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

Teckchandani A, Toida N, Goodchild J, Henderson C, Watts J, Wollscheid B, Cooper JA - J. Cell Biol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
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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Localization of integrin β1 and Dab2. (A) Confluent HeLa cells plated on collagen IV–coated coverslips were treated with anti–integrin β1 antibody for 1 h at 4°C and then fixed, permeabilized, and stained with anti-Dab2 or anticlathrin antibody. Single 0.2-µm sections at the ventral or dorsal surface are shown. (B) The number of Dab2 puncta that colocalize with integrin β1 (arrowheads in A) at the ventral and dorsal surfaces were counted. Mean values and standard errors (two independent experiments and ∼12 cells/treatment/experiment) are shown. (C) Confluent HeLa cells were fixed, permeabilized, and stained with anti-Dab2 antibodies. Antivinculin antibodies were used to visualize focal adhesions (arrowhead). Single 0.2-µm sections at the ventral surfaces of cells are shown. (A and C) The white boxes indicate the enlarged images shown in the insets.
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fig5: Localization of integrin β1 and Dab2. (A) Confluent HeLa cells plated on collagen IV–coated coverslips were treated with anti–integrin β1 antibody for 1 h at 4°C and then fixed, permeabilized, and stained with anti-Dab2 or anticlathrin antibody. Single 0.2-µm sections at the ventral or dorsal surface are shown. (B) The number of Dab2 puncta that colocalize with integrin β1 (arrowheads in A) at the ventral and dorsal surfaces were counted. Mean values and standard errors (two independent experiments and ∼12 cells/treatment/experiment) are shown. (C) Confluent HeLa cells were fixed, permeabilized, and stained with anti-Dab2 antibodies. Antivinculin antibodies were used to visualize focal adhesions (arrowhead). Single 0.2-µm sections at the ventral surfaces of cells are shown. (A and C) The white boxes indicate the enlarged images shown in the insets.

Mentions: Dab2 localizes to AP2-containing clathrin-coated pits distributed over the cell surface (Morris and Cooper, 2001). However, very little integrin β1 colocalized with Dab2 in these structures (unpublished data). Because coated pits only have a brief half-life (1–3 min; Puthenveedu and von Zastrow, 2006; Loerke et al., 2009) and only ∼1% of integrin β1 is internalized per minute, only a tiny fraction of the surface integrin may be in coated pits at any given time. To trap integrins in coated pits, we cooled cells to 4°C (Sorkin, 2004). Under these conditions, inactive integrin β1 labeled with antibody P5D2 extensively colocalized with Dab2 and clathrin (Fig. 5, A and B). Surprisingly, the Dab2-containing structures were not located close to focal adhesions, as visualized by staining fixed, permeabilized cells with antibodies to vinculin (Fig. 5 C). Rather, Dab2 colocalized with inactive integrin β1 at distant sites, predominantly on the dorsal surface of the cell (Fig. 5 A and Fig. S3 A). Thus, Dab2 is localized appropriately to mediate endocytosis of free but not engaged integrins.


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

Teckchandani A, Toida N, Goodchild J, Henderson C, Watts J, Wollscheid B, Cooper JA - J. Cell Biol. (2009)

Localization of integrin β1 and Dab2. (A) Confluent HeLa cells plated on collagen IV–coated coverslips were treated with anti–integrin β1 antibody for 1 h at 4°C and then fixed, permeabilized, and stained with anti-Dab2 or anticlathrin antibody. Single 0.2-µm sections at the ventral or dorsal surface are shown. (B) The number of Dab2 puncta that colocalize with integrin β1 (arrowheads in A) at the ventral and dorsal surfaces were counted. Mean values and standard errors (two independent experiments and ∼12 cells/treatment/experiment) are shown. (C) Confluent HeLa cells were fixed, permeabilized, and stained with anti-Dab2 antibodies. Antivinculin antibodies were used to visualize focal adhesions (arrowhead). Single 0.2-µm sections at the ventral surfaces of cells are shown. (A and C) The white boxes indicate the enlarged images shown in the insets.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712992&req=5

fig5: Localization of integrin β1 and Dab2. (A) Confluent HeLa cells plated on collagen IV–coated coverslips were treated with anti–integrin β1 antibody for 1 h at 4°C and then fixed, permeabilized, and stained with anti-Dab2 or anticlathrin antibody. Single 0.2-µm sections at the ventral or dorsal surface are shown. (B) The number of Dab2 puncta that colocalize with integrin β1 (arrowheads in A) at the ventral and dorsal surfaces were counted. Mean values and standard errors (two independent experiments and ∼12 cells/treatment/experiment) are shown. (C) Confluent HeLa cells were fixed, permeabilized, and stained with anti-Dab2 antibodies. Antivinculin antibodies were used to visualize focal adhesions (arrowhead). Single 0.2-µm sections at the ventral surfaces of cells are shown. (A and C) The white boxes indicate the enlarged images shown in the insets.
Mentions: Dab2 localizes to AP2-containing clathrin-coated pits distributed over the cell surface (Morris and Cooper, 2001). However, very little integrin β1 colocalized with Dab2 in these structures (unpublished data). Because coated pits only have a brief half-life (1–3 min; Puthenveedu and von Zastrow, 2006; Loerke et al., 2009) and only ∼1% of integrin β1 is internalized per minute, only a tiny fraction of the surface integrin may be in coated pits at any given time. To trap integrins in coated pits, we cooled cells to 4°C (Sorkin, 2004). Under these conditions, inactive integrin β1 labeled with antibody P5D2 extensively colocalized with Dab2 and clathrin (Fig. 5, A and B). Surprisingly, the Dab2-containing structures were not located close to focal adhesions, as visualized by staining fixed, permeabilized cells with antibodies to vinculin (Fig. 5 C). Rather, Dab2 colocalized with inactive integrin β1 at distant sites, predominantly on the dorsal surface of the cell (Fig. 5 A and Fig. S3 A). Thus, Dab2 is localized appropriately to mediate endocytosis of free but not engaged integrins.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus