The role of early endosomal sorting in integrin function

Abstract

Integrins are transmembrane heterodimers composed of an α and a β subunit that mediate attachment between the cell and its environment, which is a requirement for cell survival in many cell types. Cells maintain adhesion by trafficking integrins to the cell surface through endosomal sorting pathways; however, the molecular mechanisms governing this process are relatively undefined. Accumulating evidence suggests that integrin dysfunction results in a range of disease states; thus, it is critical that we expand our understanding of the spatio-temporal control of integrins. I hypothesized that integrin recycling through the endosomal compartment is important for maintenance of cell adhesion. In order to test this, HeLa cells were depleted of factors with known roles at the early and recycling endosome. Depletion of a long-loop recycling factor, EHD1 (Eps15 Homology Domain), did not lead to loss of adhesion. However, depletion of factors that have established roles at the early endosome, AAK1L (Adaptor-Associated Kinase 1 long form) and EHD3, led to cell rounding and an eventual loss of cell adhesion. Measurements of recycling kinetics revealed that AAK1L and EHD3 depletion led to a reduction in the rapid, or short-loop, recycling of β3 integrin. Commensurate with this observation, β3 integrin was redistributed to tubular endosomes near the cell surface in cells depleted of either AAK1L or EHD3. To test whether focal adhesion formation was disrupted in cells with rapid recycling defects, a TIRF live cell imaging approach was used to visualize the dynamics of β3 integrin-GFP-positive focal adhesions. This revealed a reduction in the rate of β3 integrin incorporation into focal adhesions in cells depleted of short-loop, but not long-loop, recycling factors. Moreover, structure-function analysis of AAK1L demonstrates that kinase activity and its carboxy-terminal domain (CBD2) were essential for maintaining cell adhesion. Additional loss-of-function experiments revealed that AAK1L depletion leads to an increase in β1 integrin transport to the lysosome and that AAK1L maintains activated β1 integrin levels at the plasma membrane. These data demonstrate an important role for AAK1L and EHD3 in maintaining cell function by promoting integrin rapid-recycling from the early endosome.