RESUMO
The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.
Assuntos
Caderinas/metabolismo , Caseína Quinase II/metabolismo , Caseína Quinase I/metabolismo , Adesão Celular , Drosophila melanogaster/metabolismo , alfa Catenina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Western Blotting , Caderinas/genética , Caseína Quinase I/genética , Caseína Quinase II/genética , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cães , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Ovário/citologia , Ovário/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , alfa Catenina/química , alfa Catenina/genéticaRESUMO
Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using α-catenin (α-cat) knock-out Madin Darby Canine Kidney (MDCK) cells reconstituted with wild-type and mutant forms of α-cat as a model system, we find that an established α-cat actin-binding domain unfolding mutant designed to reduce force-sensitive binding to F-actin (α-cat-H0-FABD+) can promote cytokinesis failure, particularly along epithelial wound-fronts. Enhanced α-cat coupling to cortical actin is neither sufficient nor mitotic cell-autonomous for cytokinesis failure, but critically requires the mechanosensitive Middle-domain (M1-M2-M3) and neighboring cells. Disease relevant α-cat M-domain missense mutations known to cause a form of retinal pattern dystrophy (α-cat E307K or L436P) are associated with elevated binucleation rates via cytokinesis failure. Similar binucleation rates are seen in cells expressing an α-cat salt-bridge destabilizing mutant (R551A) designed to promote M2-M3 domain unfurling at lower force thresholds. Since binucleation is strongly enhanced by removal of the M1 as opposed to M2-M3 domains, cytokinetic fidelity is most sensitive to α-cat M2-M3 domain opening. To identify α-cat conformation-dependent proximity partners that contribute to cytokinesis, we used a biotin-ligase approach to distinguished proximity partners that show enhanced recruitment upon α-cat M-domain unfurling (R551A). We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), an abscission factor previously implicated in cytokinesis. We confirm that LZTS2 enriches at the midbody, but discover it also localizes to tight and tricellular junctions. LZTS2 knock-down promotes binucleation in both MDCK and Retinal Pigmented Epithelial (RPE) cells. α-cat mutants with persistent M2-M3 domain opening showed elevated junctional enrichment of LZTS2 from the cytosol compared α-cat wild-type cells. These data implicate LZTS2 as a mechanosensitive effector of α-cat that is critical for cytokinetic fidelity. This model rationalizes how persistent mechano-activation of α-cat may drive tension-induced polyploidization of epithelia post-injury and suggests an underlying mechanism for how pathogenic α-cat mutations drive macular dystrophy.
RESUMO
Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.
Assuntos
Microtúbulos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Animais , Técnicas Biossensoriais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Microtúbulos/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteína rhoA de Ligação ao GTP/genética , Quinases da Família src/genéticaRESUMO
STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.
Assuntos
Transtornos Plaquetários/genética , Cálcio/química , Dislexia/genética , Ictiose/genética , Transtornos de Enxaqueca/genética , Miose/genética , Proteínas de Neoplasias/química , Proteína ORAI1/química , Mutação Puntual , Baço/anormalidades , Molécula 1 de Interação Estromal/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transtornos Plaquetários/metabolismo , Transtornos Plaquetários/patologia , Cálcio/metabolismo , Dislexia/metabolismo , Dislexia/patologia , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/patologia , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ictiose/metabolismo , Ictiose/patologia , Transporte de Íons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/patologia , Miose/metabolismo , Miose/patologia , Modelos Moleculares , Fadiga Muscular/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Baço/metabolismo , Baço/patologia , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
A unique feature of α-catenin localized outside the cadherin-catenin complex is its capacity to form homodimers, but the subcellular localization and functions of this form of α-catenin remain incompletely understood. We identified a cadherin-free form of α-catenin that is recruited to the leading edge of migrating cells in a phosphatidylinositol 3-kinase-dependent manner. Surface plasmon resonance analysis shows that α-catenin homodimers, but not monomers, selectively bind phosphatidylinositol-3,4,5-trisphosphate-containing lipid vesicles with high affinity, where three basic residues, K488, K493, and R496, contribute to binding. Chemical-induced dimerization of α-catenin containing a synthetic dimerization domain promotes its accumulation within lamellipodia and elaboration of protrusions with extended filopodia, which are attenuated in the α-cateninKKR<3A mutant. Cells restored with a full-length, natively homodimerizing form of α-cateninKKR<3A display reduced membrane recruitment, altered epithelial sheet migrations, and weaker cell-cell adhesion compared with WT α-catenin. These findings show that α-catenin homodimers are recruited to phosphoinositide-activated membranes to promote adhesion and migration, suggesting that phosphoinositide binding may be a defining feature of α-catenin function outside the cadherin-catenin complex.
Assuntos
Adesão Celular , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , alfa Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Cães , Humanos , Células Madin Darby de Rim Canino , Mutação , Fosfatidilinositol 3-Quinase/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Pseudópodes/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , alfa Catenina/genéticaRESUMO
STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.