Dynein synergises with EB1 to facilitate cortex-microtubule encounter and proper spindle positioning in metaphase

To rotate the bulky mitotic spindle, astral microtubules are pulled by cortex-bound dynein. We investigate whether dynein regulates astral microtubules, independent of its classical microtubule-pulling role by combining super-resolution live-cell microscopy with optogenetic tools to disrupt and measure astral microtubule distributions and spindle movements. Localised photoinactivation of EB1 reveals that microtubule ends reaching distinct cortical regions differently influence spindle movements. Using a dynein inhibitor gradient, we separate dynein’s role in spindle positioning (maintaining pole-to-cortex distance) from its role in directing spindle movements. EB1 photoinactivation of whole spindles uncovers a previously unrecognised synergistic role for dynein and EB1 in maintaining astral microtubules reaching the cortex. We demonstrate the EB1-dependent role for dynein in maintaining astral microtubule growth is independent of its classical cortical roles by analysing dynamic changes in spindle position, displacement rates and cortical dynein localisation and by depleting dynein’s cortical platform, LGN. Our finding that Dynein synergises with EB1 to regulate astral microtubule, independent of cortical pulling, has implications for how spindle positioning is correctly established in cells.