The goal of this research is to determine how the assembly dynamics and architecture of the actin cytoskeleton are controlled by formins and the cellular mechanisms regulating their activities. We are studying this question in budding yeast, where formins assemble actin cables of a characteristic length, architecture, and dynamics required for polarized cell growth. Further, we extend this work to mammalian formin regulation. The proposal focuses on several new multi-component mechanisms discovered during the previous funding cycle, which control formin-mediated actin nucleation, or the duration and speed of formin-mediated actin filament elongation events. The project combines genetics, live- cell imaging, biochemistry, and novel multi-wavelength single molecule TIRF microscopy.
The Aims are: (1) Test the hypothesis that formin-mediated actin cable nucleation is spatially and temporally controlled by the combinatorial effects of Bud6, profilin, Tpm1, and Tpm2; (2) Test the hypothesis that actin cable length, velocity, and architecture are controlled by dynamic interplay at filament barbed ends involving formins, Bud14-Kel1-Kel2 complex, capping protein, and Smy1; and (3) Test the hypothesis that human CLIP-170 interacts with mDia1 to form a novel barbed end- tracking complex that supports ultrafast actin filament elongation in vitro and in vivo.
This grant investigates the mechanisms by which the actin cytoskeleton is controlled by a family of protein?s called formins, and their interactions with cellular binding partners (Bud6, Smy1, Bud14, Hof1, CLIP-170, and capping protein). Yeast Bud6 is the functional counterpart of human Apc, mutations in which cause > 80% of all human colorectal cancers. Yeast Smy1 is the homologue of Kinesin-1, which is linked via interacting partners to Alzheimer?s, Huntington?s, and Parkinson?s diseases. Further, CLIP-170 is linked to Hodgkin?s Lymphoma. For these reasons, the research in this proposal has the potential to provide new insights into the underlying mechanisms of human disease. In addition, the work is designed to uncover basic mechanisms of cellular biochemistry that will contribute to an understanding of disease states in as-yet-unanticipated ways.
| Garabedian, Mikael V; Stanishneva-Konovalova, Tatiana; Lou, Chenyu et al. (2018) Integrated control of formin-mediated actin assembly by a stationary inhibitor and a mobile activator. J Cell Biol 217:3512-3530 |
| Sokolova, Olga S; Chemeris, Angelina; Guo, Siyang et al. (2017) Structural Basis of Arp2/3 Complex Inhibition by GMF, Coronin, and Arpin. J Mol Biol 429:237-248 |
| 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 |
| Henty-Ridilla, Jessica L; Rankova, Aneliya; Eskin, Julian A et al. (2016) Accelerated actin filament polymerization from microtubule plus ends. Science 352:1004-9 |
| Alioto, Salvatore L; Garabedian, Mikael V; Bellavance, Danielle R et al. (2016) Tropomyosin and Profilin Cooperate to Promote Formin-Mediated Actin Nucleation and Drive Yeast Actin Cable Assembly. Curr Biol 26:3230-3237 |
| Mohapatra, Lishibanya; Goode, Bruce L; Jelenkovic, Predrag et al. (2016) Design Principles of Length Control of Cytoskeletal Structures. Annu Rev Biophys 45:85-116 |
| Eskin, Julian A; Rankova, Aneliya; Johnston, Adam B et al. (2016) Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo. Mol Biol Cell 27:828-37 |
| 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 |
| Mohapatra, Lishibanya; Goode, Bruce L; Kondev, Jane (2015) Antenna Mechanism of Length Control of Actin Cables. PLoS Comput Biol 11:e1004160 |
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