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In Vitro Reconstitution of Centralspindlin‐Mediated Pebble/RhoGEF Transport
Author(s) -
Warecki Brandt,
Tao Li
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09346
Subject(s) - cytokinesis , microbiology and biotechnology , biology , cleavage furrow , cell division , genetics , cell
Cytokinesis is the last step of cell division. Failure in regulation of cytokinesis is the cause of several human diseases, including cancer. Appropriate formation of the cleavage furrow during cytokinesis requires accurate regulation of the small GTPase Rho. Multiple experiments have demonstrated that Rho activity is modulated at the central spindle by RhoGEF (Pebble in Drosophila ) and the conserved motor complex centralspindlin. However, perhaps due to the necessity of these proteins in in vivo systems, the precise mechanism for how centralspindlin and RhoGEF cooperate to activate Rho at the central spindle remains poorly understood. Here, we use the baculovirus expression system to generate recombinant Drosophila versions of the centralspindlin components Pavarotti/Kinesin‐6 and Tumbleweed/RacGAP as well as Pebble/RhoGEF. Using these purified proteins, we reconstitute key events in cytokinesis and test models for Rho activation that have been based on in vivo studies. We observe Pavarotti/Kinesin‐6‐mediated microtubule bundling and show that the recombinant centralspindlin holoenzyme recruits Pebble/RhoGEF to microtubules in vitro . Furthermore, we demonstrate that centralspindlin transports Pebble/RhoGEF along microtubules in a plus‐end directed fashion. Taken together, our studies support a model in which centralspindlin motor activity delivers Pebble/RhoGEF to the central spindle where Pebble/RhoGEF activates Rho, leading to cleavage furrow formation and proper cytokinesis. Support or Funding Information NIH INBRE P20GM103466‐17