Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics.

The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. Here we report a novel role for the multifunctional adaptor protein ALG-2-interacting protein X (ALIX) in regulating MS orientation in addition to its well-established role in cytokinesis. We show that ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing stem cells and epithelial cells and symmetrically dividing and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of γ-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, our results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division.