FRAP analysis of molecular diffusion inside sea-urchin spermatozoa
FRAP analysis of molecular diffusion inside sea-urchin spermatozoa
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DOI：
10.1242/jeb.021923
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发表时间：
2008-11-15
影响因子：
2.8
通讯作者：
Kamimura, Shinji
中科院分区：
文献类型：
--
作者：
Takao, Daisuke;Kamimura, Shinji
在海胆精子中，鞭毛运动所需的能量由位于鞭毛近端的线粒体以及肌酸穿梭系统的 ATP 扩散提供。然而，迄今为止尚未对鞭毛内部的扩散速率进行直接分析。使用FRAP（光漂白后荧光恢复）技术，我们确定荧光素衍生物（钙黄绿素、羧基荧光素和俄勒冈绿）的扩散系数为63-64 μ m(2) s(-1)。尽管这些值大约是之前用于理论计算的估计值的三分之一，但我们得出的结论是，如果肌酸穿梭系统正常工作，精子内部 ATP 扩散速率足够高，足以支持海胆精子鞭毛的持续运动。我们还研究了通过头部和尾部之间的“颈部”区域的扩散速率。当负载钙黄绿素的精子的头部区域被光漂白时，观察到头部荧光缓慢恢复以及尾部区域荧光信号的减少。这表明钙黄绿素（M-r，622.54）等小分子可以移动到头部和鞭毛之间的边界之外。我们期望这些关于精子内部扩散特性的发现将为我们提供对真核纤毛和鞭毛内部被动扩散的能量平衡和物质运输的更一般的见解。
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 mu m(2) 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 (M-r, 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.