The endosomal sorting complexes required for transport (ESCRT) play a conserved and essential role in cytokinetic abscission, the final step of cell division that physically separates daughter cells and can go awry during cancer development. The homologous family of ESCRT-III proteins plays key roles in this process by forming filaments that appear to constrict the intercellular bridge and promote abscission. ESCRT-III filaments also recruit MIT domain-containing proteins that we hypothesize provide a host of critical, but largely uncharacterized activities required for cytokinesis. To define these processes in molecular detail, we will analyze the structures, interactions and cytokinesis functions of the 12 human ESCRT-III and 21 MIT proteins. We have determined the crystal structure of the ESCRT-III protein, IST1, in its closed conformation and obtained a high resolution cryoEM structure of a helical co-polymer formed by IST1 and its ESCRT-III binding partner, CHMP1B. This is the first such reconstruction, and it reveals a remarkable assembly in which the two ESCRT-III subunits adopt radically different structures. The double-stranded helical filament comprises an outer strand of closed IST1 subunits and an inner strand of highly extended CHMP1B subunits. We will now test a series of structure-based mechanistic hypotheses for ESCRT-III filament formation and function, focusing on the importance of subunit conformational flexibility. IST1 can also form membrane-associated, double-stranded helical filaments that extrude membrane tubules from liposomes, and we will characterize this membrane remodeling reaction in biochemical detail. The MIT ATPases VPS4 and Spastin localize to intercellular bridges during cytokinesis by binding to MIT interaction motifs (MIM) within the exposed C-terminal tails of polymerized ESCRT-III subunits. Less well characterized MIT proteins have diverse effector domains that include kinases, proteases, and deubiquitinases, and we propose that a complex network of MIM-MIT interactions recruits these activities to the intercellular bridge to function in cytokinesis. In support of this idea, we have determined solution structures of three quite distinct ESCRT-III-MIT complexes and shown that an MIT kinase, ULK3, functions in cytokinesis, is recruited to intercellular bridges, and can phosphorylate ESCRT-III subunits. We have crystallized the ULK3 MIT-IST1 interaction complex and will now characterize its structure and associated biochemistry and then expand our studies to include global analyses of the structures and ESCRT-III interactions of all human MIT proteins. Finally, we will use cell-based assays to validate and extend our biochemical and structural studies and to screen the entire set of human MIT proteins for cytokinesis functions. Together, these studies will characterize the fundamental activities of ESCRT-III proteins;i.e., forming filaments, recruiting MIT proteins to function in cytokinesis, and facilitating membrane fission. Our studies will also identify the subset of MIT proteins that function in cytokinesis, elucidate their structural biology, and reveal how they are recruited.

Public Health Relevance

Dividing cells must accurately segregate their genetic information before separating into two discrete cells. The ESCRT pathway plays a central role in coordinating and facilitating cell separation, and our research will help reveal precisely how the ESCRT machinery functions in these fundamental processes that can go awry during cancer development.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01GM112080-01
Application #
8768131
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112