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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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Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B. (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test.
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fig3: Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B. (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test.

Mentions: To test whether other endocytic proteins regulate integrin surface levels, we compared the effects of removing Dab2, clathrin, AP2, ARH, or Numb. Numb was recently reported to regulate integrin endocytosis in HeLa cells (Nishimura and Kaibuchi, 2007). Removal of Dab2, AP2, or clathrin but not ARH increased the surface levels of integrins β1 and α1 to a similar extent (Fig. 3 A). Combined removal of Dab2 and AP2 caused little further increase in integrin β1 or α1, suggesting that Dab2 requires AP2 (and/or vice versa) to regulate integrin levels. Integrin α5 levels depended more on AP2 or clathrin than Dab2 (Fig. 3 A). In mirror image to Dab2, Numb removal had a greater effect on integrin α5 than integrin β1 or α1 (Fig. 3 B). These results suggest that clathrin and AP2 regulate β1, α1, and α5, but Dab2 and Numb regulate partly overlapping subsets with Dab2 dominant for β1 and α1 and Numb dominant for α5. Combined removal of Dab2 and Numb had a less than additive effect on surface levels of integrin β1 (Fig. 3 C), suggesting either that they are partially redundant or that maximum integrin levels have been reached. Neither Dab2 nor Numb affected TfnR levels.


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)

Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B. (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B. (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test.
Mentions: To test whether other endocytic proteins regulate integrin surface levels, we compared the effects of removing Dab2, clathrin, AP2, ARH, or Numb. Numb was recently reported to regulate integrin endocytosis in HeLa cells (Nishimura and Kaibuchi, 2007). Removal of Dab2, AP2, or clathrin but not ARH increased the surface levels of integrins β1 and α1 to a similar extent (Fig. 3 A). Combined removal of Dab2 and AP2 caused little further increase in integrin β1 or α1, suggesting that Dab2 requires AP2 (and/or vice versa) to regulate integrin levels. Integrin α5 levels depended more on AP2 or clathrin than Dab2 (Fig. 3 A). In mirror image to Dab2, Numb removal had a greater effect on integrin α5 than integrin β1 or α1 (Fig. 3 B). These results suggest that clathrin and AP2 regulate β1, α1, and α5, but Dab2 and Numb regulate partly overlapping subsets with Dab2 dominant for β1 and α1 and Numb dominant for α5. Combined removal of Dab2 and Numb had a less than additive effect on surface levels of integrin β1 (Fig. 3 C), suggesting either that they are partially redundant or that maximum integrin levels have been reached. Neither Dab2 nor Numb affected TfnR levels.

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