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First published online 28 February 2006
doi: 10.1242/jcs.02824

Journal of Cell Science 119, 1071-1079 (2006)
Published by The Company of Biologists 2006
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Single-molecule analysis of chemoattractant-stimulated membrane recruitment of a PH-domain-containing protein

Satomi Matsuoka1, Miho Iijima2, Tomonobu M. Watanabe1, Hidekazu Kuwayama1, Toshio Yanagida1, Peter N. Devreotes2 and Masahiro Ueda1,*

1 Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan
2 Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., 114 WBSB, Baltimore, Maryland, 21205, USA


Figure 1
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  		Fig. 1. Translocation of Crac-GFP to membranes in D. discoideum cells after stimulation with cAMP. Images in A and C show one cell. (A) D. discoideum cells were stimulated at t=0 seconds with a uniform concentration of cAMP. Standard epifluorescence images were taken at 30 images per second. Before stimulation with cAMP Crac-GFP was distributed in the cytosol. After stimulation it translocated uniformly and almost entirely to the cell surface. (B) Time course of the Crac-GFP recruitment shown in A. Fluorescence intensities of Crac-GFP on membranes were measured and plotted over time. Fluorescence intensities were calibrated by using the photobleaching rates of GFP. (C) Localization of Crac-GFP at the leading-edge pseudopod of cells in gradients of cAMP.

 

Figure 2
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  		Fig. 2. Single-molecule imaging of Crac-GFP in living D. discoideum cells. (A) Configuration of a total internal reflection fluorescence microscope (TIR-FM) for single-molecule imaging attached to an inverted microscope (IX-70, Olympus Inc, Japan). (B) Schematic illustration of single-molecule imaging of Crac-GFP bound to the basal membranes of a D. discoideum cell that is illuminated by evanescent fields generated by TIR of excitation light on the surface of a coverslip. (C) Typical images of Crac-GFP visualized by TIR-FM (left panel). Fluorescent spots were hardly detected in a cell expressed GFP alone (right panel). (D) Histogram of fluorescence intensities of the spots of Crac-GFP observed in living cells (dark gray bars) and on the surface of coverslips (light gray bars). Each distribution was fitted to an Gaussian function. (E) An example of a single-step photobleaching of Crac-GFP on coverslips. (F) An intensity profile of a fluorescent spot representing a single Crac-GFP molecule attached to a coverslip. (G) An example of a single-step photobleaching of Crac-GFP in fixed cells. (H) An intensity profile of a fluorescent spot representing a single Crac-GFP molecule in a fixed cell. The background fluorescence was heterogeneous when compared to (F), which came from unbound Crac-GFP molecules present in the cytosol.

 

Figure 3
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  		Fig. 3. Single-molecule imaging of the transient translocation of Crac-GFP to membranes upon stimulation with cAMP. (A) Sequential images of a cell expressing Crac-GFP observed under TIR-FM (see supplementary material Movie 1). The cell was stimulated at 0 seconds with cAMP. (B) Temporal changes in the number of Crac-GFP spots on membrane. After stimulation with cAMP, the number of the Crac-GFP spots increased transiently ~10 second. (C) Dissociation of Crac-GFP from membranes of cells stimulated with cAMP. Lifetimes of Crac-GFP between initial observation and disappearance on membrane were measured and plotted as a cumulative curve, giving rise to the dissociation curve. The data can be fitted to single exponential curve with a constant ({tau}) of 120 milliseconds, indicating rapid exchanges of individual Crac molecules during Crac recruitments. Dissociation curve of Crac-GFP in living cells with {tau}=120 milliseconds (bullet) and photobleaching curve of Crac-GFP absorbed on the surface of coverslip with {tau}=3800 milliseconds ({blacktriangleup}).

 

Figure 4
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  		Fig. 4. Two binding sites for Crac-GFP in living cells. (A) Three states of cells; cells before stimulation, 0 to 20 seconds after stimulation, and more than 20 seconds after stimulation were referred as unstimulated, stimulated and adapted, respectively. (B) Dissociation curves of Crac-GFP and PHCrac-GFP bound to the membranes. Lifetime of Crac-GFP was measured in the cells before stimulation ({circ}), at 0-20 seconds ({triangleup}) and more than 20 seconds ({square}) after stimulation with cAMP. The curves were fitted to a sum of exponentials with the time constant {tau} and the relative ratios (see Results). The lines represent the fitting curves. Dissociation curve of PHCrac-GFP (bullet) bound to the membrane of resting cells demonstrates that the fast-dissociation site is PtdIns(3,4,5)P3.

 

Figure 5
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  		Fig. 5. Single-molecules of Crac-GFP in polarized cells undergoing chemotaxis. (A) Chemotactic cells expressing Crac-GFP were observed under TIR-FM (see supplementary material Movie 2). Crac molecules on membranes were predominantly observed at the leading edge pseudopod (white arrow), as expected from previous works (Parent et al., 1998Go). Arrowheads represent Crac-GFP found at the rear end of cells. The gray arrow indicates the direction of cell movement. (B) Dissociation curves of Crac-GFP bound to the pseudopod ({circ}) or posterior region ({triangleup}) were fitted to a single exponential curve with a constant of 110 milliseconds and a sum of two exponentials with constants of 110 milliseconds (72%) and 890 milliseconds (28%), respectively.

 

Figure 6
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  		Fig. 6. Membrane binding of Crac-GFP in cells lacking ACA. Dissociation curves of Crac-GFP in wild-type ({triangleup}) and aca-null cells ({circ}). Cells that lack ACA have only a fast-dissociation site but lack the slow-dissociation site for Crac-GFP, suggesting that the slow-dissociation site is either ACA itself or an ACA-dependent molecule on membranes.

 

Figure 7
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  		Fig. 7. Overview of the results presented here. (A) Behavior of Crac in cells uniformly stimulated with cAMP. In resting cells, Crac can bind PtdIns(3,4,5)P3 and ACA-dependent sites. Upon stimulation with cAMP, the amount of PtdIns(3,4,5)P3 is transiently increased by PI 3-K and PTEN, leading to the transient increments of Crac localisation on membranes and its rapid recycling. (B) Crac undergoing chemotaxis in polarized cells. PtdIns(3,4,5)P3 and ACA-dependent sites are localized at the pseudopod and the tail of cells, respectively. Since the dissociation of Crac from PtdIns(3,4,5)P3 is fast, cells can respond rapidly and flexibly to directional changes of gradients. Crac might transmit some signals by freely diffusing into the cytosol from a pseudopod to a tail where ACA is localized.

 

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