RESUMO
Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.
Assuntos
Dinamina I/metabolismo , Rim/metabolismo , Microtúbulos/metabolismo , Podócitos/metabolismo , Animais , Células Cultivadas , Endocitose/fisiologia , Células Epiteliais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Tubulina (Proteína)/metabolismoRESUMO
The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic ß-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in ß-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse ß-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic ß-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.Key words: endocytosis, GluR2, AMPA, cortactin, MIN6.
Assuntos
Células Secretoras de Insulina/metabolismo , Receptores de AMPA/metabolismo , Linhagem Celular , Clatrina/genética , Clatrina/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Receptores de AMPA/genéticaRESUMO
Subsequently to the publication of the above article, the authors have realized that the secondlisted author, The Mon La, had not been properly credited as one of the cowriters of the paper. Therefore, the Authors' Contributions of the Declarations section of the article should have read as follows: Authors' contributions HY, KTa and TML designed the research and wrote the paper. HY, TA, YM, EO and TT performed mutant protein construction, protein purification and actin bundling experiments. TA and YM performed electron microscopy. EO, TML, KS and KF performed immunofluorescent microscopy, cell migration assay and analyzed data. FYW and KTo identified phosphorylation sites by MALDIMS. All authors read and approved the final manuscript. The authors apologize to the readership of the Journal for the misinformation in this regard, and for any inconvenience caused. [the original article was published in International Journal of Oncology 54: 550558, 2019; DOI: 10.3892/ijo.2018.4663].
RESUMO
Dynamin copolymerizes with cortactin to form a ringlike complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamincortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclindependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamincortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for Factin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ringlike complex with dynamin 1. Consistently, the dynamin 1phosphomimetic cortactin complexes exhibited decreased actinbundling activity. Expression of the phosphomimetic mutants resulted in not only aberrant lamellipodia and short filopodia but also cell migration in NG10815 gliomaderived cells. These results indicate that phosphorylation of cortactin by CDK5 regulates formation of lamellipodia and filopodia by modulating dynamin 1/cortactindependent actin bundling. Taken together, these findings suggest that CDK5 is a potential molecular target for anticancer therapy.