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      Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

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          Abstract

          It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.

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          Most cited references12

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          The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails

          WS Sale (1986)
          Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.
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            Observations of the structural components of flagellar axonemes and central pair microtubules from rat sperm.

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              Rotational movement of a spermatozoon around its long axis.

              The rotational movement of a spermatozoon around its longitudinal axis was investigated by two methods: by observing a spermatozoon attached vertically to a coverslip by the tip of its head, and by observing a spermatozoon freely swimming in a medium by means of 'double-focal microscopy', which yielded simultaneous images at two different focal planes. Similar results were obtained by these two methods. Sea urchin, starfish, medaka, human, golden hamster and bull spermatozoa rolled in both clockwise and counterclockwise directions, although there was a large difference in the proportion of spermatozoa rolling in each direction in the different species. The majority of sea urchin and starfish spermatozoa rolled in a clockwise direction when an observer viewed the cell from its anterior end, whereas the majority of medaka, golden hamster, human and bull spermatozoa rolled in a counterclockwise direction relative to the same observer. Moreover, some spermatozoa occasionally changed their rotational direction. These results suggest that the mechanism regulating the direction of rotation of the spermatozoa is lax. As rotational movement of a spermatozoon around its longitudinal axis is due to the three-dimensional component of the beat of the flagellum, the direction of the three-dimensional movement presumably changes as the spermatozoa swim.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 February 2016
                2016
                : 11
                : 2
                : e0148880
                Affiliations
                [001]Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan
                University of Luebeck, GERMANY
                Author notes

                Competing Interests: The author has decared that no competing interests exist.

                Conceived and designed the experiments: SI. Performed the experiments: SI. Analyzed the data: SI. Contributed reagents/materials/analysis tools: SI. Wrote the paper: SI.

                Article
                PONE-D-15-20940
                10.1371/journal.pone.0148880
                4749303
                26863204
                0c678efe-d47e-45d4-9d0b-d2d39e357e5c
                © 2016 Sumio Ishijima

                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.

                History
                : 25 May 2015
                : 25 January 2016
                Page count
                Figures: 8, Tables: 1, Pages: 13
                Funding
                The author received no specific funding for this work.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cytoskeleton
                Microtubules
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                Sperm
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Flagella
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Virulence Factors
                Pathogen Motility
                Flagella
                Research and Analysis Methods
                Model Organisms
                Animal Models
                Sea Urchins
                Biology and Life Sciences
                Organisms
                Animals
                Invertebrates
                Echinoderms
                Sea Urchins
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Biology and Life Sciences
                Cell Biology
                Cell Motility
                Ciliary Movement
                Biology and Life Sciences
                Biochemistry
                Bioenergetics
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Cell Biology
                Cell Motility
                Flagellar Motility
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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