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      Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii

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          Abstract

          Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

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          Most cited references39

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          Mechanism of blebbistatin inhibition of myosin II.

          Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.
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            The way things move: looking under the hood of molecular motor proteins.

            The microtubule-based kinesin motors and actin-based myosin motors generate motions associated with intracellular trafficking, cell division, and muscle contraction. Early studies suggested that these molecular motors work by very different mechanisms. Recently, however, it has become clear that kinesin and myosin share a common core structure and convert energy from adenosine triphosphate into protein motion using a similar conformational change strategy. Many different types of mechanical amplifiers have evolved that operate in conjunction with the conserved core. This modular design has given rise to a remarkable diversity of kinesin and myosin motors whose motile properties are optimized for performing distinct biological functions.
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              Anomalous subdiffusion is a measure for cytoplasmic crowding in living cells.

              Macromolecular crowding dramatically affects cellular processes such as protein folding and assembly, regulation of metabolic pathways, and condensation of DNA. Despite increased attention, we still lack a definition for how crowded a heterogeneous environment is at the molecular scale and how this manifests in basic physical phenomena like diffusion. Here, we show by means of fluorescence correlation spectroscopy and computer simulations that crowding manifests itself through the emergence of anomalous subdiffusion of cytoplasmic macromolecules. In other words, the mean square displacement of a protein will grow less than linear in time and the degree of this anomality depends on the size and conformation of the traced particle and on the total protein concentration of the solution. We therefore propose that the anomality of the diffusion can be used as a quantifiable measure for the crowdedness of the cytoplasm at the molecular scale.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                30 June 2015
                2015
                : 5
                : 11690
                Affiliations
                [1 ]Institute for Materials Science, Biocompatible Nanomaterials, Christian-Albrechts-Universität zu Kiel, Kaiserstr. 2 , D-24143 Kiel, Germany
                [2 ]School of Physics, Korea Institute for Advanced Study , Seoul 130-722, Republic of Korea
                [3 ]Zoological Institute, Comparative Immunobiology, Christian-Albrechts-Universität zu Kiel, Olshausenstr.40 , D-24098 Kiel, Germany
                [4 ]Institute of Physics & Astronomy, University of Potsdam , D-14776 Potsdam-Golm, Germany
                [5 ]Department of Physics, Tampere University of Technology , FI-30101 Tampere, Finland
                Author notes
                Article
                srep11690
                10.1038/srep11690
                5155589
                26123798
                3eafbe24-49d2-4cf0-bfa6-1a9fb92dc50f
                Copyright © 2015, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 23 February 2015
                : 01 June 2015
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