QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Takao, D. Articles by Kamimura, S. Search for Related Content PubMed PubMed Citation Articles by Takao, D. Articles by Kamimura, S. Social Bookmarking                         What's this?

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

View larger version (42K): [in this window] [in a new window]   Fig. 1 Pseudo-color fluorescence images of calcein-loaded spermatozoa. Each image was obtained by averaging five frames (5 ms/frame). The resultant images of brightness maps are displayed in pseudo-color (white is the brightest and black is the faintest) to clarify the detail of photobleached area. The numbers shown above each image indicate the time course after the photobleaching laser pulse was applied (units: ms). The leftmost image was obtained just before photobleaching. The white circle indicates the region where the photobleaching laser pulse was directed. Scale bar, 10 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 2 Recovery curves in FRAP experiments. Fluorescence intensities normalized by the initial value just before photobleaching were plotted against time. The data points and error bars indicate the mean ± s.d. For simplicity, error bars are indicated only for every five points. Theoretical curves (solid lines) obtained by fitting data to Eqn 1 are superimposed. Arrows indicate the time points of photobleaching. (A–C) Recovery curves of flagella loaded with (A) calcein, (B) carboxyfluorescein and (C) Oregon Green (N=6). (D) A typical curve of FRAP in aqueous solution in the case where calcein in 10 mmol l–1 HEPES (pH 7.0) and 20% glycerol (N=3) was used. (E) Fluorescence recovery in heads (open squares) and concomitant fluorescence decrease of whole flagella (open circles) when a photobleaching laser pulse was directed to the head region (N=5). Note that the data are shown with a longer time scale than for the other four.

View larger version (7K): [in this window] [in a new window]   Fig. 3 Diffusion coefficients in aqueous solutions of various viscosity. Reciprocals of obtained diffusion coefficients versus viscosity. As expected from the Einstein–Stokes equation, they were linear relationships. The results of least-squares linear regression are shown by the broken lines. For the regression, data points at 10.8 mPa s were eliminated (see text for detail). The data points and error bars indicate the mean ± s.d. (N=3).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Simulated profiles of the concentrations of intraflagellar materials (solid lines) and dynein ATPase activity (broken lines; relative values) along flagella. The simulation was executed based on the model by Tombes et al. (Tombes et al., 1987). The lengths of flagella were set to 40 µm (A,C) and 100 µm (B,D). The ATP diffusion coefficients of 150 (A,B) and 60 µm2 s–1 (C,D) were used. The diffusion coefficients of other molecules are shown in Table 2. In A and B, where we used D=150 µm2 s–1, we obtained almost the same results as those previously reported by Tombes et al. (Tombes et al., 1987).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter