Par3 and Dynein Associate to Regulate Local Microtubule Dynamics and Centrosome Orientation during Migration

Background: Centrosome orientation toward the leading edge of migrating cells depends on dynein and microtubules (MTs), as well as a number of signaling factors at the leading edge. However, centrosomes are maintained at the cell center during orientation in fibroblasts, suggesting that factors working at sites other than the leading edge may also be involved. Results: In a search for factors that function with dynein in centrosome orientation, we found that the polarity protein Par3 associated with dynein and that knockdown of Par3 inhibited centrosome orientation by disrupting the position of the centrosome at the cell center; this disrupted centrosome positioning is the same phenotype as that observed with dynein inhibition. Par3 associated with dynein through its N-terminal dimerization and PDZ1 domains and interacted specifically with dynein light intermediate chain 2 (LIC2). siRNA knockdown of LIC2, but not LIC1, or overexpression of LIC2 or the N-terminal domain of Par3, also inhibited centrosome orientation by disrupting centrosome position. In wound-edge fibroblasts, Par3 specifically localized to cell-cell contacts where it overlapped with MT ends and dynein puncta in a LIC2-dependent fashion. Live imaging showed that MTs exhibited increased pausing at cell-cell contacts compared to the leading edge and that this elevated pausing was dependent on Par3 and LIC2. Conclusions: Par3 associates with dynein and contributes to the local regulation of MT dynamics at cell-cell contacts and proper positioning of the centrosome at the cell center. We propose that Par3 acts as a cortical factor that tethers MTs through its association with LIC2 dynein.