Identifying New Cofactors for Cytokinetic Abscission

Abstract

A single cell division in the presence of incompletely segregated chromatin can result in chromosome beaks, triggering a chain of events that culminate in chromosomal damage and rapid genome evolution, which are classic hallmarks of cancer. In order to maintain genomic integrity, the cell has evolved a cell cycle checkpoint known as the abscission checkpoint. This checkpoint detects persistent mitotic errors such as incompletely segregated chromosomes, misformed nuclear pores, under-replicated DNA and tension at the intercellular bridge. Once detected, these errors trigger an abscission arrest to allow cells a chance to correct and prevent their transmission to daughter cells. How the cell integrates these diverse signals into a concerted response to delay abscission is not well understood. However, it is well-known that progression through abscission is regulated in part through the sequential action of several key kinases including Aurora B, that inhibit the membrane fission activity of ESCRT-III proteins, which localize to the intercellular bridge connecting daughter cells and are required for completing cell division. In addition to providing the mechanical force necessary for membrane constriction and fission, ESCRT-III proteins recruit essential cofactors via a complex set of interactions between their C-terminal MIT-Interacting Motifs (MIMs) and proteins that contain Microtubule Interacting and Trafficking (MIT) domains. In order to discover new activities required to correctly perform abscission, we mapped the complete network of ESCRT-III binding partners for ~20 human MIT domain-containing proteins. We then focused on three of these MIT proteins: a protease, Calpain-7, and two microtubule severing AAA-ATPases, Katanin, and Spastin. For each protein, we mapped the binding site for their respective ESCRT-III binding partners and tested whether they function in abscission or the abscission checkpoint. Our studies reveal Spastin and Calpain-7 are required for maintaining the abscission checkpoint, with Calpain-7 having an additional role in abscission. These studies biochemically describe how ESCRT-III proteins can recruit multiple different proteins to the intercellular bridge and highlight the importance of these interactions in supplying additional enzymatic activities that promote faithful abscission.