Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts

1-POS Board 1 Force Dependence of Phagosome Trafficking in RPE Cells David Altman , Willamette University, Salem, OR, USA.

Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of an external force through the use of an optical trap. To quantify applied forces, the extracellular environment was index-matched to the interior of the cell, allowing for in vitro measurements of the trap stiffness. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking. 3-POS Board 3 Single Molecule Dynamics and Hidden Markov Models for P2X1 Receptors Adam O. Barden 1 , Brian N. Webb 1 , Andrew J. Thompson 2 , James A. Brozik 1 . 1 Washington State University, Pullman, WA, USA, 2 Cambridge University, Cambridge, United Kingdom. Purinergic receptors are ubiquitous throughout the human body and participate in the regulation of vast numbers of physiological processes. In particular, ATP binds to P2X1 receptors causing a two stage allosteric modulation for each bound ATP. This modulation opens an integral pore causing the entry of calcium into cells and initiates numerous downstream processes. We will present results from stochastic single molecule fluorescence studies that show multiple discrete ATP binding states associated with individual P2X1 receptors. These observations have been incorporated into a Hidden Markov Model(s) that: (1) takes into account discrete ATP binding states, (2) accounts for the photophysical properties of the probe molecules, (3) extracts the most likely elementary rate constants, and (4) predicts the most likely state occupancies. These results have been used to create a minimal potential energy surface that describes the operation P2X1 as a molecular machine. Some recent super-resolution data that maps out the spatial arrangement of ATP on individual receptors will also be presented.

40