The goal of this research is to determine how the dynamics of the actin and microtubule cytoskeletons are coordinated by a group of three interacting mammalian proteins: APC, Dia1, and EB1. This work will define the functions and mechanisms of these proteins, and provide a deeper understanding of the activities and interactions that underlie such processes as cell migration and morphogenesis. This project uses bulk biochemical experiments combined with a novel multi-wavelength single molecule biophysics method tailored to elucidate the mechanisms of complex, multi-component regulatory systems in vitro. In addition, the mechanisms deduced from the experiments in vitro will be tested in vivo to verify that they are important for relevant biological functions of these proteins in living cells (e.g., directed cell migration).
The Specific Aims are: (1) Test two key hypotheses about the mechanism by which Dia1 and APC synergize in promoting actin assembly, involving formation of proposed physical complexes among components;(2) Test the additional hypothesis that Dia1 and APC synergize to stimulate actin assembly by a """"""""rocket launcher"""""""" mechanism;and (3) Define the mechanisms by which EB1 alone and EB1 plus microtubules regulate and/or organize APC/Dia-induced actin assembly.
This grant is an investigation of the organization and regulation of actin and microtubule cytoskeletons by the combined effects of three proteins: APC, Dia1, and EB1. APC (Adenomatous polyposis coli) is a tumor suppressor. Mutation of the human Apc gene is considered to be an early step in the progression of over 80% of colorectal cancers, inherited and sporadic. In addition, fusion of the EB1 and MLL genes can lead to acute lymphoblastic leukemia. We anticipate that this research will provide new insights into the underlying mechanisms of tumorigenesis.
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|Juanes, M Angeles; Bouguenina, Habib; Eskin, Julian A et al. (2017) Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover. J Cell Biol 216:2859-2875|
|Shekhar, Shashank (2017) Microfluidics-Assisted TIRF Imaging to Study Single Actin Filament Dynamics. Curr Protoc Cell Biol 77:12.13.1-12.13.24|
|Henty-Ridilla, Jessica L; Juanes, M Angeles; Goode, Bruce L (2017) Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis. Curr Biol 27:3535-3543.e4|
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|Bombardier, Jeffrey P; Eskin, Julian A; Jaiswal, Richa et al. (2015) Single-molecule visualization of a formin-capping protein 'decision complex' at the actin filament barbed end. Nat Commun 6:8707|
|Jansen, Silvia; Collins, Agnieszka; Chin, Samantha M et al. (2015) Single-molecule imaging of a three-component ordered actin disassembly mechanism. Nat Commun 6:7202|
|Smith, Benjamin A; Gelles, Jeff; Goode, Bruce L (2014) Single-molecule studies of actin assembly and disassembly factors. Methods Enzymol 540:95-117|
|Daou, Pascale; Hasan, Salma; Breitsprecher, Dennis et al. (2014) Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration. Mol Biol Cell 25:658-68|
|Breitsprecher, Dennis; Goode, Bruce L (2013) Formins at a glance. J Cell Sci 126:1-7|
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