TPPP acts downstream of RhoA-ROCK-LIMK2 to regulate astral microtubule organization and spindle orientation.

The actin cytoskeleton in eukaryotic cells undergoes drastic rearrangement during mitosis. The changes to the actin cytoskeleton are most obvious in the adherent cells, where the actin stress fibres are disassembled, and the cortical actin network becomes more prominent with concomitant increase in cell rigidity as cells round up and enter mitosis. Although the regulatory connections between the actin cytoskeleton and the early mitotic events are apparent, the mechanisms that govern these links are not well understood. Here, we report that LIMK1 and LIMK2, the downstream effectors of RhoA and ROCK, regulate centrosome integrity and astral microtubule organization, respectively. Surprisingly, LIMK1 and cofilin are not involved downstream of RhoA and ROCK in the regulation of astral microtubule organization. Instead, we find that LIMK2 acts through TPPP in the regulation of astral microtubule organization, whereas both LIMK1 and LIMK2 affect centrosome focusing. Both phenotypes are tightly coupled to spindle orientation in the mitotic cells. Thus, our results reveal a new regulatory link between the actin cytoskeleton and the mitotic spindle during the early stages of mitosis.