Migrating human foreskin fibroblasts stained for Dab2 (red) and actin (green). Dab2-containing pits are situated where they can take up free integrins, away from the leading edge, which is at the lower left.

Image courtesy of Dr Jonathan A. Cooper, Fred Hutchinson Cancer Research Center, Seattle, USA.

Membrane proteins undergoing clathrin-mediated endocytosis bind to adaptors that interact with clathrin and assemble coated pits, which invaginate to form intracellular vesicles. In the Journal of Cell Biology, Anjali Teckchandani, Jonathan Cooper and colleagues now report that the adaptor protein Dab2 internalizes cell surface integrins to ensure that an internal pool of integrins is readily available for the formation of cell adhesions during migration.

The authors set out to identify proteins exhibiting altered membrane expression levels in the absence of Dab2. Using an approach whereby surface glycoproteins are tagged, purified, then quantified by mass spectrometry, they identified 13 glycoproteins that were reproducibly increased on the surface of Dab2-deficient Hela cells. Five of these 13 glycoproteins were integrins — integrin 1, 1, 2, 3 and 5. Further investigation into how Dab2 affected the surface levels of integrin 1 showed that the total amount of integrin 1 was unaltered in the cells, suggesting that increased surface integrin 1 results from altered trafficking between the surface and intracellular pools.

Integrin 1 was initially localized in a dispersed population of tiny vesicles and later in larger, perinuclear recycling endosomes. Thus, the routing of internalized integrin 1 was not altered by Dab2 loss. However, Dab2-deficient cells did have a reduction in internal integrin 1 of approximately 65%, suggesting impaired endocytosis, which was confirmed by analysing the speed of 1 internalization. Dab2 colocalized with integrin 1, but not in the proximity of focal adhesions. Colocalization was observed only at distant sites, suggesting that it mediates the endocytosis of free but not engaged integrins.

Is Dab2-mediated integrin endocytosis linked to cell migration? Removing Dab2 inhibited migration specifically on collagen, a ligand for integrin 11. Furthermore, a Dab2 mutant protein that does not support endocytosis was unable to rescue either the integrin levels or migration defects present in Dab2-deficient Hela cells. Together, these results show that Dab2 regulates migration through integrin endocytosis. Interestingly, lowering the concentration of collagen or normalizing the surface levels of integrin 1 in Dab2-deficient cells did not restore migration speed, showing that slow migration was not due to excessive adhesion. Importantly, migration did not correlate with surface integrin levels, whereas it did strongly correlate with internal levels. The importance of Dab2-mediated endocytosis for trafficking integrins to the leading edge was confirmed in a scratch-wound assay where Dab2-depleted cells failed to polarise and accumulate integrin 1 at the front.

The results from Teckchandani and colleagues show for the first time that an adaptor protein in the clathrin endocytic pathway regulates migration by promoting the internalization of free membrane integrins and ensuring the maintenance of an internal pool. Thus, endocytosis appears to indirectly affect the formation of new adhesion sites at the leading edge.

Author: Kim Baumann - Copyright © 2009 Nature Publishing Group, a division of MacMillan Publishers Limited; used with permission