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First published online October 31, 2008
Journal of Experimental Biology 211, 3594-3600 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.021923
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FRAP analysis of molecular diffusion inside sea-urchin spermatozoa

Daisuke Takao and Shinji Kamimura*

Department of Life Science, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan

* Author for correspondence at present address: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo 112, Japan (e-mail: kam{at}myad.jp)

Accepted 18 September 2008

In sea-urchin spermatozoa, energy required for flagellar motility is provided by ATP diffusion from mitochondria located at the proximal ends of the flagella along with the creatine shuttle system. However, no direct analysis of the diffusion rates inside flagella has been carried out thus far. Using a FRAP (fluorescence recovery after photobleaching) technique, we determined the diffusion coefficients of fluorescein-derivatives (calcein, carboxyfluorescein and Oregon Green) to be 63–64 µm2 s–1. Although these values are about one third of the estimates that were previously used for theoretical calculations, we concluded that the rate of ATP diffusion inside spermatozoa was high enough to support the continuous motility of sea-urchin sperm flagella if the creatine shuttle system is working. We also investigated the diffusion rate through the `neck' region between the head and tail. When the head region of a calcein-loaded spermatozoon was photobleached, slow recovery of head fluorescence along with the decrease of fluorescence signal in the tail region was observed. It suggests that small molecules such as calcein (Mr, 622.54) can move beyond the boundary between the head and the flagellum. We expect that these findings regarding the diffusion properties inside spermatozoa will provide us with more general insights into the energy equilibrium and material transportation by passive diffusion inside eukaryotic cilia and flagella.

Key words: FRAP, diffusion constants, intra-flagellar transport, molecular mobility


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