epithelial integrity. CNFS showed a moderate impairment of the endothelial barrier in HUVEC (human umbilical vein endothelial cells) cells. In addition to the functional characterization of the toxin a focus was given to the toxin-host cell interaction. By using flow cytometry, native gelelectrophoresis, immunoblotting, and knockout-cell lines, Heparansulfate proteoglycans (HSPG) were identified as crucial host cell factors for a sufficient intoxication by CNFS. In HSPG-deficient cells diminished binding of the toxin to the cell surface was observed and no responsivity to the Rho Deamidation of CNFS was detectable. The second part of the thesis deals with the main virulence factor Pasteurella multocida toxin (PMT) of the animal pathogen Pasteurella multocida. As a typical AB-toxin PMT consists of a N-terminal receptor binding domain and a C-terminal catalytic domain. The N-terminus mediates the binding to the cell surface and the subsequent receptor-mediated endocytosis, whereas the C-terminus deamidates the α-subunit of heterotrimeric G-proteins. Via a genome-wide CRISPR-Knockout screen the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP1) was found to act as host cell receptor for PMT. In cells lacking LRP1 expression PMT showed no deamidation. By ectopic reexpression of single domains of LRP1, the role of the receptor was confirmed and the binding domain was found to be located in the cluster IV of the protein. On the basis of the residual binding of the DyLight488-labeled toxin to the LRP1-KO cells the necessity of other structures on the cell surface for an appropriate binding can be assumed. In summary in this work the new virulence factor CNFS of a human pathogenic Salmonella enterica strain was identified and characterized. Moreover, LRP1 was found as a crucial host cell receptor of PMT. The results of this work provide the basis for a better understanding of the pathogenesis of salmonellosis and pasteurellosis.
... In contrast, similar oncogenic properties were not apparent for the RHO GTPases. Instead, several, by now classical, papers from the research group of Alan Hall during the early 1990s demonstrated that the RHO GTPases are key regulators in the signaling cascades that control actin dynamics in eukaryotic cells [15][16][17]. These studies paved the way for a paradigm, stating that only three RHO members, RHOA, RAC1, and CDC42, were sufficient to regulate the organization and dynamics of the actin filament system, and thereby complex cellular processes such as cell morphogenesis and cell migration [18]. ...
Article
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The RHO GTPases comprise a subfamily within the RAS superfamily of small GTP-hydrolyzing enzymes and have primarily been ascribed roles in regulation of cytoskeletal dynamics in eukaryotic cells. An oncogenic role for the RHO GTPases has been disregarded, as no activating point mutations were found for genes encoding RHO GTPases. Instead, dysregulated expression of RHO GTPases and their regulators have been identified in cancer, often in the context of increased tumor cell migration and invasion. In the new landscape of cancer genomics, activating point mutations in members of the RHO GTPases have been identified, in particular in RAC1, RHOA, and CDC42, which has suggested that RHO GTPases can indeed serve as oncogenes in certain cancer types. This review describes the current knowledge of these cancer-associated mutant RHO GTPases, with a focus on how their altered kinetics can contribute to cancer progression.
Article
Cdc42 is a key factor in directed cell migration and accumulates at the leading edge of migrating cells. However, what kind of proteins control Cdc42 and when is unclear. After mechanical wounding, protein kinase C α (PKCα), a conventional PKC isozyme, begins to accumulate at the edges of cells adjacent to the wounded cells (WCs). In this study, we hypothesized that PKCα may be implicated in directed cell migration at an early stage before Cdc42 controls the migration. We focused on the spatiotemporal distribution of PKCα, Cdc42, and Rac1 before cell migration. After wounding, at the edges of cells adjacent to the WCs, PKCα accumulation, Cdc42 accumulation, Rac1 accumulation, and filopodia formation occurred in that order. The PKCα inhibitor suppressed Cdc42 accumulation at the cell edges. These results suggest that inhibition of PKCα activity inhibits cell migration. In addition, it is not Cdc42 but PKCα that may decide the direction of cell migration.
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Septic arthritis (SA) is a life-threatening condition in horses, and identifying eradication of infection in equine SA is challenging. This study explored the discovery of putative biomarkers for the eradication of joint infection in horses. We performed proteomics analysis of synovial fluid (SF) and plasma from horses with experimental SA, non-septic lipopolysaccharide-induced arthritis, and controls. The point of eradication of infection in horses with SA was determined previously. We compared spectral intensities between groups as well as before and after the eradication of infection. Twenty-six differentially abundant proteins were identified, which were upregulated in SF of horses with SA compared to the other groups, as well as compared to the same horses post-eradication of infection. In plasma, we did not identify differentially abundant proteins. Differentially abundant proteins in SF were of cellular origin and their biological functions included ubiquitination, signal transduction, apoptosis etc. The difference in their relative abundance between experimental groups was ≥10-fold compared to the abundance expected based on the difference in cell count alone (2-fold). Since most of cells in joints with bacterial infection are neutrophils, we suggest that the variable abundance of neutrophil- and cell-associated proteins represent potential biomarkers of eradication of infection in equine SA. Significance Septic arthritis is an important condition in horses, which can be life-threatening. At present, identifying eradication of infection in cases of equine septic arthritis is challenging. In this study, we performed a global proteomics analysis of synovial fluid and plasma in horses with experimental septic arthritis and identified 26 differentially expressed proteins compared to non-septic arthritis and post eradication of infection. The results of this study provide the basis for further characterization of the differentially expressed proteins and identification of clinically relevant biomarkers of septic arthritis in horses.
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Metastasis is the main cause of cancer patients' death despite tremendous efforts invested in developing the related molecular mechanisms. During cancer cell migration, cells undergo dynamic regulation of filopodia, focal adhesion, and endosome trafficking. Cdc42 is imperative for maintaining cell morphology and filopodia, regulating cell movement. Integrin beta1 activates on the endosome, the majority of which distributes itself on the plasma membrane, indicating that endocytic trafficking is essential for this activity. In cancers, high expression of Lysosome-Associated Protein Transmembrane 4B (LAPTM4B) is associated with poor prognosis. LAPTM4B-35 has been reported as displaying plasma-membrane distribution and being associated with cancer cell migration. However, the detailed mechanism of its isoform-specific distribution and whether it relates to cell migration remain unknown. Here we first report and quantify the filopodia localization of LAPTM4B-35: mechanically, that specific interaction with Cdc42 promoted its localization to the filopodia. Furthermore, our data show that LAPTM4B-35 stabilized filopodia, and regulated integrin beta1 recycling via interaction and co-trafficking on the endosome. In our zebrafish xenograft model, LAPTM4B-35 stimulated the formation and dynamics of focal adhesion, as well as further promoting cancer cell dissemination, whereas in skin-cancer patients, LAPTM4B level correlated with poor prognosis. In short, this study establishes an insight into the mechanism of LAPTM4B-35 filopodia distribution, as well as into its biological effects and its clinical significance, providing a novel target for cancer therapeutics development.
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The ADP-ribosylation factors (Arfs) comprise a family of regulatory GTP binding proteins. The Arfs regulate membrane trafficking and cytoskeleton remodeling, processes critical for eukaryotes and which have been the focus of most studies on Arfs. A more limited literature describes a role in signaling and in integrating several signaling pathways to bring about specific cell behaviors. Here, we will highlight work describing function of Arf1, Arf6 and several effectors and regulators of Arfs in signaling.
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Posterior capsular opacification (PCO) can cause postoperative visual loss after cataract surgery. Residual human lens epithelial cell (HLEC) proliferation, migration, epithelial-mesenchymal transition (EMT) and synthesis of extracellular matrix (ECM) are the entitative reasons for PCO. Low expression of Ral-binding protein 1-associated Eps domain-containing 2 (REPS2) and high levels of basic fibroblast growth factor (b-FGF) were observed in the lens and postoperative aqueous humor of cataract patients. REPS2 was identified as a negative regulator in growth factor signaling; however, its function in HLECs is unknown. This was first investigated in the present study by evaluating REPS2 expression in anterior lens capsules from cataract patients, a mouse cataract model, and HLE-b3 cells. The biological function of REPS2 in HLE-B3 cells was assessed by REPS2 silencing and Cell Counting Kit 8, wound healing, Transwell migration, F-actin staining, G-protein pulldown and western blot assays. In the present study, REPS2 was significantly downregulated in human and mouse cataract capsules and H2O2-treated HLE-B3 cells. REPS2 knockdown increased fibronectin, type I collagen, and α-smooth muscle actin expression levels and stimulated HLECs proliferation and migration; these effects were enhanced by FGF treatment and accompanied with focal adhesion kinase (FAK) phosphorylation, cell division cycle 42 (Cdc42) activation, focal adhesion protein upregulation, and F-actin cytoskeleton reorganization. However, treatment with the FAK inhibitor PF573228 abolished these effects. Thus, REPS2 downregulation in cataract HLECs induces their proliferation and facilitates FGF-induced ECM synthesis, EMT, cell adhesion and migration by activating FAK/Cdc42 signaling, which may underlie PCO pathogenesis.
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One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a FRET technique in conjunction with a machine learning-assisted cell segmentation method to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment using a RhoA biosensor. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.
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Significance New imaging-based approaches are incorporating new concepts to our knowledge of biological processes. The analysis of receptor dynamics involved in cell movement using single-particle tracking demonstrates that cells require chemokine-mediated receptor clustering to sense appropriately chemoattractant gradients. Here, we report that this process does not occur in T cells expressing CXCR4 R334X , a mutant form of CXCR4 linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis). The underlaying molecular mechanism involves inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4 R334X , which alters its lateral mobility and spatial organization. These defects, associated to CXCR4 R334X expression, contribute to the retention of hematopoietic precursors in bone marrow niches and explain the severe immunological symptoms associated with WHIM syndrome.
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Nuclear receptor coactivator 6 (Ncoa6), a modulator of several nuclear receptors and transcription factors, is essential for the decidualization of endometrial stromal cells in mice. However, the function of Ncoa6 in the human endometrium remains unclear. We investigated its function in the decidualization of human endometrial stromal cells (HESCs) isolated from resected uteri. Knockdown of Ncoa6 was performed using two independent small interfering RNAs. Decidualization was induced in vitro via medroxyprogesterone and cyclic adenosine monophosphate. We compared decidualized cellular morphology between the Ncoa6 knockdown cells and control cells. Messenger RNA (mRNA) sequencing was performed to determine the Ncoa6 target genes in undecidualized HESCs. We found that the knockdown of Ncoa6 caused the failure of morphological changes in decidualized HESCs compared to that in the control cells. mRNA sequencing revealed that Ncoa6 regulates the expression of genes associated with the regulation of actin fibers. Ncoa6 knockdown cells failed to reorganize actin fibers during the decidualization of HESCs. Ncoa6 was shown to play an essential role in decidualization via the appropriate regulation of actin fiber regulation in HESCs. Herein, our in vitro studies revealed a part of the mechanisms involved in endometrial decidualization. Future research is needed to investigate these mechanisms in women with implantation defects. Human endometrial stromal cells (HESCs) transfected with nontarget small interfering RNA (siRNA) (control) showed the typical cobblestone pattern of decidualized endometrial stromal cells after medroxyprogesterone acetate (MPA)/cyclic adenosine monophosphate (cAMP) treatment. Decidualized HESCs transfected with nuclear receptor coactivator 6 (Ncoa6)‐specific siRNAs (Ncoa6 knockdown) showed spindle shape even after MPA/cAMP treatment.
Chapter
Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.
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Rho GTPases are critically important and are centrally positioned regulators of the actomyosin cytoskeleton. By influencing the organization and architecture of the cytoskeleton, Rho proteins play prominent roles in many cellular processes including adhesion, migration, intra‐cellular transportation, and proliferation. The most important method of Rho GTPase regulation is via the GTPase cycle; however, post‐translational modifications (PTMs) also play critical roles in Rho protein regulation. Relative to other PTMs such as lipidation or phosphorylation that have been extensively characterized, protein oxidation is a regulatory PTM that has been poorly studied. Protein oxidation primarily occurs from the reaction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), with amino acid side chain thiols on cysteine (Cys) and methionine (Met) residues. The versatile redox modifications of cysteine residues exemplify their integral role in cell signalling processes. Here we review prominent members of the Rho GTPase family and discuss how lipidation, phosphorylation, and oxidation on conserved cysteine residues affects their regulation and function. Reactive oxygen species (ROS) can oxidize Rho GTPases on redox‐sensitive cysteine residues, resulting in reduced affinity for guanine nucleotides and consequent increased GDP dissociation rates. Binding of cellular GTP to the nucleotide‐free protein, independent of guanine nucleotide exchange factors (GEF), transitions the GTPases to active conformations that enable downstream signalling.
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NK cells are contained in the ILC1 group; they are recognized for their antiviral and antitumor cytotoxic capacity; NK cells also participate in other immune response processes through cytokines secretion. However, the mechanisms that regulate these functions are poorly understood since NK cells are not as abundant as other lymphocytes, which has made them difficult to study. Using public databases, we identified that NK cells express mRNA encoding class I myosins, among which Myosin 1g and Myosin 1f are prominent. Therefore, this mini-review aims to generate a model of the probable participation of Myosin 1g and 1f in NK cells, based on information reported about the function of these myosins in other leukocytes.
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Tumor-resident intracellular microbiota is an emerging tumor component that has been documented for a variety of cancer types with unclear biological functions. Here, we explored the functional significance of these intratumor bacteria, primarily using a murine spontaneous breast-tumor model MMTV-PyMT. We found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth. During metastatic colonization, intratumor bacteria carried by circulating tumor cells promoted host-cell survival by enhancing resistance to fluid shear stress by reorganizing actin cytoskeleton. We further showed that intratumor administration of selected bacteria strains isolated from tumor-resident microbiota promoted metastasis in two murine tumor models with significantly different levels of metastasis potential. Our findings suggest that tumor-resident microbiota, albeit at low biomass, play an important role in promoting cancer metastasis, intervention of which might therefore be worth exploring for advancing oncology care.
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Epithelial to mesenchymal transition (EMT) is a well-defined cellular process that was discovered in chicken embryos and described as “epithelial to mesenchymal transformation” [1]. During EMT, epithelial cells lose their epithelial features and acquire mesenchymal character with migratory potential. EMT has subsequently been shown to be essential for both developmental and pathological processes including embryo morphogenesis, wound healing, tissue fibrosis and cancer [2]. During the past 5 years, interest and study of EMT especially in cancer biology have increased exponentially due to the implied role of EMT in multiple aspects of malignancy such as cell invasion, survival, stemness, metastasis, therapeutic resistance and tumor heterogeneity [3]. Since the process of EMT in embryogenesis and cancer progression shares similar phenotypic changes, core transcription factors and molecular mechanisms, it has been proposed that the initiation and development of carcinoma could be attributed to abnormal activation of EMT factors usually required for normal embryo development. Therefore, developmental EMT mechanisms, whose timing, location, and tissue origin are strictly regulated, could prove useful for uncovering new insights into the phenotypic changes and corresponding gene regulatory control of EMT under pathological conditions. In this review, we initially provide an overview of the phenotypic and molecular mechanisms involved in EMT and discuss the newly emerging concept of epithelial to mesenchymal plasticity (EMP). Then we focus on our current knowledge of a classic developmental EMT event, neural crest cell (NCC) delamination, highlighting key differences in our understanding of NCC EMT between mammalian and non-mammalian species. Lastly, we highlight available tools and future directions to advance our understanding of mammalian NCC EMT.
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The design of an animal’s body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.
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F-Actin remodeling is important for the spread of HIV via cell–cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell–cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell–cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.
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Hepatocellular carcinoma (HCC) is the second most common cancer worldwide, demonstrating aggressiveness and mortality more frequently in men than in women. Despite reports regarding the inhibitory ability of estrogen receptor alpha (ERα, ESR1) in certain cancer progression, targets and the basis of underlying gender disparity in HCC worsening remain elusive. Here, we report the ability of ERα to transcriptionally inhibit G protein subunit alpha 12 (Gα12) responsible for HCC worsening. First, using human samples and public database, the expression of ERα and Gα12 in HCC was examined. Then, quantitative real-time PCR, chromatin immunoprecipitation-assay, luciferase assay, and immunoblottings of liver cancer cell lines confirmed the inhibitory ability of ERα on Gα12 and HCC progression. Gα12 promoted mesenchymal characteristics and amoeboidal movement, which was antagonized by ERα overexpression. Additionally, we found microRNA-141 and -200a as downstream targets of the Gα12 signaling axis for cancer malignancy regulation under the control of ERα. As for in-depth mechanism, PTP4A1 was found to be directly inhibited by microRNA-141 and -200a. Moreover, we found the inhibitory effect of ERα on amoeboidal movement by analyzing the morphology and blebbing of liver cancer cells and the active form of MLC levels. The identified targets and ESR1 levels are inversely correlated in human specimens, as well as with sex-biased survival rates of HCC patients. Collectively, ERα-dependent repression of Gα12 and consequent changes in the Gα12 signaling may explain the gender disparity in HCC, providing pharmacological clues for the control of metastatic HCC. This article is protected by copyright. All rights reserved.
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Mechanotransduction arises from information encoded in the shape of materials such as curvature. It induces activation of small GTPase signaling affecting cell phenotypes including differentiation. We carried out a set of preliminary experiments to test the hypothesis that curvature (1/radius) would also affect cell motility due to signal pathway crosstalk. High molecular weight poly (methyl methacrylate) straight nanofibers were electrospun with curvature ranging from 41 to 1 μm−1 and collected on a passivated glass substrate. The fiber curvature increased mouse mesenchymal stem cell aspect ratio (P < 0.02) and decreased cell area (P < 0.01). Despite little effect on some motility patterns such as polarity and persistence, we found selected fiber curvatures can increase normalized random fibroblastic mouse embryonic cell (MEF) migration velocity close to 2.5 times compared with a flat surface (P < 0.001). A maximum in the velocity curve occurred near 2.5 μm−1 and may vary with the time since initiation of attachment to the surface (range of 0–20 h). In the middle range of fiber curvatures, the relative relationship to curvature was similar regardless of treatment with Rho-kinase inhibitor (Y27632) or cdc42 inhibitor (ML141), although it was decreased on most curvatures (P < 0.05). However, below a critical curvature threshold MEFs may not be able to distinguish shallow curvature from a flat surface, while still being affected by contact guidance. The preliminary data in this manuscript suggested the large low curvature fibers were interpreted in a manner similar to a non-curved surface. Thus, curvature is a biomaterial construct design parameter that should be considered when specific biological responses are desired. Statement of integration, innovation, and insight Replacement of damaged or diseased tissues that cannot otherwise regenerate is transforming modern medicine. However, the extent to which we can rationally design materials to affect cellular outcomes remains low. Knowing the effect of material stiffness and diameter on stem cell differentiation, we investigated cell migration and signaling on fibrous scaffolds. By investigating diameters across orders of magnitude (50–2000 nm), we identified a velocity maximum of ~800 nm. Furthermore, the results suggest large fibers may not be interpreted by single cells as a curved surface. This work presents insight into the design of constructs for engineering tissues.
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Background Plenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury. Methods Immunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the Lyz Cre + /− ; RhoA flox/flox (cKO) mice or Lyz2 Cre + /− (Cre) mice, regardless of sex. Macrophages’ migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages. Results 1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages. Conclusions Our findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.
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The interference reflexion microscope may be used to examine the pattern of cell-substratum separation of cultured cells. Earlier studies on chick fibroblasts using this technique identified 3 types of separation: focal contacts, representing approx. 10-14 nm separation and which are sites of cell-substratum adhesion and form at the leading edge of a cell during locomotion; close contacts, representing approx. 30 nm separation; and regions of greater separation, generally 100-140 nm. Using light microscopy these studies described the association of the focal contacts with the distal ends of cytoplasmic fibres and noted their similarity to the adhesion plaques described in EM studies of thin vertical sections of fibroblasts. In this investigation the authors have directly confirmed these earlier observations by using a correlated interference-reflexion and high-voltage stereo electron-microscopic study of cultured chick heart fibroblasts. The results show that focal contacts are always associated with the distal ends of linear bundles of microfilaments which pass centripetally and often obliquely through the cytoplasm. At their proximal ends the bundles insert into a loose matrix of microfilaments and organelles surrounding the nucleus. The close contacts are often associated with a meshwork of microfilaments. The evidence supports the hypothesis that the microfilament bundles of a fibroblast form synchronously with the focal adhesions to the substratum. The results are discussed with reference to the current evidence for the role of contractile proteins in cell locomotion.
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Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.
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Expression of the Rous sarcoma virus-encoded oncoprotein, pp60v-src, subverts the normal regulation of cell growth, which results in oncogenic transformation. This process requires the intrinsic protein-tyrosine kinase activity of pp60v-src and is associated with an increase in tyrosine phosphorylation of a number of cellular proteins, candidate substrates for pp60v-src. We report here the isolation of a cDNA encoding a protein, pp125, that is a major phosphotyrosine-containing protein in untransformed chicken embryo cells and exhibits an increase in phosphotyrosine in pp60v-src-transformed chicken embryo cells. This cDNA encodes a cytoplasmic protein-tyrosine kinase which, based upon its predicted amino acid sequence and structure, is the prototype for an additional family of protein-tyrosine kinases. Immunofluorescence localization experiments show that pp125 is localized to focal adhesions; hence, we suggest the name focal adhesion kinase.
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Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.
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The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F-actin at the membrane.
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Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.
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Cytokinesis of animal cells involves the formation of the circumferential actin filament bundle (contractile ring) along the equatorial plane. To analyze the assembly mechanism of the contractile ring, we microinjected a small amount of rhodamine-labeled phalloidin (rh-pha) or rhodamine-labeled actin (rh-actin) into dividing normal rat kidney cells. rh-pha was microinjected during prometaphase or metaphase to label actin filaments that were present at that stage. As mitosis proceeded into anaphase, the labeled filaments became associated with the cortex of the cell. During cytokinesis, rh-pha was depleted from polar regions and became highly concentrated into the equatorial region. The distribution of total actin filaments, as revealed by staining the whole cell with fluorescein phalloidin, showed a much less pronounced difference between the polar and the equatorial regions. The sites of de novo assembly of actin filaments during the formation of the contractile ring were determined by microinjecting rh-actin shortly before cytokinesis, and then extracting and fixing the cell during mid-cytokinesis. Injected rhodamine actin was only slightly concentrated in the contractile ring, as compared to the distribution of total actin filaments. Our results indicate that preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring. De novo assembly of filaments, on the other hand, appears to take place preferentially outside the cleavage furrow.
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We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.
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We have found that addition of a small amount of filamentous muscle actin (F-actin) to a solution of globular actin (G-actin) in a low ionic strength medium resulted in rapid polymerization of the G-actin. This reaction was inhibited by substoichiometric levels of cytochalasins (relative potency: cytochalasin D greater than cytochalasin E approximately equal to cytochalasin B greater than dihydrocytochalasin B). Binding experiments show that F-actin, but not G-actin, contains high affinity binding sites for [3H]cytochalasin B; the number of sites detected was on the order of about one per actin filament (one site/500 actin monomers). This number remained unchanged when the actin (prepared by polymerization-depolymerization cycles) was further purified by ion exchange and gel filtration chromatography. Competitive displacement experiments showed that the relative affinity of several cytochalasins for these sites corresponds to their relative effectiveness in inhibiting actin polymerization induced by F-actin. These results suggest that actin filaments can accelerate the rate of polymerization of G-actin in low ionic strength medium by providing sites onto which actin monomers can be added, and that cytochalasins inhibit actin filament elongation by binding to high affinity sites located at the polymerization end of the filaments.
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Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell behavior. We have investigated the role of protein kinase C in alpha 5 beta 1 integrin-mediated signal transduction in Chinese hamster ovary cells. Up-regulation of protein kinase C activity by phorbol esters was found to enhance cell adhesion, spreading, and migration on a fibronectin substrate, without affecting the cell surface expression or fibronectin binding of the alpha 5 beta 1 integrin, whereas inhibition of protein kinase C activity by calphostin C inhibited these functions as well in unstimulated cells. In addition, we observed that protein kinase C activity in the cell membrane fraction transiently increases preceding cell spreading on fibronectin, but not on polylysine, additionally implying a specific role of protein kinase C in alpha 5 beta 1 integrin-mediated spreading on fibronectin. Experiments with phorbol esters and calphostin C also suggested that protein kinase C activity is required for enhanced phosphorylation of the focal adhesion kinase, pp125fak, in cells plated on fibronectin. Protein kinase C-alpha was not, however, found to directly act on pp125fak, suggesting that other mechanisms are involved in this phenomenon.
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Profilin, the prototype actin-monomer-sequestering protein, has recently emerged as a multifunctional protein with several different activities. Genetic evidence in yeast and flies confirms that profilin is required for a normal actin cytoskeleton, while biochemical evidence suggests a role in regulating phosphoinositide signalling. New studies suggest that profilin may interact with other ligands, and even its role in regulating actin polymerization is now being re-evaluated.
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Freshly plated 3T3 cells send out radial projections or filopodia. We observed cells which happended to settle on glass near the borderline of a gold-plated area. When some of the filopodia contacted the gold-plated area and others the glass substratum and remained attached for a few minutes, lamellipodia then extended preferentially toward the gold-plated area. 1-2 h later, most of the cells were found in the gold-plated area. When the filopodia of a spreading 3T3 cell contacted another already spread 3T3 cell and also the glass substratum, the first lamellipodia extended preferentially towards the glass. These observations suggest a directionally differentiated extension of lamellipodia after the filopodia of a spreading 3T3 cell have contacted different substrates in their environment. Before filopodia contact a substrate, they perform a rapid "scanning" motion. Therefore, we suggest that the filopodia of a spreading 3T3 cell serve as organs which explore the nonfluid environment and react to a certain quality of the substrate that is presently unknown. Subsequently, they mediate the extension of lamellipodia into the direction in which this quality is found. The described phenomena are reversibly inhibited by Cytochalasin B at concentrations above 5 mug/ml although filopodia are produced.
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Cells in culture reveal high levels of protein tyrosine phosphorylation in their focal adhesions, the regions where cells adhere to the underlying substratum. We have examined the tyrosine phosphorylation of proteins in response to plating cells on extracellular matrix substrata. Rat embryo fibroblasts, mouse Balb/c 3T3, and NIH 3T3 cells plated on fibronectin-coated surfaces revealed elevated phosphotyrosine levels in a cluster of proteins between 115 and 130 kD. This increase in tyrosine phosphorylation was also seen when rat embryo fibroblasts were plated on laminin or vitronectin, but not on polylysine or on uncoated plastic. Integrin mediation of this effect was suggested by finding the same pattern of elevated tyrosine phosphorylation in cells plated on the cell-binding fragment of fibronectin and in cells plated on a synthetic polymer containing multiple RGD sequences. We have identified one of the proteins of the 115-130-kD cluster as pp125FAK, a tyrosine kinase recently localized in focal adhesions (Schaller, M. D., C. A. Borgman, B. S. Cobb, R. R. Vines, A. B. Reynolds, and J. T. Parsons. 1992. Proc. Natl. Acad. Sci. USA. 89:5192). A second protein that becomes tyrosine phosphorylated in response to extracellular matrix adhesion is identified as paxillin, a 70-kD protein previously localized to focal adhesions. Treatment of cells with the tyrosine kinase inhibitor herbimycin A diminished the adhesion-induced tyrosine phosphorylation of these proteins and inhibited the formation of focal adhesions and stress fibers. These results suggest a role for integrin-mediated tyrosine phosphorylation in the organization of the cytoskeleton as cells adhere to the extracellular matrix.
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Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.
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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.
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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.
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We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.
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Several new interactions have been identified and proteins characterized in focal adhesions. Together they suggest alternative or parallel linkages between actin filaments and members of the integrin family of extracellular receptors. Transformed cells continue to serve as models for studying the assembly and disassembly of these adhesions.
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The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.
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A substantial number of novel guanine nucleotide binding regulatory proteins have been identified over the last few years but the function of many of them is largely unknown. This article will discuss a particular family of these proteins, structurally related to the Ras oncoprotein. Approximately 30 Ras-related small guanosine triphosphate (GTP)-binding proteins are known, and from yeast to man they appear to be involved in controlling a diverse set of essential cellular functions including growth, differentiation, cytoskeletal organization, and intracellular vesicle transport and secretion.
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We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.
Article
We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.
Article
We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin-actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells.
Article
Evidence for a structural precursor of the focal contact in cultured fibroblasts and continuing studies on the development of the precursor and contact are discussed. The structural precursor consists of an F-actin-rich, rib-like fiber within the motile lamellipodium. The focal contact forms beneath the fiber, part of which is retained at the contact as the initial adhesion plaque. Therefore, F-actin is present at the contact from the beginning. Vinculin accumulates at the plaque during a 90-second period after the contact forms. A novel feature of the distribution of talin has been found. The protein is present along the distal margin of the lamellipodium, where it is further concentrated as a series of nodes at the tips of each precursor and between precursors. This distribution of talin is independent of that which develops at the plaque after the contact forms. The structural development of the precursor has been followed with AVEC-DIC optics. The process begins with the development of fine oblique fibers from small structural nodes at the margin of the lamellipodium, and continues with the fusion of the nodes at the margin and inward coalescence of the fibers. It is suggested that talin may function as a cross-linking protein in the convergence of actin filaments at the membrane, while other actin-bundling proteins participate in the inward coalescence of the filaments to form fibers. The F-actin core of the precursor could provide a structural framework against which differences at the external surface of the membrane develop prior to contact formation.