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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-mediated migration requires the p96-specific exon. (A and D) Migration through filters coated with collagen IV (A and D) or vitronectin (A) toward 10% FBS was measured using a Boyden chamber. Cells passing through triplicate membranes were counted and averaged. The collagen IV results in A show mean and standard error of four independent experiments. *, P < 0.01. (B) Drawing of p96 and p67 forms of Dab2 showing known binding sites. (C and D) Surface integrin β1 levels (C) and migration (D) for Dab2-deficient HeLa cells reexpressing vector alone or T7-tagged mouse p96 or p67. Mean values and standard errors from two independent experiments are shown. Dashed lines indicate the control levels. (E) Western blots show Dab2 knockdown and reexpression of T7-tagged mouse p96 or p67. Con, control.
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fig6: Dab2-mediated migration requires the p96-specific exon. (A and D) Migration through filters coated with collagen IV (A and D) or vitronectin (A) toward 10% FBS was measured using a Boyden chamber. Cells passing through triplicate membranes were counted and averaged. The collagen IV results in A show mean and standard error of four independent experiments. *, P < 0.01. (B) Drawing of p96 and p67 forms of Dab2 showing known binding sites. (C and D) Surface integrin β1 levels (C) and migration (D) for Dab2-deficient HeLa cells reexpressing vector alone or T7-tagged mouse p96 or p67. Mean values and standard errors from two independent experiments are shown. Dashed lines indicate the control levels. (E) Western blots show Dab2 knockdown and reexpression of T7-tagged mouse p96 or p67. Con, control.

Mentions: We measured surface integrin levels by staining fixed, nonpermeabilized Dab2-deficient and control cells with anti-integrin antibodies and fluorescent secondary antibodies, followed by FACS. HeLa cells express integrins α1β1, α3β1, α2β1, α5β1 (in approximate order of decreasing abundance), and αvβ5 (Riikonen et al., 1995). Consistent with the CSC proteomic results, removal of Dab2 caused ∼1.7–2.5-fold increases in surface levels of integrins β1 and α1, a small increase in α5, and no change in αvβ5 (Fig. 2, A and B). Because transferrin (Tfn) receptor (TfnR) endocytosis does not require Dab2 (Maurer and Cooper, 2006), we measured TfnR steady-state surface levels as a control. As expected, TfnR surface levels were unaltered by Dab2 depletion (Fig. 2 B). The observed effects were specific because surface integrin β1 was also increased when Dab2 was transiently depleted with siRNAs targeting different sequences in Dab2 (Fig. S1 A), and integrin levels were rescued by reexpressing Dab2 (see Fig. 6 C).


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-mediated migration requires the p96-specific exon. (A and D) Migration through filters coated with collagen IV (A and D) or vitronectin (A) toward 10% FBS was measured using a Boyden chamber. Cells passing through triplicate membranes were counted and averaged. The collagen IV results in A show mean and standard error of four independent experiments. *, P < 0.01. (B) Drawing of p96 and p67 forms of Dab2 showing known binding sites. (C and D) Surface integrin β1 levels (C) and migration (D) for Dab2-deficient HeLa cells reexpressing vector alone or T7-tagged mouse p96 or p67. Mean values and standard errors from two independent experiments are shown. Dashed lines indicate the control levels. (E) Western blots show Dab2 knockdown and reexpression of T7-tagged mouse p96 or p67. Con, control.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Dab2-mediated migration requires the p96-specific exon. (A and D) Migration through filters coated with collagen IV (A and D) or vitronectin (A) toward 10% FBS was measured using a Boyden chamber. Cells passing through triplicate membranes were counted and averaged. The collagen IV results in A show mean and standard error of four independent experiments. *, P < 0.01. (B) Drawing of p96 and p67 forms of Dab2 showing known binding sites. (C and D) Surface integrin β1 levels (C) and migration (D) for Dab2-deficient HeLa cells reexpressing vector alone or T7-tagged mouse p96 or p67. Mean values and standard errors from two independent experiments are shown. Dashed lines indicate the control levels. (E) Western blots show Dab2 knockdown and reexpression of T7-tagged mouse p96 or p67. Con, control.
Mentions: We measured surface integrin levels by staining fixed, nonpermeabilized Dab2-deficient and control cells with anti-integrin antibodies and fluorescent secondary antibodies, followed by FACS. HeLa cells express integrins α1β1, α3β1, α2β1, α5β1 (in approximate order of decreasing abundance), and αvβ5 (Riikonen et al., 1995). Consistent with the CSC proteomic results, removal of Dab2 caused ∼1.7–2.5-fold increases in surface levels of integrins β1 and α1, a small increase in α5, and no change in αvβ5 (Fig. 2, A and B). Because transferrin (Tfn) receptor (TfnR) endocytosis does not require Dab2 (Maurer and Cooper, 2006), we measured TfnR steady-state surface levels as a control. As expected, TfnR surface levels were unaltered by Dab2 depletion (Fig. 2 B). The observed effects were specific because surface integrin β1 was also increased when Dab2 was transiently depleted with siRNAs targeting different sequences in Dab2 (Fig. S1 A), and integrin levels were rescued by reexpressing Dab2 (see Fig. 6 C).

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