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Research Article
Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella
Hiroshi Yoke, Chikako Shingyoji
Journal of Experimental Biology 2017 220: 1122-1134; doi: 10.1242/jeb.147942
Hiroshi Yoke
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Chikako Shingyoji
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  • ORCID record for Chikako Shingyoji
  • For correspondence: chikako@bs.s.u-tokyo.ac.jp
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  • Fig. 1.
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    Fig. 1.

    Schematic depiction of the experimental setup. Microtubule sliding was induced by consecutive perfusion of 21S dynein and then of microtubules with 1 mmol l−1 ATP to the perfusion chamber (top panel). Following the perfusion, the upper coverslip of the perfusion chamber was slid sideways (thick arrow) with a tweezer to obtain an open surface, which enabled the micromanipulation of the sliding microtubules with a glass microneedle.

  • Fig. 2.
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    Fig. 2.

    Planar bending induced continuation of forward sliding and stoppage of sliding. (A) Sequential images taken from the original data of a microtubule (MT) showing continuation of forward (Fw) sliding after imposed planar bending. The numbers in the upper left corner of each panel are time (s) counted after the bending started, and the sliding direction of a microtubule is indicated by the arrows. The position of the tip of a glass microneedle used for bending is indicated by arrowheads in the panels in which bending was performed. The microtubule moved from the upper right corner to the left, and changed the moving track slightly downwards after imposed bending (0.8 s and thereafter). The bottom panel shows the superimposed tracings of the microtubule before (interrupted line) and after (blue continuous line) imposed bending (0 and 0.8 s), and red arrows indicate three kinds of external strain that are estimated to be imposed on the microtubule and dyneins with planar bending: lateral strain (a), pulling strain (b) and elastic strain (c). Scale bars, 10 µm. (B) Sequential images of a microtubule showing stoppage of microtubule sliding induced by planar bending. This microtubule moving towards the upper left direction showed stoppage when the posterior region was bent (0.6–0.9 s, and the bottom illustration), and became stationary (0.9–10.5 s). The arrowhead marks the position of the tip of the glass microneedle at 0.6 s. Scale bar, 10 µm.

  • Fig. 3.
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    Fig. 3.

    Decrease in sliding velocity induced by planar bending. (A) Sequential images of a microtubule (indicated with blue) that showed decrease in sliding velocity in response to planar bending. Two sequential images with an interval of 3.2 s, which was used for measuring displacement (path distance), are shown for each of the three bending states: before, during and after bending. The arrows show the anterior end of the microtubule. Fw, forward sliding. Scale bar, 10 µm. (B) The time course of the change in path distance of the anterior end of the same microtubule shown in A. The sliding velocity, which is equivalent to the slope of this plot, was decreased during bending, and recovered after bending. The arrowheads represent examples of passive motions of the anterior end caused by movements of the glass microneedle during bending of the microtubule. The periods shown by the asterisks were used for calculation of average sliding velocities plotted in C. (C) The average sliding velocities before bending, during bending, and after the bend dissolved, are plotted for nine microtubules. They showed decrease in velocity by bending. Each set of three dots connected by a continuous line indicates data from a single microtubule, and the open circles indicate the data from the microtubule shown in A and B. (D) The percentages of decrease in average sliding velocities, calculated from the average sliding velocities before bending and during bending, are plotted against the bend angles (θ) for the nine microtubules (MT). The definition of the bend angle θ is shown in the inset. Each filled circle represents a datum from a single microtubule and the open circle represents the datum from the microtubule shown in A and B.

  • Fig. 4.
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    Fig. 4.

    Behaviour of the microtubules that showed stoppage by planar bending of the first manipulation. Most of the microtubules (N=106/136) that became stationary by planar bending recovered sliding movement spontaneously (filled box) or by the second bending (grey box). The relative frequency of recovery of sliding to the number of bending-induced stoppage was approximately 80%. Remaining cases were microtubule severing (hatched box) and no further response (open box). N, number of trials of imposed bending.

  • Fig. 5.
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    Fig. 5.

    Recovery of sliding in microtubules that showed stoppage. (A) Sequential images of a microtubule that showed spontaneous recovery of sliding after stoppage. The anterior region, which stopped sliding in response to planar bending and became stationary between 2.7 and 11.4 s, recovered sliding at 11.4 s. Fw, forward sliding. Scale bar, 10 µm. (B) Distribution of the durations of stoppage induced by mechanical manipulation. The data are from the experiments with 150 µg ml−1 21S dynein from sperm of Pseudocentrotus depressus in assay buffer with an ATP regeneration system. N, number of microtubules.

  • Fig. 6.
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    Fig. 6.

    Backward strain is important for induction of stoppage. (A) Effect of regional difference of bending on the relative frequency of occurrence of stoppage; data from the experiments using 150 µg ml−1 21S dynein obtained from Pseudocentrotus depressus and assay buffer with an ATP regeneration system are shown. N, number of manipulations; *P<0.03, chi-squared test. (B) Effect of the backward displacement of the anterior region on the relative frequency of occurrence of stoppage; data from the experiments using 150 µg ml−1 21S dynein obtained from Pseudocentrotus depressus and assay buffer with an ATP regeneration system are shown. **P=1×10−5<0.01, chi-squared test. (C) Backward pushing without bending also induced stoppage. Sequential images of a microtubule that showed stoppage in response to backward pushing without bending are shown. The numbers in the upper left corner of each image indicate the time (seconds) after the beginning of backward pushing. The sliding microtubule (−4.6 to 0 s) was pushed backwards, causing head-on collision with the glass microneedle at 0–0.4 s, and became stationary (0.4–30.4 s). The positions of the anterior end of the microtubule before and after backward pushing are indicated by open and filled arrowheads, respectively. Fw, forward sliding. Scale bar, 10 µm. (D) Sequential images of a microtubule that showed stoppage in the anterior region in response to imposed bending. The microtubule was bent in the middle region (0–5.7 s) and only the anterior region of the microtubule showed stoppage, while the rest continued forward sliding (5.7–14.4 s). A sliding region and a stationary region of the microtubule are indicated in blue and red (at 5.7 and 10.1 s), respectively. Just before the stoppage of the anterior region, the anterior region was pulled backwards by the glass microneedle (5.2–5.7 s). Scale bar, 10 µm. (E) Diagram showing superimposed tracings of the same microtubule in D before and after the backward pulling of the anterior region. The blue dotted line indicates the microtubule at 5.2 s, and the blue and red continuous lines indicate the regions of the microtubule sliding and being stationary at 5.7 s.

  • Fig. 7.
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    Fig. 7.

    Backward sliding was induced by continuous backward strain. (A) Sequential images of a microtubule that showed backward sliding (Bw) in the anterior region in response to continuous bending in the posterior region. The microtubule sliding towards the lower left in the panel before bending (at −5.0 s) changed its moving direction towards upper right when the posterior region was bent. Such backward sliding of the anterior region of the microtubule at 0.7–13.7 s was induced while the glass microneedle was kept attached to the microneedle without movement of the microneedle. After the backward movement for 13 s, the anterior region of the microtubule changed its shape with decrease in the curvature of the bend (13.7–46.1 s). Finally the whole microtubule recovered forward sliding (Fw) (at 50.5 s). Scale bar, 10 µm. (B) Superimposed tracings of the same microtubule shown in A (0.7–13.7 s). The arrowhead shows the position of the tip of the glass microneedle. The arrows show the positions of the anterior end of the microtubule and the open arrow indicates the direction of backward sliding. (C) Sequential images of a microtubule showing the backward sliding over the whole length. The microtubule was bent in the posterior region, which induced backward sliding. The glass microneedle kept attached to the microtubule seemed to slowly move towards the lower direction in the panel (1.9–74.9 s, the tip of the glass microneedle indicated by the arrowheads). The microtubule recovered forward sliding (original direction of movement) at 74.9 s. Scale bar, 10 µm. (D) Superimposed tracings of the same microtubule shown in C. Lines in black, blue, red and an interrupted line show, respectively, microtubule during the backward sliding at 1.9, 28.6, and 74.9 s and during the forward sliding at 81.3 s. The arrows indicate the positions of the both ends of the microtubule, and the open arrows indicate the direction of sliding.

  • Table 1.
  • Fig. 8.
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    Fig. 8.

    Three-dimensional bending of the sliding microtubule caused dissociation. Sequential images of a microtubule (MT) that showed dissociation in response to imposed bending (upper panels obtained from the record) and their schematic illustrations (lower panels). The glass microneedle was inserted into the space between the sliding microtubule and the glass surface, and resulted in a part of the microtubule being gradually blurred and difficult to focus on. Finally the whole length of the microtubule was out of focus. This indicates that the microtubule completely dissociated from the dynein-coated glass surface. Fw, forward sliding. Scale bar, 10 µm.

  • Fig. 9.
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    Fig. 9.

    Schematic diagrams showing the model of the regulation of dynein activities mediated by external strain. (A) Schematic depiction of the elastic strain imposed on dynein in the interbend region of a beating axoneme. Black arrows show the directions of active sliding, which mainly takes place in the bend regions to promote propagation of the bends. The upper panel shows a schematic depiction of an axoneme propagating alternate bends from the proximal (left) to the distal regions (right). As the bends propagate, dyneins in one bend region (see the middle bend, for example) eventually reach the straight interbend region (see the region indicated by the dotted rectangle), where elastic strain (red arrows) caused by the proximal bend of the axoneme is imposed on dyneins, and the reversal of the sliding direction occurs. (Note that in the leftmost bend the direction of the active sliding is the opposite to that of the middle bend.) The lower right panel shows the cross-section of the axoneme, viewed from the proximal side. The interdoublet sliding in the middle bend shown in the upper panel is thought to be produced by dyneins on the doublet number 7 (the red rectangle in the lower right panel). The lower left panel shows the directions of the active sliding (black arrows) and of the elastic strain (red arrows) imposed on the doublets 7 and 8 in the region shown by the dotted rectangle in the upper panel. (B) Schematic depiction of the backward strain that is important in inducing stoppage and backward sliding in the present experimental system. The direction of the external strain (the red arrow) relative to the direction of the active sliding (the black arrow) is the same as that of the elastic strain in the interbend region of an axoneme (A).

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Keywords

  • Mechanical signal
  • Self-regulatory response
  • Axonemal dynein

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Research Article
Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella
Hiroshi Yoke, Chikako Shingyoji
Journal of Experimental Biology 2017 220: 1122-1134; doi: 10.1242/jeb.147942
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Research Article
Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella
Hiroshi Yoke, Chikako Shingyoji
Journal of Experimental Biology 2017 220: 1122-1134; doi: 10.1242/jeb.147942

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