PROJECT SUMMARY/ABSTRACT To divide, cells must faithfully partition their duplicated genomes (mitosis) and then separate (cytokinesis). The final abscission step of cytokinesis is catalyzed by the ESCRT machinery, which severs the midbody to separate the nascent daughter cells. This step is negatively regulated by the NoCut/abscission checkpoint in response to mitotic errors such as intercellular DNA bridges or incomplete nuclear pore formation, providing cells with time to correct errors or mount protective responses. NoCut failure therefore results in DNA damage and is linked to several types of cancer. Within the midbody, ESCRT-III proteins form membrane-bound filaments that collaborate with VPS4 ATPases to constrict and cut the membrane. These filaments also bind other MIT domain-containing cofactors that participate in NoCut and abscission. We previously identified three classes of MIT enzymes with important cytokinetic functions: 1) ULK3 kinase, which phosphorylates and inactivates ESCRT-III proteins, 2) VPS4s and related “meiotic clade” AAA ATPases, and 3) the CAPN7 cysteine protease. We will now study how the latter two classes of MIT enzymes function during cytokinesis to remodel ESCRT-III filaments (VPS4s), sever spindle microtubules (SPASTIN and KATANIN), or sustain NoCut signaling and then promote abscission (CAPN7 and SPASTIN). We have also recently discovered a previously uncharacterized cytoplasmic organelle, the Abscission Checkpoint Body (ACB), that contributes to NoCut abscission delay, apparently by sequestering essential ESCRT abscission factors like ALIX away from the midbody. We will now elucidate how ACBs form and help to regulate abscission timing. Our ultimate goal is to define how cells maintain NoCut signaling to prevent premature abscission, and then sever the midbody to create separate daughter cells.