RESUMO
BACKGROUND: Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvß3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration. METHODS: Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy. RESULTS: We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and focal adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in focal adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation. CONCLUSIONS: The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced focal adhesion disassembly in mesenchymal cells. The mechanism involves focal adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in focal adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adesões Focais/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Sindecana-4/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Polaridade Celular , Fibroblastos/metabolismo , Inativação Gênica , Camundongos , Microtúbulos/metabolismo , Ligação Proteica , Ratos , Antígenos Thy-1/metabolismoRESUMO
Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neurônios/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Citoesqueleto/genética , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Neurônios/citologia , Ratos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.
Assuntos
Axônios/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cinética , Quinases Lim/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Hyaluronan (HA) modulates multidrug resistance (MDR) as well as cell migration. Tiam1 is involved in cytoskeleton reorganization during tumor invasion. In this report we show the relationship among HA, Tiam1, migration and MDR in murine lymphoma cell lines. We observed that MDR cells presented higher migratory capacity towards HA in vitro as well as higher constitutive active Tiam1 expression than the sensitive cell line. Besides, HA treatment induced migration towards HA of MDR cell lines through Tiam1 activation by a PI3K-dependent mechanism, showing that disruption of HA signaling would be useful in treatment of MDR hematological malignancies.
Assuntos
Movimento Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ácido Hialurônico/farmacologia , Linfoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Linfoma/patologia , Camundongos , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.