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      A Hidden Markov Model for Single Particle Tracks Quantifies Dynamic Interactions between LFA-1 and the Actin Cytoskeleton


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          The extraction of hidden information from complex trajectories is a continuing problem in single-particle and single-molecule experiments. Particle trajectories are the result of multiple phenomena, and new methods for revealing changes in molecular processes are needed. We have developed a practical technique that is capable of identifying multiple states of diffusion within experimental trajectories. We model single particle tracks for a membrane-associated protein interacting with a homogeneously distributed binding partner and show that, with certain simplifying assumptions, particle trajectories can be regarded as the outcome of a two-state hidden Markov model. Using simulated trajectories, we demonstrate that this model can be used to identify the key biophysical parameters for such a system, namely the diffusion coefficients of the underlying states, and the rates of transition between them. We use a stochastic optimization scheme to compute maximum likelihood estimates of these parameters. We have applied this analysis to single-particle trajectories of the integrin receptor lymphocyte function-associated antigen-1 (LFA-1) on live T cells. Our analysis reveals that the diffusion of LFA-1 is indeed approximately two-state, and is characterized by large changes in cytoskeletal interactions upon cellular activation.

          Author Summary

          Many important biological processes begin when a target molecule binds to a cell surface receptor protein. This event leads to a series of biochemical reactions involving the receptor and signalling molecules, and ultimately a cellular response. Surface receptors are mobile on the cell surface and their mobility is influenced by their interaction with intracellular proteins. We wish to understand the details of these interactions and how they are affected by cellular activation. An experimental technique called single particle tracking (SPT) uses optical microscopy to study the motion of cell-surface receptors, revealing important details about the organization of the cell membrane. In this paper, we propose a new method of analyzing SPT data to identify reduced receptor mobility as a result of transient binding to intracellular proteins. Using our analysis we are able to reliably differentiate receptor motion when a receptor is freely diffusing on the membrane versus when it is interacting with an intracellular protein. By observing the frequency of transitions between free and bound states, we are able to estimate reaction rates for the interaction. We apply our method to the receptor LFA-1 in T cells and draw conclusions about its interactions with the T cell cytoskeleton.

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          Most cited references31

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          Single-particle tracking: applications to membrane dynamics.

          Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is observed, but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.
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            Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.

            The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.
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              From fixed to FRAP: measuring protein mobility and activity in living cells.

              Experiments with fluorescence recovery after photobleaching (FRAP) started 30 years ago to visualize the lateral mobility and dynamics of fluorescent proteins in living cells. Its popularity increased when non-invasive fluorescent tagging became possible with the green fluorescent protein (GFP). Many researchers use GFP to study the localization of fusion proteins in fixed or living cells, but the same fluorescent proteins can also be used to study protein mobility in living cells. Here we review the potential of FRAP to study protein dynamics and activity within a single living cell. These measurements can be made with most standard confocal laser-scanning microscopes equipped with photobleaching protocols.

                Author and article information

                Role: Editor
                PLoS Comput Biol
                PLoS Computational Biology
                Public Library of Science (San Francisco, USA )
                November 2009
                November 2009
                6 November 2009
                : 5
                : 11
                : e1000556
                [1 ]Department of Mathematics and Institute of Applied Mathematics, University of British Columbia, Vancouver, British Columbia, Canada
                [2 ]Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
                UT Southwestern Medical Center, United States of America
                Author notes

                Conceived and designed the experiments: CC. Performed the experiments: CC. Analyzed the data: RD CC. Wrote the paper: RD CC DC. Proposed and developed the model: RD DC.

                Das et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                : 20 April 2009
                : 6 October 2009
                Page count
                Pages: 16
                Research Article
                Biophysics/Experimental Biophysical Methods
                Biophysics/Theory and Simulation
                Immunology/Leukocyte Activation

                Quantitative & Systems biology
                Quantitative & Systems biology


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