RESUMEN
Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity.
Asunto(s)
Antígenos CD13/metabolismo , Polaridad Celular , Células Epiteliales/citología , Epitelio/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD13/química , Antígenos CD13/genética , Células CACO-2 , Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Células Epiteliales/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cell migration is an important biological phenomenon involved in many homeostatic and aberrant physiological processes. Phosphorylation of the focal adhesion adaptor protein, paxillin, on serine 273 (S273) has been implicated as a key regulator of cell migration. Here, it is shown that phosphorylation on paxillin S273 leads to highly migratory cells with small dynamic adhesions. Adhesions at protrusive edges of the cell were more dynamic than adhesions at retracting edges. Temporal image correlation microscopy revealed that these dynamic adhesions undergo rapid binding of paxillin, PAK1 and ßPIX. We identified membrane proximal adhesion subdomains in protrusive regions of the cell that show rapid protein binding that is dependent on paxillin S273 phosphorylation, PAK1 kinase activity and phosphatases. These dynamic adhesion subdomains corresponded to regions of the adhesion that also show co-binding of paxillin/PAK1 and paxillin/ßPIX complexes. It is likely that parts of individual adhesions are more dynamic while others are less dynamic due to their association with the actin cytoskeleton. Variable adhesion and binding dynamics are regulated via differential paxillin S273 phosphorylation across the cell and within adhesions and are required for regulated cell migration. Dysregulation through phosphomutants, PAK1-KD or ßPIX mutants resulted in large stable adhesions, long protein binding times and slow cell migration. Dysregulation through phosphomimics or PAK1-CA led to small dynamic adhesions and rapid cell migration reminiscent of highly migratory cancer cells. Thus, phosphorylation of paxillin S273 is a key regulator of cell migration through recruitment of ßPIX and PAK1 to sites of adhesion.
Asunto(s)
Adhesión Celular , Movimiento Celular , Paxillin/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Quinasas p21 Activadas/metabolismo , Animales , Células CHO , Cricetulus , Microscopía Intravital , Microscopía Fluorescente , Mutación , Paxillin/genética , Fosforilación/genética , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Serina/genética , Serina/metabolismo , Quinasas p21 Activadas/genéticaRESUMEN
Cell migration is a tightly-regulated process that involves protein gradients formed by the Rho family of GTPases, including Rho and Rac. The front (rear) of cells is generally characterized by higher active Rac (Rho) and lower active Rho (Rac) concentrations. Protein clusters, called adhesions, that anchor cells to their external environment have been shown to be dynamic and small (stable and large) at the cell front (rear), forming the force-transmission points necessary for persistent movement. Differences in adhesion sizes and dynamics have been linked to gradients in Rac and Rho activity. Here, we study the effects of Rac activation and gradients in Rac and Rho concentrations and activities on cellular polarity and adhesion size using mathematical and experimental approaches. The former is accomplished by expanding an existing reaction-diffusion model to a 2D domain utilizing stochastic dynamics. The model revealed that a hysteresis between the induced/uninduced states (corresponding to higher/lower Rac concentrations, respectively) along with Rac and Rho activation gradients, generated by chemical cues, were vital for forming polarity. Experimentally, the induced state was generated by increasing the cellular ßPIX (a Rac-GEF) level and/or decreasing ROCK (a Rac-GAP effector protein) activity with Y-27632 (a ROCK-inhibitor). In agreement with the simulations, our results showed that cells with elevated RacGTP migrated faster, indicating more robust cellular polarization. However, the directionality of cells was not changed significantly, suggesting that external and/or internal physical or chemical cues were needed. Complementing the faster migration observed, adhesions were smaller, generating the phenotype expected with the induced state.