Asunto(s)
Integrinas , Humanos , Integrinas/metabolismo , Movimiento Celular , Transporte de Proteínas , Adhesión CelularRESUMEN
Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables ß3-integrin-mediated force generation independently of ß1 integrin. ß3-integrin-mediated forces were associated with a decrease in ß3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in ß3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.
Asunto(s)
Clatrina , Endocitosis , Adhesiones Focales , Integrina beta1 , Integrina beta3 , Clatrina/metabolismo , Endocitosis/fisiología , Integrina beta1/genética , Mecanotransducción Celular , Talina/genéticaRESUMEN
Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1 (also known as ITGB1BP1), a ß1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitylated by Smurf1, preventing ICAP-1 binding to ß1 integrin. The non-ubiquitylatable form of ICAP-1 modifies ß1 integrin focal adhesion organization and interferes with fibronectin density sensing. ICAP-1 is also required for adapting cell migration in response to substrate stiffness in a ß1-integrin-independent manner. ICAP-1 monoubiquitylation regulates rigidity sensing by increasing MRCKα (also known as CDC42BPA)-dependent cell contractility through myosin phosphorylation independently of substrate rigidity. We provide evidence that ICAP-1 monoubiquitylation helps in switching from ROCK2-mediated to MRCKα-mediated cell contractility. ICAP-1 monoubiquitylation serves as a molecular switch to coordinate extracellular matrix density and rigidity sensing thus acting as a crucial modulator of cell migration and mechanosensing.
Asunto(s)
Movimiento Celular , Matriz Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa de Distrofia Miotónica/metabolismo , Ubiquitinación , Quinasas Asociadas a rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión , Fenómenos Biomecánicos , Adhesión Celular , Línea Celular , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Humanos , Integrina beta1/química , Integrina beta1/metabolismo , Ratones , Modelos Biológicos , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
PURPOSE: To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents. METHODS: The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the sample's lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%. RESULTS: In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration. CONCLUSIONS: The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.