Daniel L. Purich - US grants

MAP-2a and MAP-2b are high-molecular-weight neuronal microtubule- associated proteins that interact with dendritic microtubules (MTs), and MAP-2c is a corresponding lower-molecular-weight MAP abundant in developing axons. Both proteins share highly conserved N-terminal and C-terminal regions of about 150 and 310 amino acids, respectively, with the latter containing the three 18-amino-acid repeated sequences thought to constitute the MT-binding domain. We propose to further define microtubule binding interactions of the MT-binding fragment, relying on new procedures for preparing the fragment and building on our studies of the three non-identical repeated amino-acid regions. We also plan to characterize m2-peptide displacement of intact MAP-2 from assembled microtubules. To define the amino acid side-chains responsible for binding of peptide-m2 to microtubules and to assess the minimal sequence needed to promote tubule assembly and/or MAP-2 displacement. We plan to study microtubule length redistribution kinetics of microtubules assembled from pure tubulin in the absence or presence of the peptide-m2. We propose to complete the determination of the amino acid sequence of the bovine MT-binding fragment using PCR-derived bovine brain cDNA clones. In a parallel effort, we will investigate phosphorylation of tubule-binding fragment of bovine MAP-2, including: (a) evaluation of the affinity of phosphorylated-tubule-binding fragment for microtubules with the peptide displacement assay; (b) characterization of m2-peptide phosphorylation effects using direct synthesis of m2 analogues containing p-Ser and p-Thr residues; and (c) localization of phosphorylated sites in the MT-binding fragment using enzymatic and chemical cleavage, followed by the diagonal electrophoresis with intervening alkaline phosphatase treatment. Finally, we will carry out microinjection experiments using the MT-binding fragment and synthetic peptides to investigate their efficacy in displacing MAP-2 and tau proteins, and their action in altering cellular distribution of MAP-2 and tau, as well as any changes in cell morphology.

DESCRIPTION (provided by applicant): 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.

PROPOSAL NO.: 0505929
PRINCIPAL INVESTIGATOR: A. Ladd
INSTITUTION NAME: University of Florida

MULTI-SCALE MODELING OF CHEMICAL-TO-MECHANICAL ENERGY CONVERSION IN ACTIN-BASED MOTILITY

This grant is to develop and validate a biologically relevant, multi-scale model of force generation by polymerization of the biopolymer, actin. Monomeric actin polymerizes into stiff filaments from surface-bound components, which crosslink and propel the surface forward. How the chemical energy involved in monomer addition is converted into mechanical work is critical in understanding cell motility, as well as for exploiting actin-based motility for micro-/nanoscale sensors and actuators. The extension of the wormlike model of the actin filaments to incorporate bending and torsion, as well as the incorporation of the hydration and gel will be studied. Intellectual merits include: incorporation of molecular-level kinetics and energetics into a mesoscale model of polymerizing and cross-linking filaments; the design of a computational framework for modeling the mechanical properties of solutions of stiff biopolymers such as actin, accounting for its resistance to bending and torsion, position and orientation-dependent chemical functionalization along the molecular backbone; and the coupling of the polymer dynamics to the surrounding solvent. Time-dependent variations in concentration of critical components of the polymerization process will also be studied. The broader impacts of the work include establishment of new collaborations between biochemists, physicists, and bioengineers studying actin networks, and chemical engineers developing numerical methods to simulate polymer solutions. New understanding of the coupling between filament elongation and force generation will be valuable in designing technological applications, such as microscale sensors and actuators using linear molecular motors based on actin motility. A small symposium will be organized to promote an objective discussion of the merits of various computational approaches to polymer simulations. The research will contribute to the education and training of graduate students in a collaborative, multidisciplinary environment and undergraduate students will participate in making specific calculations for software development and applications.