Single-cell electroporation of fluorescent probes into sea urchin sperm cells and subsequent FRAP analysis.
In sea urchin spermatozoa, the energy required for flagellar motility depends only on the diffusional supply from proximal mitochondria, and thus the diffusion rate inside flagella is one of the most crucial factors limiting the practical size and design of the motile machinery. To determine the diffusion rates of materials inside sperm cells, FRAP (fluorescence recovery after photobleaching) analysis of incorporated fluorescent probes is one of the most powerful approaches. However, the only practically possible method until now was to use the ester forms of fluorescence, and our choice was limited to those of relatively small molecular masses, such as fluorescein derivatives. In this report, we show that a modified single-cell electroporation technique can be applied as a new microinjection method for sperm cells of the sea urchin. The method was applied to FRAP analysis to determine the rate of intraflagellar diffusion.
Takao
Daisuke
D
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan.
Kamimura
Shinji
S
D000818:Animals; D018274:Electroporation / Q000379:methods*; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D008297:Male; D012617:Sea Urchins / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*
D005456:Fluorescent Dyes