Abstract
The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.
Highlights
Cytokinesis is the final stage of cell division that splits a cell into two
We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLPRacGAP centralspindlin complex to the cell equator and spindle midzone
Recent studies in mammals suggest an important role for Polo-like kinase 1 (Plk1)2 for the interaction between RacGAP and Pebble, because inhibiting the function of Plk1 in anaphase prevents the localization of Pebble/Ect2 to the equator and the subsequent ring assembly, the centralspindlin complex still localizes correctly (8 –11)
Summary
Immunohistochemistry—Brains were dissected from third instar larvae in ice-cold PBS with 4% paraformaldehyde using forceps in a two-well microscope slide. Frozen ice packs were put under the dissecting microscope stage to keep it cold. The dissected brains were moved to a clean well on the same slide and shredded with fine needles. Four brains were done at a time, and the whole dissecting and fixing procedure was carried out within 10 min. The fix was removed from the well without removing the brain pieces and replaced with PBS plus
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