Figure 1.
Figure 1. F-actin and GM1 colocalize in microdomains formed at exocytotic sites. (A) Chromaffin cells were unstimulated (U) or stimulated for 10 min with nicotine (S) in the presence of anti-DBH antibodies and Alexa Fluor 633–conjugated cholera toxin. Cells were then fixed and labeled with TRITC-phalloidin and Alexa Fluor 488–conjugated anti–rabbit antibodies to reveal DBH staining. Merged images were recorded in the same optical section. Bars, 10 µm. Masks representing the colocalization area (phalloidin/cholera toxin or phalloidin/DBH) were generated by selecting the double-labeled pixels. (B) Semiquantitative analysis of the percentage of F-actin colocalized with GM1 and exocytotic sites. Asterisks indicate statistical significance (***, P < 0.001) for medians (black line) determined using a Mann-Whitney test and the white line represents the mean (±SEM; n = 20). Similar results were obtained on two culture preparations. (C) Distribution of GM1 and actin on plasma membrane sheets visualized by immunogold labeling and electron microscopy. Membrane sheets were prepared from untreated cells or cells stimulated with 20 µM nicotine in the presence of biotinylated cholera toxin to detect external GM1. Cells were treated with 50 µM latrunculin B where indicated. Membranes were then incubated with anti-actin antibodies revealed with anti–rabbit antibodies coupled to 10 nm gold particles and streptavidin coupled to 6 nm gold particles to reveal cholera toxin/GM1. Bars, 100 nm. (D) Histogram representing the relative distribution of 6 nm and 10 nm gold particles according to their distance from a granule (error bars indicate ±SEM; n = 40 images). The distance and number of particles were determined manually. Note that GM1-bound particles and actin-bound particles are concentrated within 0.1 µm from the granule edge. A significant number of actin-bound particles were located on the granules (<25 nm away). (E) Bivariate K-function analysis of double-labeled membranes. Images obtained in unstimulated (15) and nicotine-stimulated (20) cells were analyzed. Values of L(r)-r greater than the 95% CI indicated the significant coclustering of actin and GM1. (F) Spatial point pattern analysis of GM1 labeling. For each condition, 30 images were analyzed and experiments were performed on two different cell cultures. Values of L(r)-r greater than the 95% CI indicated significant GM1 clustering in nicotine-stimulated cells, whereas a random pattern was seen in untreated cells and nicotine-stimulated cells treated with latrunculin B.

F-actin and GM1 colocalize in microdomains formed at exocytotic sites. (A) Chromaffin cells were unstimulated (U) or stimulated for 10 min with nicotine (S) in the presence of anti-DBH antibodies and Alexa Fluor 633–conjugated cholera toxin. Cells were then fixed and labeled with TRITC-phalloidin and Alexa Fluor 488–conjugated anti–rabbit antibodies to reveal DBH staining. Merged images were recorded in the same optical section. Bars, 10 µm. Masks representing the colocalization area (phalloidin/cholera toxin or phalloidin/DBH) were generated by selecting the double-labeled pixels. (B) Semiquantitative analysis of the percentage of F-actin colocalized with GM1 and exocytotic sites. Asterisks indicate statistical significance (***, P < 0.001) for medians (black line) determined using a Mann-Whitney test and the white line represents the mean (±SEM; n = 20). Similar results were obtained on two culture preparations. (C) Distribution of GM1 and actin on plasma membrane sheets visualized by immunogold labeling and electron microscopy. Membrane sheets were prepared from untreated cells or cells stimulated with 20 µM nicotine in the presence of biotinylated cholera toxin to detect external GM1. Cells were treated with 50 µM latrunculin B where indicated. Membranes were then incubated with anti-actin antibodies revealed with anti–rabbit antibodies coupled to 10 nm gold particles and streptavidin coupled to 6 nm gold particles to reveal cholera toxin/GM1. Bars, 100 nm. (D) Histogram representing the relative distribution of 6 nm and 10 nm gold particles according to their distance from a granule (error bars indicate ±SEM; n = 40 images). The distance and number of particles were determined manually. Note that GM1-bound particles and actin-bound particles are concentrated within 0.1 µm from the granule edge. A significant number of actin-bound particles were located on the granules (<25 nm away). (E) Bivariate K-function analysis of double-labeled membranes. Images obtained in unstimulated (15) and nicotine-stimulated (20) cells were analyzed. Values of L(r)-r greater than the 95% CI indicated the significant coclustering of actin and GM1. (F) Spatial point pattern analysis of GM1 labeling. For each condition, 30 images were analyzed and experiments were performed on two different cell cultures. Values of L(r)-r greater than the 95% CI indicated significant GM1 clustering in nicotine-stimulated cells, whereas a random pattern was seen in untreated cells and nicotine-stimulated cells treated with latrunculin B.

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