Ishijima S (2016), Self-Sustained Oscillatory Sliding Movement of ...

ECB-ART-44498

PLoS One 2016 Jan 01;112:e0148880. doi: 10.1371/journal.pone.0148880.

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

Ishijima S .

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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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Genes referenced: irak1bp1 LOC100887844 LOC115925415

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	Fig 1. The oscillatory sliding movement of the doublet microtubules of bull sperm models.(A) Phase-contrast video micrographs showing the oscillatory sliding movement of the doublet microtubules extruded by synchronous sliding. (B) Phase-contrast video micrographs showing the oscillatory sliding movement by metachronal sliding. The free-Ca2+ solution concentration was adjusted to 10−9 M. Time interval between successive images is 1.28 s in (A) and 0.64 s in (B). The profiles of the microtubule sliding displacement of A and B are shown in (C) and (D), respectively. The sliding velocity of the synchronous sliding movement was 3.02 μm/s, which was calculated between frames 1 and 2 in A (C). The sliding velocity of the metachronal sliding movement was 1.23 μm/s between frames 1 and 3 in B (D). Filled circles in C and D are the values of the sliding displacement obtained from the frames shown in A and B. Bars = 10 μm.
	Fig 2. The oscillatory sliding movement of the doublet microtubules having a different configuration of bull sperm models.Phase-contrast video micrographs showing the oscillatory sliding movements of the doublet microtubules extruded by synchronous sliding. The free-Ca2+ solution concentration was adjusted to 10−9 M. Time interval between successive images is 0.75 s in (A) and 0.72 s in (B). (A and B) The same spermatozoon. The oscillatory sliding movement of a fiber having a strophoid curve (A). After the oscillatory sliding movement of the fiber for approximately 5 minutes, a new fiber was extruded from the axoneme and then began its oscillatory sliding movement (B). Bar = 10 μm.
	Fig 3. Coordination between the synchronous and metachronal sliding movements of the doublet microtubules of bull sperm models.(A and B) Phase-contrast video micrographs showing two distinct oscillatory sliding movements of the doublet microtubules by synchronous and metachronal sliding on a flagellum. Free-Ca2+ solution concentration was adjusted to 10−9 M. Time interval between successive images is 0.70 s in (A) and 0.29 s in (B). The profiles of the microtubule sliding displacement of A are shown in (C). The profiles of two cycles of the microtubule sliding displacement of B are shown in (D). Sliding velocity was 5.58 μm/s between frames 1 and 3 in A for the synchronous sliding movement, and 0.40 μm/s between frames 3 and 5 in A for the metachronal sliding movement (C). Circles represent values of the sliding movement of the doublet microtubules extruded by the synchronous sliding, whereas the values shown as squares represent the metachronal sliding. Filled circles and squares in C and D are values of the sliding displacement obtained from the frames shown in A and B. Bars = 10 μm.
	Fig 4. The association of microtubule sliding with flagellar bending of bull sperm models.(A) Phase-contrast video micrographs showing oscillatory synchronous and metachronal sliding and flagellar bending. The first cycle of flagellar bending is shown. The free-Ca2+ solution concentration was adjusted to 10−9 M. Time interval between successive images is 0.15 s. The beat frequency of flagellar bending was 1.2 Hz. (B) The profiles of five cycles of the sliding displacement of synchronous and metachronal sliding movements and of the transverse displacement of the flagellum shown in A. The transverse displacement of sperm flagellum from the sperm head axis (d) was measured at a position 15 μm from the head-tail junction (A). Bar = 10 μm.
	Fig 5. The association of microtubule sliding with flagellar waveform of bull sperm models.(A) Phase-contrast video micrographs showing oscillatory synchronous and metachronal sliding and flagellar bending. A left-handed helical bend at a distal region of the flagellum was determined by differential focusing: the flagellar parts were focused on the higher focal plane. The free-Ca2+ solution concentration was adjusted to 10−9 M. Time interval between successive images is 0.94 s. The beat frequency of flagellar bending was 0.18 Hz. (B) The profiles of the sliding displacement of synchronous and metachronal sliding movements shown in A. Circles represent the synchronous sliding values and squares the metachronal sliding values. The bar denoted by letters “2D” expresses the duration of the planar flagellar bending. Filled circles and squares in B are values of the sliding displacement obtained from the frames shown in A. Bar = 20 μm.
	Fig 6. Hypothetical diagrams explaining flagellar bend formation from two distinct oscillatory microtubule sliding events.(A) Synchronous sliding between the doublet Nos. 7 and 8 generates a large fiber loop of the doublet microtubule Nos. 4–7 at the midpiece and forms an asymmetrical planar bend. The sliding displacement of the synchronous sliding is dependent on the Ca2+ concentration. Higher Ca2+ concentrations produce larger sliding displacements [1,2,4]. (B) Metachronal sliding between the doublet Nos. 3 and 4 retracts the extruded fiber of the doublets Nos. 4–7 into the flagellum, followed by metachronal sliding between the doublets Nos. 2 and 3 in two loci on the flagellum. The sliding at the base of the midpiece generates a small fiber loop for the doublets Nos. 9–2 and the sliding at the distal region of the flagellum begins to induce metachronal sliding between the doublets Nos. 1 and 2. (C) Metachronal sliding propagating towards the tip and simultaneously transferring in the sequence of doublets Nos. 1, 9, 8, and 7 leads to the generation of a left-handed helical bend at a distal region of the flagellum at low Ca2+ concentrations [1,8,9]. The numbers in the circles indicate the number of the doublet microtubules according to Afzelius [11]. The numbers indicate the sliding of corresponding doublet microtubules. The synchronous sliding superimposed on the metachronal sliding converts a helical bend into a half-funnel shape.
	Fig 7. Sliding disintegration of elastase-treated axonemes of bull sperm models.A phase-contrast video micrograph showing sliding disintegration of an elastase-treated bull sperm axoneme. Elastase of 20 μg/ml was added to the reactivation solution. The free-Ca2+ solution concentration was adjusted to 10−9 M. Bar = 10 μm.
	Fig 8. The spontaneous reversal of the sliding direction of the doublet microtubules of sea urchin sperm axonemes.(A) Dark-field video micrographs showing the sliding movements of the doublet microtubules. The sea urchin sperm axoneme still beats in the first frame. The microtubule sliding was induced with 0.5 mM MgATP2- and 20 μg/ml elastase. The numbers indicate the microtubule sliding events that occur on a flagellum. The free-Ca2+ solution concentration was adjusted to 10−9 M. Time interval between successive images is 0.25 s. (B) The profiles of microtubule sliding displacement shown in A. The numbers correspond to those of the microtubule sliding shown in A. The circles represent values of large sliding displacements and squares represent small sliding displacements. The sliding velocity was 31.9 μm/s, as calculated between frames 1 and 5 of the sliding No. 1 and 24.5 μm/s between frames 1 and 3 of the sliding No. 2. Filled circles and squares are values of the sliding displacement obtained from the frames shown in A. Bar = 15 μm.

References [+] :

AFZELIUS, Electron microscopy of the sperm tail; results obtained with a new fixative. 1959, Pubmed, Echinobase