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Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts
40
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.