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?v?3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway.

ABSTRACT: Protein transport through the endosome is critical for maintaining proper integrin cell surface integrin distribution to support cell adhesion, motility and viability. Here we employ a live-cell imaging approach to evaluate the relationship between integrin function and transport through the early endosome. We discovered that two early endosome factors, AAK1L and EHD3, are critical for ?v?3-integrin-mediated cell adhesion in HeLa cells. siRNA-mediated depletion of either factor delays short-loop ?3 integrin recycling from the early endosome back to the cell surface. Total internal reflection fluorescence-based colocalization analysis reveals that ?3 integrin transits AAK1L- and EHD3-positive endosomes near the cell surface, a subcellular location consistent with a rapid-recycling role for both factors. Moreover, structure-function analysis reveals that AAK1L kinase activity, as well as its C-terminal domain, is essential for cell adhesion maintenance. Taken together, these data reveal an important role for AAK1L and EHD3 in maintaining cell viability and adhesion by promoting ?v?3 integrin rapid recycling from the early endosome.

SUBMITTER: Waxmonsky NC

PROVIDER: S-EPMC3744025 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Αvβ3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway.

Waxmonsky Nicole C NC Conner Sean D SD

Journal of cell science 20130618 Pt 16

Protein transport through the endosome is critical for maintaining proper integrin cell surface integrin distribution to support cell adhesion, motility and viability. Here we employ a live-cell imaging approach to evaluate the relationship between integrin function and transport through the early endosome. We discovered that two early endosome factors, AAK1L and EHD3, are critical for αvβ3-integrin-mediated cell adhesion in HeLa cells. siRNA-mediated depletion of either factor delays short-loop ...[more]

PMID: 23781025

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Project description:Integrin-mediated adhesion is a central feature of cellular adhesion, locomotion, and endothelial cell mechanobiology. Although integrins are known to be transmembrane proteins, little is known about the role of membrane biophysics and dynamics in integrin adhesion. We treated human aortic endothelial cells with exogenous amphiphiles, shown previously in model membranes, and computationally, to affect bilayer thickness and lipid phase separation, and subsequently measured single-integrin-molecule adhesion kinetics using an optical trap, and diffusion using fluorescence correlation spectroscopy. Benzyl alcohol (BA) partitions to liquid-disordered (Ld) domains, thins them, and causes the greatest increase in hydrophobic mismatch between liquid-ordered (Lo) and Ld domains among the three amphiphiles, leading to domain separation. In human aortic endothelial cells, BA increased ?1-integrin-Arg-Gly-Asp-peptide affinity by 18% with a transition from single to double valency, consistent with a doubling of the molecular brightness of mCherry-tagged ?1-integrins measured using fluorescence correlation spectroscopy. Accordingly, BA caused an increase in the size of focal-adhesion-kinase/paxillin-positive peripheral adhesions and reduced migration speeds as measured using wound-healing assays. Vitamin E, which thickens Lo domains and disperses them by lowering edge energy on domain boundaries, left integrin affinity unchanged but reduced binding probability, leading to smaller focal adhesions and equivalent migration speed relative to untreated cells. Vitamin E reversed the BA-induced decrease in migration speed. Triton X-100 also thickens Lo domains, but partitions to both lipid phases and left unchanged binding kinetics, focal adhesion sizes, and migration speed. These results demonstrate that only the amphiphile that thinned Ld lipid domains increased ?1-integrin-Arg-Gly-Asp-peptide affinity and valency, thus implicating Ld domains in modulation of integrin adhesion, nascent adhesion formation, and cell migration.

Project description:RationaleCardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology.ObjectiveTo define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology.Methods and resultsWe identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes.ConclusionsThese data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.

Dataset Information

?v?3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway.

Publications

Αvβ3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway.

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