Bothrops atrox venom: Biochemical properties and cellular phenotypes of three highly toxic classes of toxins.

Snake venoms have a complex mixture of compounds that are conserved across species and act synergistically, triggering severe local and systemic effects. Identification of the toxin classes that are most damaging to cell homeostasis would be a powerful approach to focus on the main activities that underpin envenomation. Here, we focus on the venom of Bothrops atrox, snake responsible for most of the accidents in Amazon region of South America. We identified the key cytotoxic toxin fractions from B. atrox venom and mapped their biochemical properties, protein composition and cell damage. Five fractions were obtained by mass exclusion chromatography and contained either a single class of enzymatic activity (i.e., L-amino acid oxidases or Hyaluronidases) or different activities co-distributed in two or more protein fractions (e.g., Metalloproteinases, Serine Proteases, or Phospholipases A2). Only three protein fractions reduced cell viability of primary human cells. Strikingly, such activity is accompanied by disruption of cell attachment to substratum and to neighbouring cells. Such strong perturbation of morphological cell features indicates likely defects in tissue integrity in vivo. Mass spectrometry identified the main classes of toxins that contribute to these phenotypes. We provide here a strategy for the selection of key cytotoxic proteins for targeted investigation of their mechanism of action and potential synergism during snakebite envenomation. Our data highlights putative toxins (or combinations of) that may be the focus of future therapeutic interference.

Intrinsic cell rheology drives junction maturation.

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.

Rac1-PAK1 regulation of Rab11 cycling promotes junction destabilization.

Rac1 GTPase is hyperactivated in tumors and contributes to malignancy. Rac1 disruption of junctions requires its effector PAK1, but the precise mechanisms are unknown. Here, we show that E-cadherin is 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 a core Rab regulator, RabGDIß, but not RabGDIα. Phosphorylated RabGDIß preferentially associates with Rab5 and Rab11, which is predicted to promote Rab retrieval from membranes. Consistent with this hypothesis, Rab11 is activated by Rac1, and inhibition 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 counteracts Rab11-dependent E-cadherin delivery to junctions (recycling and/or exocytosis). This unique small GTPase crosstalk has an impact on Rac1 and PAK1 regulation of membrane remodeling during epithelial dedifferentiation, adhesion, and motility.

Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency.

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.

Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.

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.

L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes.

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.

Vascular Permeability: Flow-Mediated, Non-canonical Notch Signalling Promotes Barrier Integrity.

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.

So far, yet so close: α-Catenin dimers help migrating cells get together.

Epithelial cells in tissues use their actin cytoskeletons to stick together, whereas unattached cells make active plasma membrane protrusions to migrate. In this issue, Wood et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201612006) show that the junction component α-catenin is critical in freely moving cells to promote adhesion and migration.

Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.

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.

Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

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

The TBC/RabGAP Armus coordinates Rac1 and Rab7 functions during autophagy.

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.

Cycling around cell-cell adhesion with Rho GTPase regulators.

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.

Hold on tightly: how to keep the local activation of small GTPases.

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.

Mtss1 promotes cell-cell junction assembly and stability through the small GTPase Rac1.

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.

ROCK1 and ROCK2 regulate epithelial polarisation and geometric cell shape.

BACKGROUND INFORMATION: Cell-cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear. RESULTS: Upon induction of cell-cell adhesion, we find that human keratinocytes acquire specific geometric shapes favouring hexagons, by re-modelling junction length/orientation and thus neighbour allocation. Acquisition of geometric shape correlates temporally with epithelial polarisation, as shown by an increase in lateral height. ROCK1 and ROCK2 are important regulators of myosin II contraction, but their specific role in epithelial cell shape has not been addressed. Depletion of ROCK proteins interferes with the correct proportion of hexagonal cell shapes and full elongation of lateral domain. Interestingly, ROCK proteins are not essential for maintenance of circumferential thin bundles, the main contractile epithelial F-actin pool. Instead, ROCK1 or ROCK2 regulates thin bundle contraction and positioning along the lateral domain, an important event for the stabilisation of the elongating lateral domain. Mechanistically, E-cadherin clustering specifically leads to ROCK1/ROCK2-dependent inactivation of myosin phosphatase and phosphorylation of myosin regulatory light chain. These events correlate temporally with the increase in lateral height and thin bundle compaction towards junctions. CONCLUSION: We conclude that ROCK proteins are necessary for acquisition of elongated and geometric cell shape, two key events for epithelial differentiation.

Illuminating the functional and structural repertoire of human TBC/RABGAPs.

The Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.

Quantitative and robust assay to measure cell-cell contact assembly and maintenance.

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).

Ajuba is required for Rac activation and maintenance of E-cadherin adhesion.

Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.

RhoG is required for both FcγR- and CR3-mediated phagocytosis.

Phagocytosis is a highly ordered process orchestrated by signalling through Rho GTPases to locally organise the actin cytoskeleton and drive particle uptake. Specific Rho family members that regulate phagocytosis are not known, as the majority of studies have relied on the use of dominant-negative mutants and/or toxins, which can inactivate multiple Rho GTPases. To identify the relevant GTPases for phagocytosis through the Fcγ receptor (FcγR) and complement receptor 3 (CR3), we depleted 20 Rho proteins individually in an RNA interference (RNAi) screen. We find that distinct GTPase subsets are required for actin polymerisation and uptake by macrophages: FcγR-dependent engulfment requires Cdc42 and Rac2 (but not Rac1), whereas CR3 requires RhoA. Surprisingly, RhoG is required for particle uptake through both FcγR and CR3. RhoG has been previously linked to Rac and Cdc42 signalling in different model systems, but not to RhoA. Interestingly, we find that RhoG is also recruited and activated at phagocytic cups downstream of FcγR and CR3, irrespective of their distinct actin structures and mechanisms of internalisation. Thus, the functional links between RhoG and RhoA downstream of CR3-dependent phagocytosis are new and unexpected. Our data suggest a broad role for RhoG in consolidating signals from multiple receptors during phagocytosis.

Armus is a Rac1 effector that inactivates Rab7 and regulates E-cadherin degradation.

BACKGROUND: Cell-cell adhesion and intracellular trafficking are regulated by signaling pathways from small GTPases of the Rho, Arf, and Rab subfamilies. How signaling from distinct small GTPases are integrated in a given process is poorly understood. RESULTS: We find that a TBC/RabGAP protein, Armus, integrates signaling between Arf6, Rac1, and Rab7 during junction disassembly. Armus binds specifically to activated Rac1 and its C-terminal TBC/RabGAP domain inactivates Rab7. Thus, Armus is a novel Rac1 effector and a bona fide GAP for Rab7 in vitro and in vivo, a unique and previously unreported combination. Arf6 activation efficiently disrupts cell-cell contacts and is known to activate Rac1 and Rab7. Arf6-induced E-cadherin degradation is efficiently blocked by expression of Armus C-terminal domain or after Armus RNAi. Coexpression of Arf6 with dominant-negative Rab7 or Rac1 also inhibits junction disassembly. Importantly, Armus RabGAP expression also prevents EGF-induced scattering in keratinocytes, a process shown here to require Arf6, Rac1, and Rab7 function. To our knowledge, this is the first report to demonstrate a molecular and functional link between Rac1 and Rab7. CONCLUSIONS: Our data indicate that active Rac1 recruits Armus to locally inactivate Rab7 and facilitate E-cadherin degradation in lysosomes. Thus, the integration of Rac1 and Rab7 activities by Armus provides an important regulatory node for E-cadherin turnover and stability of cell-cell contacts.