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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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Dab2 regulates integrin β1 endocytosis. (A–C) Control (Con) and Dab2-deficient HeLa cells were incubated with anti–integrin β1 antibody (P5D2) for 30 min at 4°C, followed by warming at 37°C for 0, 15, or 30 min. Mean values and standard errors (∼20 cells/treatment) of pixel intensities were measured. (A) Surface antibody on fixed, nonpermeabilized cells was detected with fluorescent secondary antibody. 6-µm flattened z projections of the entire cell are shown. (B) Surface antibody was removed by acid stripping, and internalized antibody was detected. 6-µm flattened z projections of the entire cell are shown. (C) Internalized antibody colocalizes with EEA1 at 15 min and with Tfn at 30 min. Dab2-deficient cells internalize less integrin β1 antibody but the same amount of Tfn compared with control cells. (D–G) Control and Dab2-deficient HeLa cells were surface labeled with sulpho-NHS-SS-biotin at 4°C for 30 min and then warmed to 37°C in the absence (D and E) or presence (F and G) of 2 mM primaquine for the indicated times. Biotin was removed from surface receptors with MesNa treatment, and cells were lysed and incubated with an anti–integrin β1 antibody. Immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting with peroxidase-conjugated streptavidin. (E and G) Quantification of three independent experiments. For each time point, mean values and standard errors are shown.
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fig4: Dab2 regulates integrin β1 endocytosis. (A–C) Control (Con) and Dab2-deficient HeLa cells were incubated with anti–integrin β1 antibody (P5D2) for 30 min at 4°C, followed by warming at 37°C for 0, 15, or 30 min. Mean values and standard errors (∼20 cells/treatment) of pixel intensities were measured. (A) Surface antibody on fixed, nonpermeabilized cells was detected with fluorescent secondary antibody. 6-µm flattened z projections of the entire cell are shown. (B) Surface antibody was removed by acid stripping, and internalized antibody was detected. 6-µm flattened z projections of the entire cell are shown. (C) Internalized antibody colocalizes with EEA1 at 15 min and with Tfn at 30 min. Dab2-deficient cells internalize less integrin β1 antibody but the same amount of Tfn compared with control cells. (D–G) Control and Dab2-deficient HeLa cells were surface labeled with sulpho-NHS-SS-biotin at 4°C for 30 min and then warmed to 37°C in the absence (D and E) or presence (F and G) of 2 mM primaquine for the indicated times. Biotin was removed from surface receptors with MesNa treatment, and cells were lysed and incubated with an anti–integrin β1 antibody. Immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting with peroxidase-conjugated streptavidin. (E and G) Quantification of three independent experiments. For each time point, mean values and standard errors are shown.

Mentions: Control or Dab2-deficient HeLa cells were grown on collagen-coated coverslips, incubated with P5D2 anti–integrin β1 antibody at 4°C, and washed, and surface integrin β1 was detected with fluorescent secondary antibody. As predicted by CSC and FACS, Dab2-deficient HeLa cells bound ∼70–90% more P5D2 than control cells (Fig. 4 A, time 0; and Fig. S1 C). Most integrin β1 was spread over the cell surface as small puncta, as noted previously (Powelka et al., 2004). Focal adhesions were not detected, which is consistent with the ability of P5D2 to bind to integrin β1 and block adhesion (Dittel et al., 1993). Similar results were obtained with noninhibitory anti–integrin α1 antibody TS2/7 (Fig. S2 A). Parallel coverslips that had been incubated with P5D2 or integrin α1 antibody were warmed to 37°C and allowed to recycle antibody for various times. By 30 min (Fig. 4) or 2 h (Fig. S2 A), the amount of integrin β1 or α1 antibody on the surface of control cells had decreased by ∼50–55%, whereas that on the surface of Dab2-deficient cells had decreased by ∼15–20%, which is consistent with either decreased endocytosis or increased exocytosis in the absence of Dab2.


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)

Dab2 regulates integrin β1 endocytosis. (A–C) Control (Con) and Dab2-deficient HeLa cells were incubated with anti–integrin β1 antibody (P5D2) for 30 min at 4°C, followed by warming at 37°C for 0, 15, or 30 min. Mean values and standard errors (∼20 cells/treatment) of pixel intensities were measured. (A) Surface antibody on fixed, nonpermeabilized cells was detected with fluorescent secondary antibody. 6-µm flattened z projections of the entire cell are shown. (B) Surface antibody was removed by acid stripping, and internalized antibody was detected. 6-µm flattened z projections of the entire cell are shown. (C) Internalized antibody colocalizes with EEA1 at 15 min and with Tfn at 30 min. Dab2-deficient cells internalize less integrin β1 antibody but the same amount of Tfn compared with control cells. (D–G) Control and Dab2-deficient HeLa cells were surface labeled with sulpho-NHS-SS-biotin at 4°C for 30 min and then warmed to 37°C in the absence (D and E) or presence (F and G) of 2 mM primaquine for the indicated times. Biotin was removed from surface receptors with MesNa treatment, and cells were lysed and incubated with an anti–integrin β1 antibody. Immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting with peroxidase-conjugated streptavidin. (E and G) Quantification of three independent experiments. For each time point, mean values and standard errors are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Dab2 regulates integrin β1 endocytosis. (A–C) Control (Con) and Dab2-deficient HeLa cells were incubated with anti–integrin β1 antibody (P5D2) for 30 min at 4°C, followed by warming at 37°C for 0, 15, or 30 min. Mean values and standard errors (∼20 cells/treatment) of pixel intensities were measured. (A) Surface antibody on fixed, nonpermeabilized cells was detected with fluorescent secondary antibody. 6-µm flattened z projections of the entire cell are shown. (B) Surface antibody was removed by acid stripping, and internalized antibody was detected. 6-µm flattened z projections of the entire cell are shown. (C) Internalized antibody colocalizes with EEA1 at 15 min and with Tfn at 30 min. Dab2-deficient cells internalize less integrin β1 antibody but the same amount of Tfn compared with control cells. (D–G) Control and Dab2-deficient HeLa cells were surface labeled with sulpho-NHS-SS-biotin at 4°C for 30 min and then warmed to 37°C in the absence (D and E) or presence (F and G) of 2 mM primaquine for the indicated times. Biotin was removed from surface receptors with MesNa treatment, and cells were lysed and incubated with an anti–integrin β1 antibody. Immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting with peroxidase-conjugated streptavidin. (E and G) Quantification of three independent experiments. For each time point, mean values and standard errors are shown.
Mentions: Control or Dab2-deficient HeLa cells were grown on collagen-coated coverslips, incubated with P5D2 anti–integrin β1 antibody at 4°C, and washed, and surface integrin β1 was detected with fluorescent secondary antibody. As predicted by CSC and FACS, Dab2-deficient HeLa cells bound ∼70–90% more P5D2 than control cells (Fig. 4 A, time 0; and Fig. S1 C). Most integrin β1 was spread over the cell surface as small puncta, as noted previously (Powelka et al., 2004). Focal adhesions were not detected, which is consistent with the ability of P5D2 to bind to integrin β1 and block adhesion (Dittel et al., 1993). Similar results were obtained with noninhibitory anti–integrin α1 antibody TS2/7 (Fig. S2 A). Parallel coverslips that had been incubated with P5D2 or integrin α1 antibody were warmed to 37°C and allowed to recycle antibody for various times. By 30 min (Fig. 4) or 2 h (Fig. S2 A), the amount of integrin β1 or α1 antibody on the surface of control cells had decreased by ∼50–55%, whereas that on the surface of Dab2-deficient cells had decreased by ∼15–20%, which is consistent with either decreased endocytosis or increased exocytosis in the absence of Dab2.

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