An IFT20 mechanotrafficking axis is required for integrin recycling, focal adhesion dynamics, and polarized cell migration.

Directional cell migration drives embryonic development, cancer metastasis, and tissue repair and regeneration. Here, we examine the role of intraflagellar transport (IFT) 20 (Ift20) during polarized migration of epidermal cells. IFT20 is implicated in regulating cell migration independently of the primary cilium, but how IFT proteins integrate with the cell migration machinery is poorly understood.

The ciliary GTPase Arl3 maintains tissue architecture by directing planar spindle orientation during epidermal morphogenesis.

Arl/ARF GTPases regulate ciliary trafficking, but their tissue-specific functions are unclear. Here, we demonstrate that ciliary GTPase Arl3 is required for mitotic spindle orientation of mouse basal stem cells during skin development. Arl3 loss diminished cell divisions within the plane of the epithelium, leading to increased perpendicular divisions, expansion of progenitor cells and loss of epithelial integrity.

A Presenilin-2-ARF4 trafficking axis modulates Notch signaling during epidermal differentiation.

How primary cilia impact epidermal growth and differentiation during embryogenesis is poorly understood. Here, we show that during skin development, Notch signaling occurs within the ciliated, differentiating cells of the first few suprabasal epidermal layers. Moreover, both Notch signaling and cilia disappear in the upper layers, where key ciliary proteins distribute to cell-cell borders.

FAK, talin and PIPKIγ regulate endocytosed integrin activation to polarize focal adhesion assembly.

Integrin endocytic recycling is critical for cell migration, yet how recycled integrins assemble into new adhesions is unclear. By synchronizing endocytic disassembly of focal adhesions (FAs), we find that recycled integrins reassemble FAs coincident with their return to the cell surface and dependent on Rab5 and Rab11. Unexpectedly, endocytosed integrins remained in an active but unliganded state in endosomes.

A role for the primary cilium in Notch signaling and epidermal differentiation during skin development.

Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar transport proteins (IFTs) and conditional ablation of Ift-88 and Kif3a during embryogenesis. In both cultured keratinocytes and embryonic epidermis, all of these eliminated cilia, and many (not Kif3a) caused hyperproliferation.

Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells.

Focal adhesion disassembly is regulated by microtubules (MTs) through an unknown mechanism that involves dynamin. To test whether endocytosis may be involved, we interfered with the function of clathrin or its adaptors autosomal recessive hypercholesteremia (ARH) and Dab2 (Disabled-2) and found that both treatments prevented MT-induced focal adhesion disassembly. Surface labeling experiments showed that integrin was endocytosed in an extracellular matrix-, clathrin-, and ARH- and Dab2-dependent manner before entering Rab5 endosomes.

Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase.

Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions.