No abstract

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies, for the budding of some enveloped viruses, and for daughter cell scission in cytokinesis. Here we found that the ESCRT-III proteins CHMP2A and CHMP3 could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule while the AAA-type ATPase VPS4 could bind on the inside of the tubule and disassemble the tubes upon ATP hydrolysis. CHMP2A and CHMP3 co-polymerized in solution and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly-budding vesicle, catalyzing late steps in budding under the control of VPS4.