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First published online 4 March 2003
doi: 10.1242/jcs.00336

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Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes

Izumi Nakano, Takeshi Kobayashi^*, Misako Yoshimura and Chikako Shingyoji

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan
^* Present address: ERATO Kusumi Membrane Organizer Project, JST, Kumazaki bldg. 5-11-33 Chiyoda, Naka-Ku, Nagoya 460-0012, Japan

View larger version (39K): [in a new window]   Fig. 1. (A,B) Elastase-treated axonemes before (left) and after (right) ATP application. 20 µM ATP at <10–9 M Ca2+ induced more than two sliding events, causing sliding disintegration of axonemes into individual doublets (A), whereas 1 mM ATP at 10–4 M Ca2+ induced one sliding event that caused splitting of the axoneme into a pair of doublet bundles (B). The thinner bundle slid over the thicker bundle to the left (arrow in B). (C) Proportions of three types of sliding, categorized by the total number of sliding events (1, 2 and 3). Left, <10–9 M Ca2+, 20 µM ATP; middle, <10–9 M Ca2+, 1 mM ATP; right, 10–4 M Ca2+, 1 mM ATP.

View larger version (18K): [in a new window]   Fig. 2. Sliding velocities of elastase-treated axonemes at 1 mM ATP (0.6 mM MgATP). Distributions of the velocity at <10–9 M Ca2+ (A) and 10–4 M Ca2+ (B) were similar to each other, and there was no significant difference between the two.

View larger version (81K): [in a new window]   Fig. 3. Ultrastructural analysis of the effects of Ca2+ on four splitting patterns of the elastase-treated axonemes. (A) Electron micrographs showing the four patterns of the thicker bundles. Cross-sectional views of the axonemes are shown as seen from the base to the tip of the axonemes. Arrowheads in c and d indicate the 5-6 bridges. Scale bar, 50 nm. (B) Schematic diagrams of the thicker bundles corresponding to the electron micrographs shown in A with those of the thinner bundles obtained by the splitting. (C) Relative frequencies of eight splitting patterns of the doublet bundles that contained the central pair (CP) at <10–9-10–3 M Ca2+. Boxes from left to right for each concentration of Ca2+ indicate: one doublet with CP (grey boxes), 7-4 pattern (dark grey boxes), 7-3 pattern (dotted boxes), 8-4 pattern, 8-3 pattern, two doublets connected with a 5-6 bridge and with CP (open boxes), 4-8 pattern, and 3-8 pattern. Numbers (n) shown to the right of the columns are the numbers of cross-sections counted for each concentration of Ca2+. Data obtained from three experiments were averaged because there was no significant difference in the pattern frequency.

View larger version (118K): [in a new window]   Fig. 4. Sliding of a thicker bundle over a thinner bundle in an elastase-treated flagellar axoneme with a head. Dark-field micrographs of a sperm flagellum before (top) and after (bottom) induced sliding. A flagellum was cut in two with a glass needle (arrowhead, top) and treated with elastase. Application of 1 mM ATP at 10–3 M Ca2+ induced lengthwise splitting into two bundles with the thicker bundle sliding to the right (bottom, arrow) (i.e. away from the head), leaving a thinner bundle attached to the glass surface.

View larger version (80K): [in a new window]   Fig. 5. Sliding of singlet microtubules along the thinner (A) and the thicker (B) bundles. (A) <10–9 M Ca2+. (B) 10–5 M Ca2+. Similar sliding was observed both on the thinner and the thicker bundles regardless of the concentration of Ca2+. The top panels in each case were recorded under stronger illumination to observe the paired bundles before singlet microtubules were added. The following three panels in each case, which were recorded under decreased illumination to record clearer image of singlet microtubules, are sequential images of the movements of singlet microtubules along the bundles. Arrowheads indicate the starting positions of the left ends of the singlet microtubules that interacted with the bundles (at 0 seconds). Arrows indicate the direction of sliding of the singlet microtubules.

View larger version (17K): [in a new window]   Fig. 6. (A) Behaviour of microtubules on thinner (Thin) and thicker (Thick) bundles at various concentrations of Ca2+. The microtubules that attached to the bundles showed sliding movement (solid boxes), back-and-forth movement (hatched boxes) or no movement (open boxes). The numbers of bundles with which microtubules interacted are shown in the right-hand column. (B) The ratio of the number of occurrences of microtubule sliding on the thinner bundles to that on the thicker bundles at various concentrations of Ca2+.

View larger version (13K): [in a new window]   Fig. 7. Time courses of microtubule sliding movement at 1 mM ATP in the low (A) and high (B) concentrations of Ca2+. The filled circles and crosses indicate the microtubules that slid along thinner and thicker bundles, respectively.

View larger version (47K): [in a new window]   Fig. 8. Distribution of microtubule sliding velocities on thinner (left) and thicker (right) bundles at 1 mM ATP (0.9 mM MgATP) and low (A) and high (B) concentrations of Ca2+. The sliding velocities are shown at <10–9 M Ca2+(open boxes), 10–7 M Ca2+ (filled boxes), 10–5 M Ca2+ (grey boxes) and 10–4 M Ca2+ (dotted boxes). Mean sliding velocities ± s.d. (n=number of microtubules) are shown in each graph. Velocities of sliding between the two doublet bundles (Fig. 2) were larger than those of microtubule sliding on bundles shown in this figure. This difference might be due to a different ionic strength between the reactivating solution (for Fig. 2) and the sliding assay buffer (for Fig. 8).

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