JCB_201709005 861..876

The mitotic spindle checkpoint delays anaphase onset until all kinetochores are stably attached to microtubules emanating from opposing spindle poles (Lara-Gonzalez et al., 2012). This mechanism provides more time for the cell to correct erroneous kinetochore–microtubule attachments and avoid possible chromosome missegregation and aneuploidy. Conserved components of the spindle checkpoint pathway include the Mad (Mad1, Mad2, and BubR1) and the Bub (Bub1 and Bub3) proteins. Accumulation of Mad and Bub proteins on unattached kinetochores is essential to prevent activation of the anaphase-promoting complex/cyclosome and delay mitotic exit, but the molecular details are incompletely understood (Funabiki and Wynne, 2013; London and Biggins, 2014b). Kinetochore localization of the Mad1-Mad2 heterotetramer is a major determinant of the spindle checkpoint activity (Maldonado and Kapoor, 2011; Kuijt et al., 2014). Bub1 interacts with Mad1 to activate the spindle checkpoint in budding yeast and Caenorhabditis elegans (London and Biggins, 2014a; Moyle et al., 2014). However, Mad1-Mad2 recruitment to kinetochores also requires the activity of the Rod–ZW10– Zwilch (RZZ) complex in metazoans (Buffin et al., 2005; Kops et al., 2005; Caldas et al., 2015; Silió et al., 2015). The RZZ complex is mainly present in metazoans (Karess, 2005; Vleugel et al., 2012), and cells lacking RZZ proteins are spindle checkpoint deficient (Basto et al., 2000; Chan et al., 2000; Williams et al., 2003). Despite its important role in spindle checkpoint signaling, how RZZ is targeted to kinetochores remains unclear. The Knl1-Zwint1 protein complex was initially proposed to recruit RZZ to kinetochores, and RZZ localization may be regulated by Aurora B–dependent phosphorylation of Zwint1 (Wang et al., 2004; Kops et al., 2005; Kasuboski et al., 2011; Varma et al., 2013). Recently, it was shown that Knl1-Bub1 is also required for localization of the RZZ to kinetochores in HeLa cells independently of Zwint1 (Caldas et al., 2015). Furthermore, there is evidence of a Knl1and Bub1-independent mechanism for RZZ and Mad1-Mad2 kinetochore targeting in HeLa and human retinal pigment epithelial cells suggesting additional factors are involved (Caldas et al., 2015; Silió et al., 2015). After chromosome biorientation at the metaphase plate, dynein transports RZZ and Mad1-Mad2 from kinetochores to the spindle poles via microtubules in a process called stripping, to silence the spindle checkpoint (Howell et al., 2001; Wojcik et al., 2001). The endosomal sorting complex required for transport (ESC RT) machinery is required for biogenesis of multivesicular endosomes, viral budding, cytokinetic abscission, and sealing of the newly formed nuclear envelope during cell division (Hurley, 2015; Campsteijn et al., 2016). These functions require membrane remodeling and scission and involve assembly of cytosolic components of the ESC RT-III subcomplex into helical filaments that constrict and cut membrane invaginations in conjunction with the ATPase Vps4 that is essential for disassembly of ESC RT-III spirals and is also likely to provide energy input for membrane neck constriction (Adell et al., 2014). Chmp4c, an ESC RT-III component, is one of three human orthologues The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod–ZW10–Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore–microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport– dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores. The ESC RT protein Chmp4c regulates mitotic spindle checkpoint signaling