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      Single-molecule imaging of cytoplasmic dynein in vivo.

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          Abstract

          While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior.

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          Author and article information

          Journal
          Methods Cell Biol
          Methods in cell biology
          Elsevier BV
          0091-679X
          0091-679X
          2015
          : 125
          Affiliations
          [1 ] Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
          [2 ] Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
          Article
          S0091-679X(14)00002-8
          10.1016/bs.mcb.2014.10.001
          25640420
          028d6eac-ed78-4e4b-837b-e4001973b088
          History

          Diffusion,Microtubules,Motor proteins,Single-molecule observation,TIRF,Total internal reflection fluorescence microscopy,Dynein,Fission yeast,HILO,Live-cell imaging

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