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First published online March 17, 2006
Journal of Experimental Biology 209, 1336-1343 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02131
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Requirement of the fixed end for spontaneous beating in flagella

Miki Fujimura and Makoto Okuno*

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan


Figure 1
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  		Fig. 1. Relationship between UV intensity and irradiation period for complete suppression of reactivated flagella. UV irradiation covered the entire flagellum and flagellar movement stopped completely within this period. The intensity of UV irradiation was defined as a relative value. Irradiation without the ND filter was defined as 100% (see Materials and methods). The concentration of PRODAN was 0.02 µmol l–1 (diamonds), 1 µmol l–1 (squares), 5 µmol l–1 (triangles) and 10 µmol l–1 (crosses).

 

Figure 2
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  		Fig. 2. Dark-field micrographs of flagella treated with PRODAN-UV `locally' in the presence of ATP and trypsin. Limited disintegration of flagella was observed in the region without PRODAN-UV treatment. (A) Basal region of the flagellum treated with PRODAN-UV (the region between two arrowheads). (B) Distal region of a flagellum treated with PRODAN-UV (the region between two arrowheads). The arrow marks microtubules that came out loose.

 

Figure 3
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  		Fig. 3. Dark-field micrographs (A–C) and shear curves (D–F) of reactivated sea urchin sperm flagella. ATP concentration was 15 µmol l–1. (A) Dark-field micrographs of symmetrical bending waves of the control sperm flagellum before UV irradiation. Beat frequency was 4.8 Hz. Bar, 10 µm. (B) Dark-field micrographs of `proximal region inhibition step 1'. Approximately 5 µm of the proximal region was inhibited (between the two arrowheads). Beat frequency was 5.0 Hz. Bar, 10 µm. (C) Dark-field micrographs of `proximal region inhibition step 2'. Approximately 12 µm of the proximal region was inhibited (between the two arrowheads). Beat frequency was 8.0 Hz. Bar, 10 µm. (D–F) Results obtained from the series of seven frames in A–C are superimposed in D–F, respectively. Every shear curve represents every 1/30 s. (D) Shear curves corresponding A. (E) Shear curves corresponding B. (F) Shear curves corresponding C.

 

Figure 4
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  		Fig. 4. Relationship between beat frequency and `movable length' of the flagellum. The flagellum was inhibited in its proximal region in the presence of 10 µmol l–1 ATP.

 

Figure 5
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  		Fig. 5. UV irradiation of flagella reactivated with a high concentration of ATP (0.2 mmol l–1). Shear curves from seven successive frames are superimposed. Every curve represents every 1/60 s. (A) Distal region (approximately 10 µm) inhibited. (B) Proximal region (approximately 10 µm) inhibited.

 

Figure 6
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  		Fig. 6. Effect of cAMP on flagella inhibited by PRODAN-UV at the basal region (5–10 µm) in various concentrations of ATP and cAMP. MgATP concentrations were 10 µmol l–1, 200 µmol l–1 and 1 mmol l–1. cAMP concentrations were 0 µmol l–1, 5 µmol l–1, 50 µmol l–1 and 200 µmol l–1. Key: move, intact distal portion shows sinusoidal, vigorous movement; tremble, intact portion does not show sinusoidal movement but does show slight vibration; stop, intact portion shows no movement. N=20 flagella examined for each condition.

 

Figure 7
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  		Fig. 7. Spontaneous beating of basal region dissected flagella. A head-attached, reactivated spermatozoon was treated with PRODAN-UV at the basal region of flagellum. Then, the PRODAN-UV treated basal region (between the arrows) was dissected out by a microglass needle. The dissected flagellum continued to show sinusoidal wave movement without a head and base of flagellum. This experiment was repeated more than 20 times. ATP concentration was 15 µmol l–1. Bar, 10 µm. (A) Micrograph 1, the inhibited reactivated flagellum before its head was dissected out. Micrograph 2, the process of dissection. Micrograph 3–7, phase-contrast micrographs of the reactivated flagellum whose basal region (between two arrows) was PRODAN-UV treated and dissected out. Each micrograph represents 1/30 s. (B) Superimposed shear curves of the panels in A.

 




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