Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis

Abstract

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.

Cytokinesis in Animal Cells

“…PI(4,5)P2 interacts with the contractile ring components anillin and septins and regulates F-actin polymerization by modulating the activity of the actin-binding proteins profilin and cofilin (Yin and Janmey 2003;Bertin et al 2010;Liu et al 2012). Finally, recent studies have shown that the RhoGEF ECT2 and the centralspindlin subunit MgcRacGAP con-tain protein domains that bind to polyanionic phosphoinositide lipids (Su et al 2011;Frenette et al 2012;Lekomtsev et al 2012). Consistent with these observations, enrichment of PI(4,5) P2 is restricted to the cleavage furrow to maintain contractile ring structure (Ben El Kadhi et al 2011).…”

“…4B), and its interaction with septins has been proposed to be required for the formation of these constrictions (Renshaw et al 2014). Finally, both anillin and the RacGAP component of centralspindlin have been shown to link directly the midbody ring to the plasma membrane (Kechad et al 2012;Lekomtsev et al 2012;Liu et al 2012).…”

Cytokinesis in Metazoa and Fungi

“…Indeed, a second, smaller pool of centralspindlin that localizes to the plasma membrane has recently been shown to play a role (Basant et al 2015). The Cyk4 subunit of centralspindlin contains a weak membranebinding domain (Lekomtsev et al 2012). During anaphase, centralspindlin undergoes regulated oligomerization.…”

“…The mechanism of Aurora B localization is incompletely understood, although the kinesins of the Kif20 family (also called MKLP2) and Kif4 are required in vertebrate systems Nguyen et al 2014). Although the membrane-bound pool of centralspindlin is important when the midzone and cortex are far apart, in cells in which they are closer, or once the furrow has begun to ingress, in larger cells, the midzone pool of centralspindlin and associated ECT-2 likely sustain furrow ingression (Yüce et al 2005;Lekomtsev et al 2012). …”

“…Thus, there might not be a single, specific tether for the contractile ring, but rather a constellation of diverse contributing factors. Finally, late in cytokinesis, the membrane-binding C1 domain of the centralspindlin subunit Cyk4 is required for membrane linkage to the central spindle at the midbody stage (Lekomtsev et al 2012), which is also stabilized by the scaffold protein anillin (D'Avino et al 2008;Kechad et al 2012). …”