Chemo-mechanical energy transduction is the fundamental process by which motor molecules develop force and cause shortening (e.g., kinesin- tubulin, myosin-actin). Several theoretical models exist to describe mechanical events at the molecular level. Most of the experimental testing of these theories is based on indirect measurements. Although significant progress has been made in measuring motion at molecular level (in-vitro motility assays), limited data exist regarding direct, high- fidelity measurements of molecular motile force. The overall goal of this pilot project is to develop a technique to measure force at molecular level using the atomic force microscope (AFM). Recent studies have convincingly demonstrated the feasibility of AFM for imaging three- dimensional surface topology of biological samples (e.g., receptors, ion channels) at molecular resolution. Most of these imaging studies have focused on forces normal to the sample surface. In contrast, the technique proposed here relies on the measurement of lateral force, i.e., parallel to the sample surface. Specifically, purified kinesin or myosin molecules (one or few), attached to the scanning tip of the AFM, will interact with a tubulin microtubule or an actin filament absorbed onto a glass coverslip. Following activation with ATP, the lateral force (i.e., along the microtubule or actin filament) will be measured by quantifying the torsion of the cantilever that holds the tip. The specific developmental steps to accomplish the stated goal are: (i) To develop reliable methods to attach kinesin or myosin molecules to the AFM scanning tip and to adsorb microtubules or actin filaments to a glass coverslip; (ii) To develop an optical system for better visualization and integrate it with the AFM; and (iii) To integrate a flash-photolysis system for delivering ATP into the AFM fluid cell using caged compounds. This methodology will be tested by addressing two questions under near- isometric conditions; (i) What are the frequency and the duty cycle of force generation and how do they vary with temperature? and (ii) How does the force development vary with the location on the microtubule or actin filament? AFM-based force measurement can potentially be combined with recently developed """"""""optical tweezers or traps"""""""" to study force generation under various shortening conditions. The successful development of the proposed technique will be useful in studying mechanisms and determinants of force generation in various biological motor molecules. These studies compliment our long-standing interest in linking the mechanical and energetic behavior of the whole heart to the underlying structural and biochemical processes.
| Shroff, S G; Saner, D R; Lal, R (1995) Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy. Am J Physiol 269:C286-92 |
