RESUMEN
The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.
Asunto(s)
Proteínas de Drosophila , Vía de Señalización Hippo , Animales , Humanos , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Neurofibromina 2/metabolismo , Drosophila melanogaster/metabolismo , Mamíferos , Proteínas Serina-Treonina Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. Here we show that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib - similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a 'picket fence' diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.
Asunto(s)
Uniones Adherentes , Proteínas de Drosophila , Uniones Adherentes/metabolismo , Animales , Polaridad Celular/fisiología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células EpitelialesRESUMEN
Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E-cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. We show that these pseudostratified epithelial cells downregulate E-cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho-kinase-mediated actomyosin contractility blocks downregulation of E-cadherin during mitosis. Mitotic activation of Rho-kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity.
Asunto(s)
Uniones Adherentes , Huso Acromático , Animales , Drosophila/genética , Células Epiteliales , MitosisRESUMEN
Apical-basal polarity is a fundamental property of animal tissues. Drosophila embryos provide an outstanding model for defining mechanisms that initiate and maintain polarity. Polarity is initiated during cellularization, when cell-cell adherens junctions are positioned at the future boundary of apical and basolateral domains. Polarity maintenance then involves complementary and antagonistic interplay between apical and basal polarity complexes. The Scribble/Dlg module is well-known for promoting basolateral identity during polarity maintenance. Here, we report a surprising role for Scribble/Dlg in polarity initiation, placing it near the top of the network-positioning adherens junctions. Scribble and Dlg are enriched in nascent adherens junctions, are essential for adherens junction positioning and supermolecular assembly, and also play a role in basal junction assembly. We test the hypotheses for the underlying mechanisms, exploring potential effects on protein trafficking, cytoskeletal polarity or Par-1 localization/function. Our data suggest that the Scribble/Dlg module plays multiple roles in polarity initiation. Different domains of Scribble contribute to these distinct roles. Together, these data reveal novel roles for Scribble/Dlg as master scaffolds regulating assembly of distinct junctional complexes at different times and places.
Asunto(s)
Uniones Adherentes/fisiología , Polaridad Celular/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Proteínas del Dominio Armadillo/metabolismo , Biotinilación , Citoesqueleto/metabolismo , Perros , Proteínas de Drosophila/metabolismo , Ectodermo/metabolismo , Células Epiteliales/metabolismo , Femenino , Gástrula/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Morfogénesis , Mutación , Fenotipo , Interferencia de ARN , Complejo Shelterina , Transducción de Señal , Proteínas de Unión a Telómeros/metabolismo , Uniones Estrechas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Key events ranging from cell polarity to proliferation regulation to neuronal signaling rely on the assembly of multiprotein adhesion or signaling complexes at particular subcellular sites. Multidomain scaffolding proteins nucleate assembly and direct localization of these complexes, and the protein Scribble and its relatives in the LAP protein family provide a paradigm for this. Scribble was originally identified because of its role in apical-basal polarity and epithelial integrity in Drosophila melanogaster It is now clear that Scribble acts to assemble and position diverse multiprotein complexes in processes ranging from planar polarity to adhesion to oriented cell division to synaptogenesis. Here, we explore what we have learned about the mechanisms of action of Scribble in the context of its multiple known interacting partners and discuss how this knowledge opens new questions about the full range of Scribble protein partners and their structural and signaling roles.
Asunto(s)
Adhesión Celular/fisiología , Polaridad Celular/fisiología , Proliferación Celular/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Drosophila melanogaster , Complejos Multiproteicos/metabolismoRESUMEN
Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Complejo de Señalización de la Axina/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Animales , Animales Modificados Genéticamente , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Proteína Axina/química , Proteína Axina/genética , Proteína Axina/metabolismo , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/genética , Línea Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. Defining the mechanisms underlying polarity establishment remains a key issue. Drosophila embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction positioning. Here we define mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane localization, recruitment to nascent junctions and macromolecular assembly at tricellular junctions. Our data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy (PDZ-GEF) is crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe mediate adherens junction and tricellular junction recruitment but are dispensable for membrane localization. Our data also support a role for Canoe multimerization. These multifactorial inputs shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. We integrate the existing data into a new polarity establishment model.
Asunto(s)
Polaridad Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Uniones Adherentes/metabolismo , Animales , Animales Modificados Genéticamente , Polaridad Celular/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Gastrulación , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Modelos Biológicos , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Interferencia de ARN , Complejo Shelterina , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genéticaRESUMEN
Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and in vitro The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification (i.e. substitution with Asp or phosphorylation) "undocks" and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilin's ability to weaken longitudinal filament subunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filament mechanical properties and promote severing.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Imitación Molecular , Factores Despolimerizantes de la Actina/química , Microscopía por Crioelectrón , Microscopía Fluorescente , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Serina/metabolismoRESUMEN
Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between â¼0.1-5 µM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed â¼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.
Asunto(s)
Citoesqueleto de Actina/química , Actinas/química , Exones , Tropomiosina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Peso Molecular , Polimerizacion , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Isoformas de Proteínas/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/química , Animales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Multimerización de Proteína/efectos de los fármacos , Conejos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tropomiosina/antagonistas & inhibidores , Tropomiosina/químicaRESUMEN
α-Smooth Muscle Actin (α-SMA), a widely characterized cytoskeletal protein, represents the hallmark of myofibroblast differentiation. Transforming growth factorß1 (TGFß1) stimulates α-SMA expression and incorporation into stress fibers, thus providing an increased myofibroblast contractile force that participates in tissue remodeling. We have addressed the molecular mechanism by which α-SMA is stably incorporated into stress fibers in human myofibroblasts following exposure to TGFß1. The unique N-terminal sequence AcEEED, which is critical for α-SMA incorporation into stress fibers, was used to screen for AcEEED binding proteins. Tropomyosins were identified as candidate binding proteins. We find that after TGFß1 treatment elevated levels of the Tpm1.6/7 isoforms, and to a lesser extent Tpm2.1, precede the increase in α-SMA. RNA interference experiments demonstrate that α-SMA fails to stably incorporate into stress fibers of TGFß1 treated fibroblasts depleted of Tpm1.6/7, but not other tropomyosins. This does not appear to be due to exclusive interactions between α-SMA and just the Tpm1.6/7 isoforms. We propose that an additional AcEEED binding factor may be required to generate α-SMA filaments containing just Tpm1.6/7 which result in stable incorporation of the resulting filaments into stress fibers.
Asunto(s)
Fibroblastos/metabolismo , Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Isoformas de Proteínas/metabolismo , Tropomiosina/metabolismo , Humanos , Proteómica , Fibras de EstrésRESUMEN
The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Ratones , Células 3T3 NIH , Neoplasias/patología , Neuroblastoma/tratamiento farmacológico , Tropomiosina/antagonistas & inhibidores , Tropomiosina/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The actin cytoskeleton is indispensable for normal cellular function. In particular, several actin-based structures coordinate cellular motility, a process hijacked by tumor cells in order to facilitate their propagation to distant sites. The actin cytoskeleton, therefore, represents a point for chemotherapeutic intervention. The challenge in disrupting the actin cytoskeleton is in preserving actin-driven contraction of cardiac and skeletal muscle. By targeting actin-binding proteins with altered expression in malignancy, it may be possible to achieve tumor-specific toxicity. A number of actin-binding proteins act cooperatively and synergistically to regulate actin structures required for motility. The actin cytoskeleton is characterized by a significant degree of plasticity. Targeting specific actin-binding proteins for chemotherapy will only be successful if no other compensatory mechanisms exist.