The Microtubule Cytoskeleton

The microtubule cytoskeleton also participates in the maintenance of epithelial planar polarity by modulating mitotic spindle orientation. The organization of epithelial cells to form hollow organs with a single lumen entails that each cell divides symmetrically within the epithelial plane, so that both resulting daughter cells remain in the same plane. Hence, the mitotic spindles must orient within the planar axis. Perpendicular divisions, on the other hand, are necessary to create stratified epithelia. Defective spindle orientation flaws the axis of cell division and could eventually disrupt epithelial organization and generate daughter cells unrestrained by contact with neighbors. Mitotic spindle positioning in most epithelia is controlled by astral microtubules that are nucleated at the spindle poles and orient their plus ends towards the cell cortex.

There are two primary events that determine spindle orientation:

• establishment of a polarity axis by the biased distribution of polarity proteins at the cell cortex; and
• alignment of the mitotic spindle with respect to this polarity axis by microtubule plus ends “capture” at these cortical spots.

The cortical proteins that determine the spindle alignment generate a biased orientation of the spindle by establishing physical connections with astral microtubules plus-ends binding proteins and stabilizing these otherwise highly dynamic structures. The cortical protein complexes that interact with astral microtubule plus ends include the Gai/LGN complex, which is recruited to the lateral membrane by Dlg and E-cadherin, and excluded from the apical membrane by aPKC phosphorylation. In turn, LGN interacts with “nuclear mitotic apparatus” (NuMA), which binds to dynein/dynactin, the motor protein responsible for the astral-microtubule pulling force that directs the movement of the spindle. Another cortical protein complex composed by cdc42 and the junctional adhesion molecule-A (JAM-A) that promotes dynein/dynactin accumulation at the lateral membrane have also been identified. Far less is known about the molecular actors that regulate spindle orientation at the spindle apparatus.

The end-binding protein 1 (EB1) is an autonomously plus end binding protein that regulates microtubule dynamic instability by increasing the periods of microtubule growth, and decreasing those of microtubule severing. In addition to its role in the formation and stabilization of spindle microtubules, studies in drosophila indicated that EB1 is a crucial factor for spindle orientation during symmetric planar division in epithelial cells. Our own studies performed in three dimensional epithelial cell cultures showed that EB1 is loaded on astral microtubules at the spindle poles, and that its presence at these structures is essential for spindle orientation and accurate lumen formation. Although the exact mechanism governing EB1 directed spindle orientation has not been elucidated, EB1 is a scaffold that recruits specific proteins to the microtubule plus ends, including the dynactin subunit p150 glued and the polarity protein Par1, both involved in spindle orientation. P150 glued itself regulates spindle orientation, probably by enhancing dynein processivity.