Our recently introduced actoclampin motor model describes how force is generated in actin-based motility through clamped-filament elongation. We proposed a three-step mechanoenzymatic process (called the """"""""Lock, Load & Fire"""""""" or """"""""LLF"""""""" mechanism): during locking, an affinity-modulated clamp protein (also tethered to the surface of a motile object) binds onto ATP-containing actin monomers situated at a filament's barbed-end; in the loading step, new actin ATP monomers bind onto the barbed end of a clamped-filament, an event that triggers the firing step, whereto ATP hydrolysis on the clamped actin subunit greatly attenuates the clamp's affinity for the filament. ATP hydrolysis initiates clamp translocation and re-locking to the new ATP-containing terminus, starting another LLF cycle. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface, and stochastic simulations reproduce the signature motions of motile Listeria. This elongation motor exploits actin's intrinsic ATPase activity to provide a simple, high fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. Our research proposal addresses model-based hypotheses designed to test specific features of the LLF mechanism of actin polymerization motors.
Specific Aim -1 includes experiments aimed at differentiating the LLF model from Brownian Ratchet-type mechanisms (a) by conducting motility studies near and below the (+)-end critical concentration, and (b) by using covalently cross-linked profilin-actin that cannot release profilin after each LLF cycle.
Specific Aim -2 focuses on (a) the role of ATP hydrolysis in motility using slowly hydrolyzing ATP analogues pp(NH)pA and ATPgammaS to identify the likely force-producing step(s) in the LLF mechanism.
Specific Aim -3 deals with profilin's potential role in a kinetic proofreading pathway to suppress loading of actin-ADP into clamped-filament motors.
In Specific Aim -4, we will apply sedimentation and fluorescence anisotropy measurements to learn if and how ATP hydrolysis modulates the strength of binding interactions of VASP's clamping domain with the ends of actin filaments. Together, these investigations promise to shed new light on actin-based motility by testing fundamental mechanistic properties.
| Dickinson, Richard B (2009) Models for actin polymerization motors. J Math Biol 58:81-103 |
| Dickinson, Richard B; Purich, Daniel L (2007) Nematode sperm motility: nonpolar filament polymerization mediated by end-tracking motors. Biophys J 92:622-31 |
| Dickinson, Richard B; Purich, Daniel L (2006) Diffusion rate limitations in actin-based propulsion of hard and deformable particles. Biophys J 91:1548-63 |
| Zeile, William L; Zhang, Fangliang; Dickinson, Richard B et al. (2005) Listeria's right-handed helical rocket-tail trajectories: mechanistic implications for force generation in actin-based motility. Cell Motil Cytoskeleton 60:121-8 |
| Dickinson, Richard B; Caro, Luzelena; Purich, Daniel L (2004) Force generation by cytoskeletal filament end-tracking proteins. Biophys J 87:2838-54 |
