Preparation of centralspindlin as an active heterotetramer of kinesin and GTPase activating protein subunits for in vitro structural and functional assays

Abstract Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the central spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the central spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission). A unique and functionally important feature of centralspindlin as a kinesin-containing protein complex is that the nonmotor component, CYK4, is not a passive cargo of the MKLP1 motor, but an integrated component of a microtubule-organizing machinery. Thus, for in vitro structural and functional assays, it is pivotal to prepare active stoichiometric complexes of the two components. Discussed here are two complimentary approaches, (1) reconstitution of the complex in bacterial extracts ( in extract reconstitution) and (2) purification of a native complex from a mammalian cell line using a localization and affinity purification (LAP) tag.