Imperial College London

ProfessorVaniaBraga

Faculty of MedicineNational Heart & Lung Institute

Professor of Cellular Signalling
 
 
 
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Contact

 

+44 (0)20 7594 3233v.braga

 
 
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Location

 

Office no. 105Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

84 results found

Dong Y, Xu Y, Lian C, Prak K, Leo HL, Tetley TD, Braga V, Emerson M, Ahnström J, Yap CHet al., 2024, Safe and efficacious near superhydrophobic hemostat for reduced blood loss and easy detachment in traumatic wounds, ACS Applied Materials and Interfaces, Vol: 16, Pages: 4307-4320, ISSN: 1944-8244

Hemorrhage is the leading cause of trauma death, and innovation in hemostatic technology is important. The strongly hydrophobic carbon nanofiber (CNF) coating has previously been shown to have excellent hemostatic properties. However, the understanding of how CNF coating guides the coagulation cascade and the biosafety of CNF as hemostatic agents has yet to be explored. Here, our thrombin generation assay investigation showed that CNF induced fast blood coagulation via factor (F) XII activation of the intrinsic pathway. We further performed studies of a rat vein injury and demonstrated that the CNF gauze enabled a substantial reduction of blood loss compared to both the plain gauze and kaolin-imbued gauze (QuikClot). Analysis of blood samples from the model revealed no acute toxicity from the CNF gauze, with no detectable CNF deposition in any organ, suggesting that the immobilization of CNF on our gauze prevented the infiltration of CNF into the bloodstream. Direct injection of CNF into the rat vein was also investigated and found not to elicit overt acute toxicity or affect animal survival or behavior. Finally, toxicity assays with primary keratinocytes revealed minimal toxicity responses to CNF. Our studies thus supported the safety and efficacy of the CNF hemostatic gauze, highlighting its potential as a promising approach in the field of hemostatic control.

Journal article

Sri-Ranjan K, Sanchez-Alonso JL, Swiatlowska P, Rothery S, Novak P, Gerlach S, Koeninger D, Hoffmann B, Merkel R, Stevens M, Sun S, Gorelik J, Braga Vet al., 2022, Intrinsic cell rheology drives junction maturation, Nature Communications, Vol: 13, ISSN: 2041-1723

A fundamental property of higher eukaryotes that underpins their evolutionary success is stable cell-cell cohesion. Yet, how intrinsic cell rheology and stiffness contributes to junction stabilization and maturation is poorly understood. We demonstrate that localized modulation of cell rheology governs the transition of a slack, undulated cell-cell contact (weak adhesion) to a mature, straight junction (optimal adhesion). Cell pairs confined on different geometries have heterogeneous elasticity maps and control their own intrinsic rheology co-ordinately. More compliant cell pairs grown on circles have slack contacts, while stiffer triangular cell pairs favour straight junctions with flanking contractile thin bundles. Counter-intuitively, straighter cell-cell contacts have reduced receptor density and less dynamic junctional actin, suggesting an unusual adaptive mechano-response to stabilize cell-cell adhesion. Our modelling informs that slack junctions arise from failure of circular cell pairs to increase their own intrinsic stiffness and resist the pressures from the neighbouring cell. The inability to form a straight junction can be reversed by increasing mechanical stress artificially on stiffer substrates. Our data inform on the minimal intrinsic rheology to generate a mature junction and provide a springboard towards understanding elements governing tissue-level mechanics.

Journal article

Erasmus JC, Smolarczyk K, Brezovjakova H, Mohd-Naim NF, Lozano E, Matter K, Braga Vet al., 2021, Rac1-PAK1 regulation of Rab11 cycling promotes junction destabilization, The Journal of Cell Biology, Vol: 220, Pages: 1-19+, ISSN: 0021-9525

Rac1 GTPase is hyper-activated in tumours and contributes to malignancy.Rac1 disruption of junctions requires its effector PAK1, but the precise mechanisms are unknown. Here we identify that E-cadherinis internalized via micropinocytosis in a PAK1–dependent manner, without catenin dissociation and degradation. In addition to internalization, PAK1 regulates E-cadherin transport by fine-tuning Rab small GTPase function. PAK1 phosphorylates RabGDI(a core Rab regulator), but not RabGDI. Phosphorylated RabGDIpreferentiallyassociateswith Rab5 and Rab11, which is predicted to promote Rab retrieval from membranes. Consistent with this hypothesis, Rab11is activated by hyper-activeRac1andinhibition of Rab11 function partially rescues E-cadherin destabilization. Thus, Rac1 activation reduces surface cadherin levels as a net result of higher bulk flow of membrane uptake that counteractsRab11-dependentE-cadherin delivery to junctions (recycling and/or exocytosis). This unique small GTPase crosstalk has impact onRac1 and PAK1regulation of membrane remodelling during epithelial de-differentiation, adhesion and motility.

Journal article

Brezovjakova H, Tomlinson C, Mohd Naim N, Swiatlowska P, Erasmus JC, Huveneers S, Gorelik J, Bruche S, Braga VMet al., 2019, Junction Mapper is a novel computer vision tool to decipher cell-cell contact phenotypes., Elife, Vol: 8

Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.

Journal article

Lopes-de-Souza L, Costal-Oliveira F, Stransky S, Fonseca de Freitas C, Guerra-Duarte C, Braga VMM, Chávez-Olórtegui Cet al., 2019, Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency, Toxicon, Vol: 170, Pages: 68-76, ISSN: 0041-0101

Accidents with venomous snakes are a major health hazard in tropical countries. Bothrops genus is responsible for almost 80% of snakebites in Brazil. Immunotherapy is the only approved specific treatment against snake toxins and the production of therapeutic antivenoms requires quality control tests to determine their neutralizing potency. Currently, these controls are performed by in vivo lethality neutralization, however, the inhibition of particular events produced by bothropic venoms such as coagulopathy, hemorrhage, edema or cytotoxic effects are also required. The aim of this work is to develop an in vitro alternative assay for antivenom pre-clinical evaluation. In this sense, we designed a cell viability assay using different amounts (0.2–10 μL/well) of low and high potency anti-bothropic sera, previously classified by the traditional in vivo test, for assessing the antivenom capacity to protect the cells against B. jararaca venom cytotoxicity (5xEC50 = 58.95 μg/mL). We found that high potency sera are more effective in neutralizing B. jararaca venom cytotoxicity when compared to low potency sera, which is in accordance to their pre-determined in vivo potency. Considering sera in vitro inhibitory concentration able to prevent 50% cell death (IC50) and their known in vivo potency, a cut-off point was determined to discriminate low and high potency sera. Our data provide insights for the development of an in vitro method which can determine the anti-bothropic antivenom potency during its production.

Journal article

Schultz F, Swiatlowska P, Alvarez-Laviada A, Sanchez-Alonso JL, Song Q, de Vries AAF, Pijnappels DA, Ongstad E, Braga VMM, Entcheva E, Gourdie RG, Miragoli M, Gorelik Jet al., 2019, Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43., FASEB Journal, Pages: 1-16, ISSN: 0892-6638

Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte-myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.-Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.

Journal article

Braga V, Costal-Oliveiraa F, Stransky S, Guerra-Duarte C, Souza DLND, Vivas-Ruiz DE, Yarlequé A, Sanchez EF, Chávez-Olórtegui Cet al., 2019, L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes, Scientific Reports, Vol: 9, Pages: 1-14, ISSN: 2045-2322

Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC50) of 5.1 μg/mL. Cytotoxicity caused by LAAO was mediated by H2O2 and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms.

Journal article

Braga V, 2018, Signaling by small GTPases at cell-cell junctions: protein interactions building control and networks, Cold Spring Harbor perspectives in biology, Vol: 10, ISSN: 1943-0264

A number of interesting reports highlight the intricate network of signaling proteins that coordinate formation and maintenance of cell-cell contacts. We have much yet to learn about how the in vitro binding data is translated into protein association inside the cells and whether such interaction modulates the signaling properties of the protein. What emerges from recent studies is the importance to carefully consider small GTPase activation in the context of where its activation occurs, which upstream regulators are involved in the activation/inactivation cycle and the GTPase interacting partners that determine the intracellular niche and extent of signaling. Data discussed here unravel unparalleled cooperation and coordination of functions among GTPases and their regulators in supporting strong adhesion between cells.

Journal article

Stransky S, Costal-Oliveira F, Lopes-de-Souza L, Guerra-Duarte C, Chávez-Olórtegui C, Braga VMMet al., 2018, In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom, PLoS Neglected Tropical Diseases, Vol: 12, ISSN: 1935-2727

Envenomation by the bushmaster snake Lachesis muta muta is considered severe, characterized by local effects including necrosis, the main cause of permanent disability. However, cellular mechanisms related to cell death and tissue destruction, triggered by snake venoms, are poorly explored. The purpose of this study was to investigate the cytotoxic effect caused by L. m. muta venom in normal human keratinocytes and to identify the cellular processes involved in in cellulo envenomation. In order to investigate venom effect on different cell types, Alamar Blue assay was performed to quantify levels of cellular metabolism as a readout of cell viability. Apoptosis, necrosis and changes in mitochondrial membrane potential were evaluated by flow cytometry, while induction of autophagy was assessed by expression of GFP-LC3 and analyzed using fluorescence microscopy. The cytotoxic potential of the venom is shown by reduced cell viability in a concentration-dependent manner. It was also observed the sequential appearance of cells undergoing autophagy (by 6 hours), apoptosis and necrosis (12 and 24 hours). Morphologically, incubation with L. m. muta venom led to a significant cellular retraction and formation of cellular aggregates. These results indicate that L. m. muta venom is cytotoxic to normal human keratinocytes and other cell lines, and this toxicity involves the integration of distinct modes of cell death. Autophagy as a cell death mechanism, in addition to apoptosis and necrosis, can help to unravel cellular pathways and mechanisms triggered by the venom. Understanding the mechanisms that underlie cellular damage and tissue destruction will be useful in the development of alternative therapies against snakebites.

Journal article

Fischer A, Braga VMM, 2018, Vascular permeability: flow-mediated, non-canonical notch signalling promotes barrier integrity, Current Biology, Vol: 28, Pages: R119-R121, ISSN: 1879-0445

The vascular permeability barrier must be maintained in response to changes to vessel calibre, shear stress and blood pressure. A new study reveals a remarkable mechanism for flow-mediated regulation of permeability: Notch1 activation leads to the assembly of GTPase signalling complexes at VE-cadherin contacts and a strengthening of the endothelial barrier.

Journal article

Machesky L, Braga VMM, 2017, So far, yet so close: alpha-Catenin dimers help migrating cells get together, Journal of Cell Biology, Vol: 216, Pages: 3437-3439, ISSN: 0021-9525

Journal article

Braga VMM, McCormack JJ, Bruche S, Ouadda ABD, Ishii H, Lu H, Garcia-Cattaneo A, Chavez-Olortegi C, Lamarche-Vane Net al., 2017, The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts, Scientific Reports, Vol: 7, ISSN: 2045-2322

Levels of active Rac1 at epithelial junctions are partially modulated via interaction with Ajuba, an actin binding and scaffolding protein. Here we demonstrate that Ajuba interacts with the Cdc42 GTPase activating protein CdGAP, a GAP for Rac1 and Cdc42, at cell-cell contacts. CdGAP recruitment to junctions does not require Ajuba; rather Ajuba seems to control CdGAP residence at sites of cell-cell adhesion. CdGAP expression potently perturbs junctions and Ajuba binding inhibits CdGAP activity. Ajuba interacts with Rac1 and CdGAP via distinct domains and can potentially bring them in close proximity at junctions to facilitate activity regulation. Functionally, CdGAP-Ajuba interaction maintains junctional integrity in homeostasis and diseases: (i) gain-of-function CdGAP mutants found in Adams-Oliver Syndrome patients strongly destabilize cell-cell contacts and (ii) CdGAP mRNA levels are inversely correlated with E-cadherin protein expression in different cancers. We present conceptual insights on how Ajuba can integrate CdGAP binding and inactivation with the spatio-temporal regulation of Rac1 activity at junctions. Ajuba poses a novel mechanism due to its ability to bind to CdGAP and Rac1 via distinct domains and influence the activation status of both proteins. This functional interplay may contribute towards conserving the epithelial tissue architecture at steady-state and in different pathologies.

Journal article

Erasmus JC, Bruche S, Pizarro L, Maimari N, Poggioli T, Tomlinson C, Lees J, Zalivina I, Wheeler A, Alberts A, Russo A, Braga VMMet al., 2017, Corrigendum: Defining functional interactions during biogenesis of epithelial junctions, Nature Communications, Vol: 8, Pages: 14195-14195, ISSN: 2041-1723

The original version of this Article (https://doi.org/10.1038/ncomms13542) contained an error in the spelling of the author Tommaso Poggioli, which was incorrectly given as Tommaso Pogglioli. This has now been corrected in both the PDF and HTML versions of the Article.

Journal article

Braga VMM, 2016, Defining functional interactions during biogenesis of epithelial junctions, Nature Communications, Vol: 7, Pages: 1-17, ISSN: 2041-1723

In spite of extensive recent progress, a comprehensive understanding of how actin cytoskeleton remodelling supports stable junctions remains to be established. Here we design a platform that integrates actin functions with optimized phenotypic clustering and identify new cytoskeletal proteins, their functional hierarchy and pathways that modulate E-cadherin adhesion. Depletion of EEF1A, an actin bundling protein, increases E-cadherin levels at junctions without a corresponding reinforcement of cell-cell contacts. This unexpected result reflects a more dynamic and mobile junctional actin in EEF1A-depleted cells. A partner for EEF1A in cadherin contact maintenance is the formin DIAPH2, which interacts with EEF1A. In contrast, depletion of either the endocytic regulator TRIP10 or the Rho GTPase activator VAV2 reduces E-cadherin levels at junctions. TRIP10 binds to and requires VAV2 function for its junctional localization. Overall, we present new conceptual insights on junction stabilization, which integrate known and novel pathways with impact for epithelial morphogenesis, homeostasis and diseases.

Journal article

Jaber N, Mohd-Naim N, Wang Z, DeLeon JL, Kim S, Zhong H, Sheshadri N, Dou Z, Edinger AL, Du G, Braga VMM, Zong W-Xet al., 2016, Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus, JOURNAL OF CELL SCIENCE, Vol: 129, Pages: 4424-4435, ISSN: 0021-9533

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34−/− mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein–lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.

Journal article

Braga V, 2016, Spatial integration of E-cadherin adhesion, signalling and the epithelial cytoskeleton, Current Opinion in Cell Biology, Vol: 42, Pages: 138-145, ISSN: 1879-0410

The characteristic tall and elongated shape of epithelial cells requires specialized adhesive structures and a distinct organization of cytoskeletal filaments. Cytoskeletal networks coordinate a precise organization of adhesive and signalling complexes along cell-cell contacts and enable exquisite strong cohesion among epithelial cells. E-cadherin, a calcium-dependent adhesion receptor, is an essential adhesive system in epithelia and its dynamic regulation and pathways that stabilize cell-cell adhesion have been extensively studied. This review highlights the less understood mechanisms underlying how cadherin receptor signalling drives cytoskeletal rearrangements which ultimately define the epithelial cell shape. In the past two years, new insights identify specific actin-binding proteins and regulators of the epithelial cytoskeleton as a framework to support junction dynamics, plasticity and maintenance.

Journal article

Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, Adhihetty PJ, Adler SG, Agam G, Agarwal R, Aghi MK, Agnello M, Agostinis P, Aguilar PV, Aguirre-Ghiso J, Airoldi EM, Ait-Si-Ali S, Akematsu T, Akporiaye ET, Al-Rubeai M, Albaiceta GM, Albanese C, Albani D, Albert ML, Aldudo J, Algül H, Alirezaei M, Alloza I, Almasan A, Almonte-Beceril M, Alnemri ES, Alonso C, Altan-Bonnet N, Altieri DC, Alvarez S, Alvarez-Erviti L, Alves S, Amadoro G, Amano A, Amantini C, Ambrosio S, Amelio I, Amer AO, Amessou M, Amon A, An Z, Anania FA, Andersen SU, Andley UP, Andreadi CK, Andrieu-Abadie N, Anel A, Ann DK, Anoopkumar-Dukie S, Antonioli M, Aoki H, Apostolova N, Aquila S, Aquilano K, Araki K, Arama E, Aranda A, Araya J, Arcaro A, Arias E, Arimoto H, Ariosa AR, Armstrong JL, Arnould T, Arsov I, Asanuma K, Askanas V, Asselin E, Atarashi R, Atherton SS, Atkin JD, Attardi LD, Auberger P, Auburger G, Aurelian L, Autelli R, Avagliano L, Avantaggiati ML, Avrahami L, Awale S, Azad N, Bachetti T, Backer JM, Bae DH, Bae JS, Bae ON, Bae SH, Baehrecke EH, Baek SH, Baghdiguian S, Bagniewska-Zadworna A, Bai H, Bai J, Bai XY, Bailly Y, Balaji KN, Balduini W, Ballabio A, Balzan R, Banerjee R, Bánhegyi G, Bao H, Barbeau B, Barrachina MD, Barreiro E, Bartel B, Bartolomé A, Bassham DC, Bassi MT, Bast RC, Basu A, Batista MT, Batoko H, Battino M, Bauckman K, Baumgarner BL, Bayer KU, Beale R, Beaulieu JF, Beck GR, Becker C, Beckham JD, Bédard PA, Bednarski PJ, Begley TJ, Behl C, Behrends C, Behrens GM, Behrns KE, Bejarano E, Belaid A, Belleudi F, Bénard G, Berchem G, Bergamaschi D, Bergami M, Berkhout B, Berliocchi L, Bernard A, Bernard M, Bernassola F, Bertolotti A, Bess AS, Besteiro S, Bettuzzi S, Bhalla S, Bhattacharyya S, Bhutia SK, Biagosch C, Bianchi MW, Biard-Piechaczyk M, Billes V, Bincoletto C, Bingol B, Bird SW, Bitoun M, Bjedov I, Blackstone C, Blanc L, Blanco GA, Blomhoff HK, Boada-Romero E, Böckler S, Boes M, Boesze-Battagliaet al., 2016, Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)., Autophagy, Vol: 12, Pages: 1-215, ISSN: 1554-8635

Journal article

Erasmus JC, Welsh NJ, Braga VMM, 2015, Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion, Cellular Signalling, Vol: 27, Pages: 1905-1913, ISSN: 1873-3913

The precise mechanisms via which Rac1 is activated by cadherin junctions are not fully known. In keratinocytesRac1 activation by cadherin junctions requires EGFR signalling, but how EGFR does so is unclear. To addresswhich activator could mediate E-cadherin signalling to Rac1, we investigated EGFR and two Rac1 GEFs, SOS1and DOCK180. EGFR RNAi prevented junction-induced Rac1 activation and led to fragmented localization ofE-cadherin at cadherin contacts. In contrast, depletion of another EGFR family member, ErbB3, did not interferewith either process. DOCK180 RNAi, but not SOS1, prevented E-cadherin-induced Rac1 activation. However, in astrong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment orcadherin localisation at junctions. Rather, reduced DOCK180 levels impaired the resistance to mechanicalstress of pre-formed cell aggregates. Thus, within the same cell type, EGFR and DOCK180 regulate Rac1activation by newly-formed contacts, but control separate cellular events that cooperate to stabilisejunctions

Journal article

Kelly DJ, Warren SC, Alibhai D, Kumar S, Alexandrov Y, Munro I, Margineanu A, McCormack J, Welsh NJ, Serwa RA, Thinon E, Kongsema M, McGinty J, Talbot C, Murray EJ, Stuhmeier F, Neil MAA, Tate EW, Braga VMM, Lam EW-F, Dunsby C, French PMWet al., 2015, Automated multiwell fluorescence lifetime imaging for Forster resonance energy transfer assays and high content analysis, ANALYTICAL METHODS, Vol: 7, Pages: 4071-4089, ISSN: 1759-9660

Journal article

Garcia-Cattaneo A, Braga VMM, 2013, Hold on tightly How to keep the local activation of small GTPases, CELL ADHESION & MIGRATION, Vol: 7, Pages: 283-287, ISSN: 1933-6918

Signaling regulated by Rho small GTPases plays a pivotal role in cell migration, cell attachment to substratum or to their neighbors among other functions. Concerted efforts have focused on understanding how different GTPases are activated by specific stimuli and which regulator is responsible for the spatio-temporal control of their activity at particular intracellular sites. We have recently described the role of a scaffold protein, Ajuba, in adherens junction maintenance via direct stabilization of activated small GTPase Rac1 at cell–cell contacts. Ajuba binds to both active and inactive forms of Rac1. Upon junction formation, Rac1 activation initiates a positive feedback loop leading to Ajuba phosphorylation and Ajuba-mediated retention of activated Rac1 at junctions. Thus, cytoskeletal proteins may have a dual role to provide a scaffolding platform and dynamically modulate small GTPases function at a specific place, irrespective of their ability to interact with active and inactive forms. Here we discuss similar mechanisms via which cytoskeletal proteins can facilitate cellular processes downstream of Rho proteins by increasing their affinity to activated GTPases.

Journal article

Pellegrino L, Stebbing J, Braga VM, Frampton AE, Jacob J, Buluwela L, Jiao LR, Periyasamy M, Madsen CD, Caley MP, Ottaviani S, Roca-Alonso L, El-Bahrawy M, Coombes RC, Krell J, Castellano Let al., 2013, miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts, Nucleic Acids Research, Vol: 41, Pages: 5400-5412, ISSN: 1362-4962

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.

Journal article

Carroll B, Mohd-Naim N, Maximiano F, Frasa MA, McCormack J, Finelli M, Thoresen SB, Perdios L, Daigaku R, Francis RE, Futter C, Dikic I, Braga VMMet al., 2013, The TBC/RabGAP Armus Coordinates Rac1 and Rab7 Functions during Autophagy, Developmental Cell, Vol: 25, Pages: 15-28, ISSN: 1878-1551

Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking.

Journal article

McCormack J, Welsh NJ, Braga VMM, 2013, Cycling around cell-cell adhesion with Rho GTPase regulators, JOURNAL OF CELL SCIENCE, Vol: 126, Pages: 379-391, ISSN: 0021-9533

The formation and stability of epithelial adhesive systems, such as adherens junctions, desmosomes and tight junctions, rely on a number of cellular processes that ensure a dynamic interaction with the cortical cytoskeleton, and appropriate delivery and turnover of receptors at the surface. Unique signalling pathways must be coordinated to allow the coexistence of distinct adhesive systems at discrete sub-domains along junctions and the specific properties they confer to epithelial cells. Rho, Rac and Cdc42 are members of the Rho small GTPase family, and are well-known regulators of cell–cell adhesion. The spatio-temporal control of small GTPase activation drives specific intracellular processes to enable the hierarchical assembly, morphology and maturation of cell–cell contacts. Here, we discuss the small GTPase regulators that control the precise amplitude and duration of the levels of active Rho at cell–cell contacts, and the mechanisms that tailor the output of Rho signalling to a particular cellular event. Interestingly, the functional interaction is reciprocal; Rho regulators drive the maturation of cell–cell contacts, whereas junctions can also modulate the localisation and activity of Rho regulators to operate in diverse processes in the epithelial differentiation programme.

Journal article

Serva A, Knapp B, Claas C, Tsai YC, Lisauskas T, Matula P, Harder N, Kaderali L, Rohr K, Erfle H, Eils R, Braga VMM, Starkuviene Vet al., 2012, miR-17-5p regulates endocytic trafficking through targeting TBC1D2/ Armus, PLoS ONE, Vol: 7, ISSN: 1932-6203

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic clusterthat encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by thiscluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this clusterand sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytictrafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially beachieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17,which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.

Journal article

Kalaji R, Wheeler AP, Erasmus JC, Lee SY, Endres RG, Cramer LP, Braga VMMet al., 2012, ROCK1 and ROCK2 regulate epithelial polarisation and geometric cell shape, BIOLOGY OF THE CELL, Vol: 104, Pages: 435-451, ISSN: 0248-4900

Journal article

Frasa MAM, Koessmeier KT, Ahmadian MR, Braga VMMet al., 2012, Illuminating the functional and structural repertoire of human TBC/ RABGAPs, NATURE REVIEWS MOLECULAR CELL BIOLOGY, Vol: 13, Pages: 67-73, ISSN: 1471-0072

Journal article

Dawson JC, Bruche S, Spence HJ, Braga VMM, Machesky LMet al., 2012, Mtss1 promotes cell-cell junction assembly and stability through the small GTPase Rac1, PLoS One, Vol: 7, ISSN: 1932-6203

Cell-cell junctions are an integral part of epithelia and are often disrupted in cancer cells during epithelial-to-mesenchymal transition (EMT), which is a main driver of metastatic spread. We show here that Metastasis suppressor-1 (Mtss1; Missing in Metastasis, MIM), a member of the IMD-family of proteins, inhibits cell-cell junction disassembly in wound healing or HGF-induced scatter assays by enhancing cell-cell junction strength. Mtss1 not only makes cells more resistant to cell-cell junction disassembly, but also accelerates the kinetics of adherens junction assembly. Mtss1 drives enhanced junction formation specifically by elevating Rac-GTP. Lastly, we show that Mtss1 depletion reduces recruitment of F-actin at cell-cell junctions. We thus propose that Mtss1 promotes Rac1 activation and actin recruitment driving junction maintenance. We suggest that the observed loss of Mtss1 in cancers may compromise junction stability and thus promote EMT and metastasis.

Journal article

Nola S, Erasmus JC, Braga VM, 2012, Quantitative and robust assay to measure cell-cell contact assembly and maintenance, Vol: 827, Pages: 143-155, ISSN: 1940-6029

Epithelial junction formation and maintenance are multistep processes that rely on the clustering of macromolecular complexes. These events are highly regulated by signalling pathways that involve Rho small GTPases. Usually, when analysing the contribution of different components of Rho-dependent pathways to cell-cell adhesion, the localisation of adhesion receptors at junctions is evaluated by immunofluorescence. However, we find that this method has limitations on the quantification (dynamic range), ability to detect partial phenotypes and to differentiate between the participation of a given regulatory protein in assembly and/or maintenance of cell-cell contacts.In this chapter, we describe a suitable method, the aggregation assay, in which we adapted a quantitative strategy to allow objective and reproducible detection of partial phenotypes. Importantly, this methodology estimates the ability of cells to form junctions and their resistance to mechanical shearing forces (stabilisation).

Journal article

Braga VM, Nola S, Erasmus J, 2012, Quantitative and robust assay to measure cell-cell contact assembly and maintenance. Rho GTPases. Methods and Protocols, Publisher: Humana Press, Springer, Pages: 143-156

Book chapter

Nola S, Daigaku R, Smolarczyk K, Carstens M, Martin-Martin B, Longmore G, Bailly M, Braga VMMet al., 2011, Ajuba is required for Rac activation and maintenance of E-cadherin adhesion, JOURNAL OF CELL BIOLOGY, Vol: 195, Pages: 855-871, ISSN: 0021-9525

Journal article

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