The regulation of cell architecture and morphology is essential during the course of vertebrate development, organogenesis, and tissue homeostasis. The actin-associated Shroom proteins are critical determinants of epithelial cell shape, controlling the process of apical constriction and cellular contractility. Shroom proteins work by targeting the Rock-Myosin II pathway to specific regions of the cells where it causes the formation of a contractile actomyosin network. The ability of Shroom to engage the Rock-myosin pathway is dependent on a conserved domain that binds directly to Rock. Based on our work, we predict that this pathway represents an evolutionarily conserved signaling module that is required for a number of developmental events in vertebrates, including formation of the central nervous system, eye, and intestines. This proposal will investigate the assembly and function of this pathway at the structural, biochemical, and cellular levels. We will accomplish this through the following aims.
Aim I. Structural Analysis and Characterization of a Shrm SD2 domain. We will use X-ray crystallography to determine the structure for an SD2 domain as a starting point for generating hypotheses about how SD2 interacts with Rock to control contractility. We will use biochemical and cell biological approaches, combined with mutants derived from our analysis of the structure, to test these hypotheses.
Aim II. Characterization of the Shroom-Binding Domain (SBD) of Rock. We will determine the structure of SBD to give insight into its conformation and design informative mutants to determine which portions of SBD are physically interacting with Shrm. We will also examine the effects of these mutations in vivo. Coupled with the structure of SD2, we will be able to generate simple testable models that describe the SD2-SBD interaction and how it regulates Rock activity.
Aim III. Structure of the SD2-SBD complex and implications for signaling. The interaction between Shrm and Rock is sufficient to regulate downstream cytoskeletal changes and alter cell morphology. We will address the nature of the interaction by solving the structure of the SD2- SBD complex. We will expand our analysis to examine the role each domain plays in regulating cell morphology and analyze the interplay between the Shrm-Rock pathway and the Rho-Rock pathway. These studies are significant because only by elucidating how signaling complexes are assembled can we understand their manner of function and regulation. Because the Rock- myosin II pathway is central to a vast number of cellular processes, understanding how this pathway functions may provide invaluable insight into the basic mechanisms of cellular regulation under both normal and disease conditions. In addition, targeted disruption of specific Rock-dependent events may prove to be a powerful therapeutic approach to treating certain human diseases and disorders.

Public Health Relevance

Cells must be able to regulate their shape in order to form the correct body plan and functional tissues and organs. This research investigates a network of proteins that controls how cells manage this complicated task. By understanding these processes from the atomic to the cellular level we hope to learn how defects in these pathways may cause human disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Flicker, Paula F
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University of Pittsburgh
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