Quantitative proteomics identifies a Dab2/integrin module regulating cell migration. J Cell Biol

Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 08/2009; 186(1):99-111. DOI: 10.1083/jcb.200812160
Source: PubMed


Clathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins beta1, alpha1, alpha2, and alpha3 but not alpha5 or alphav chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin beta1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge. By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

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    • "We do not expect cell attachment on surface is affected by cycloheximide treatment, since attachment, unlike adhesion, is too fast to rely on protein synthesis (it takes place in some minutes). Moreover, for attachment , cells use receptors that are already exposed on cell surface or are supplied by exocytosis of integrin-recycled containing vesicles [38] [39] [40]. "
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    ABSTRACT: The development of materials that allow proper functioning of cells on solid supports is directly relevant to the construction of living-cell biosensors. Both physical and chemical properties of the surfaces have been shown to be critical in this field. Our aim is to report correlations between chemical properties of surfaces and cell behavior by studying adhesion, viability and proliferation of fibroblasts and HeLa cells. Neither fibroblasts nor HeLa cells adhered to a hydrophobic surface. Fibroblasts were able to attach and proliferate well on all other surfaces tested. In contrast, on some surfaces where HeLa cells adhered and were viable, proliferation decreased by half while on others proliferation was not affected. Proliferation was significantly correlated with the level of adsorption of serum proteins on the surface (quantified by surface plasmon resonance), but not with surface wettability (water contact angle). Interestingly, surfaces modified with COOH and HSO3 groups were the ones that favored most protein adsorption and allowed the best measures for HeLa cell proliferation. The decrease of HeLa cell proliferation on surfaces covered with poly-l-lysine (PL) was related with the profile of integrin expression. Compared to a polystyrene control surface, there was an increase in αV and αVβ3 and a decrease in α2 and α3, indicating that migration rather than proliferation could be favored on PL functionalized surfaces. These results indicate that charge is more important than wettability to determine biocompatibility. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Oct 2014 · Colloids and surfaces B: Biointerfaces
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    • "These physiological phenotypes might be attributable to the adaptor activity of Dab2 for megalin and LDL receptor, respectively. Another well-documented Dab2 binding ligand is integrin [34,35]. Though an impact of Dab2 on cell migration was observed in cultured cells, we found no related physiological phenotype in the mutant mice. "
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    ABSTRACT: Disabled-2 (Dab2) is an endocytic adaptor protein involved in clathrin-mediated endocytosis and cargo trafficking. Since its expression is lost in several cancer types, Dab2 has been suggested to be a tumor suppressor. In vitro studies indicate that Dab2 establishes epithelial cell polarity and organization by directing endocytic trafficking of membrane glycoproteins. Dab2 also modulates cellular signaling pathways by mediating the endocytosis and recycling of surface receptors and associated signaling components. Previously, two independent gene knockout studies have been reported, with some discrepancies in the observed embryonic phenotypes. To further clarify the in vivo roles of Dab2 in development and physiology, we designed a new floxed allele to delete dab2 gene. The constitutive dab2 deleted embryos showed a spectrum in the degree of endoderm disorganization in E5.5 and no mutant embryos persisted at E9.5. However, the mice were grossly normal when dab2 deletion was restricted to the embryo proper and the gene was retained in extraembryonic tissues using Meox2-Cre and Sox2-Cre. Adult Dab2-deficient mice had a small but statistically significant increase in serum cholesterol levels. The study of the new dab2 mutant allele in embryos and embryoid bodies confirms a role for Dab2 in extraembryonic endoderm development and epithelial organization. Experimental results with embryoid bodies suggest that additional endocytic adaptors such as Arh and Numb could partially compensate for Dab2 loss. Conditional deletion indicates that Dab2 is dispensable for organ development, when the vast majority of the embryonic cells are dab2 null. However, Dab2 has a physiological role in the endocytosis of lipoproteins and cholesterol metabolism.
    Full-text · Article · Oct 2013 · BMC Developmental Biology
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    • "It has been shown that Numb regulates Integrin-b turnover in vitro (Nishimura and Kaibuchi, 2007; Teckchandani et al., 2009). To determine whether Numb also functions as a regulator of focal adhesions in the retinal epithelium, we generated retinal clones overexpressing numb and monitored Integrin-b1 localization (Figures 5G–5P). "
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    ABSTRACT: Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.
    Full-text · Article · Oct 2012 · Developmental Cell
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