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1.
EMBO J ; 42(6): e112863, 2023 03 15.
Article in English | MEDLINE | ID: mdl-36807601

ABSTRACT

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.


Subject(s)
Drosophila Proteins , Hippo Signaling Pathway , Animals , Humans , Signal Transduction/physiology , Tumor Suppressor Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Neurofibromin 2/metabolism , Drosophila melanogaster/metabolism , Mammals , Protein Serine-Threonine Kinases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism
2.
EMBO Rep ; 21(4): e49700, 2020 04 03.
Article in English | MEDLINE | ID: mdl-32030856

ABSTRACT

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.


Subject(s)
Adherens Junctions , Spindle Apparatus , Animals , Drosophila/genetics , Epithelial Cells , Mitosis
3.
Development ; 146(22)2019 11 21.
Article in English | MEDLINE | ID: mdl-31628110

ABSTRACT

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.


Subject(s)
Adherens Junctions/physiology , Cell Polarity/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental , Membrane Proteins/physiology , Tumor Suppressor Proteins/physiology , Animals , Armadillo Domain Proteins/metabolism , Biotinylation , Cytoskeleton/metabolism , Dogs , Drosophila Proteins/metabolism , Ectoderm/metabolism , Epithelial Cells/metabolism , Female , Gastrula/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Madin Darby Canine Kidney Cells , Male , Morphogenesis , Mutation , Phenotype , RNA Interference , Shelterin Complex , Signal Transduction , Telomere-Binding Proteins/metabolism , Tight Junctions/metabolism , Transcription Factors/metabolism , rab5 GTP-Binding Proteins/metabolism
4.
J Cell Biol ; 218(3): 742-756, 2019 03 04.
Article in English | MEDLINE | ID: mdl-30598480

ABSTRACT

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.


Subject(s)
Cell Adhesion/physiology , Cell Polarity/physiology , Cell Proliferation/physiology , Drosophila Proteins/metabolism , Membrane Proteins/metabolism , Signal Transduction/physiology , Animals , Drosophila melanogaster , Multiprotein Complexes/metabolism
5.
PLoS Genet ; 14(4): e1007339, 2018 04.
Article in English | MEDLINE | ID: mdl-29641560

ABSTRACT

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.


Subject(s)
Armadillo Domain Proteins/metabolism , Axin Signaling Complex/metabolism , Drosophila Proteins/metabolism , Transcription Factors/metabolism , Wnt Signaling Pathway , Animals , Animals, Genetically Modified , Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome/chemistry , Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome/genetics , Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Armadillo Domain Proteins/chemistry , Armadillo Domain Proteins/genetics , Axin Protein/chemistry , Axin Protein/genetics , Axin Protein/metabolism , Axin Signaling Complex/chemistry , Axin Signaling Complex/genetics , Cell Line , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Proteolysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription, Genetic , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Wnt1 Protein/genetics , Wnt1 Protein/metabolism
6.
Development ; 145(2)2018 01 26.
Article in English | MEDLINE | ID: mdl-29361565

ABSTRACT

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.


Subject(s)
Cell Polarity/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Telomere-Binding Proteins/metabolism , Adherens Junctions/metabolism , Animals , Animals, Genetically Modified , Cell Polarity/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Female , Gastrulation , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Models, Biological , Protein Interaction Domains and Motifs , Protein Transport , RNA Interference , Shelterin Complex , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/genetics
7.
J Biol Chem ; 292(48): 19565-19579, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28939776

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Molecular Mimicry , Actin Depolymerizing Factors/chemistry , Cryoelectron Microscopy , Microscopy, Fluorescence , Molecular Dynamics Simulation , Phosphorylation , Protein Binding , Serine/metabolism
8.
Bioarchitecture ; 6(4): 61-75, 2016 Jul 03.
Article in English | MEDLINE | ID: mdl-27420374

ABSTRACT

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 ∼0.1-5 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 ∼3-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.


Subject(s)
Actin Cytoskeleton/chemistry , Actins/chemistry , Exons , Tropomyosin/chemistry , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actins/genetics , Actins/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Kinetics , Molecular Weight , Polymerization , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Tropomyosin/genetics , Tropomyosin/metabolism
9.
Sci Rep ; 6: 19816, 2016 Jan 25.
Article in English | MEDLINE | ID: mdl-26804624

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/metabolism , Protein Isoforms/metabolism , Tropomyosin/metabolism , Actin Cytoskeleton/chemistry , Animals , Humans , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding/drug effects , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Multimerization/drug effects , Rabbits , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Tropomyosin/antagonists & inhibitors , Tropomyosin/chemistry
10.
Future Med Chem ; 1(7): 1311-31, 2009 Oct.
Article in English | MEDLINE | ID: mdl-21426105

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/drug effects , Microfilament Proteins/antagonists & inhibitors , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/physiology , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Cortactin/genetics , Cortactin/metabolism , Destrin/genetics , Destrin/metabolism , Gelsolin/genetics , Gelsolin/metabolism , Humans , Microfilament Proteins/chemistry , Myosin Type II/genetics , Myosin Type II/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Signal Transduction , Tropomyosin/genetics , Tropomyosin/metabolism , Wiskott-Aldrich Syndrome Protein Family/genetics , Wiskott-Aldrich Syndrome Protein Family/metabolism
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