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1.
Figure 2

Figure 2. Release of ANF-EmGFP, and localization within the actin cytoskeleton.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

(A) 3D reconstruction of PC12 cell expressing ANF-EmGFP, before and after stimulation. Voxel dimensions are 108×108×200 nm. (B) Timelapse series showing a cell expressing ANF-EmGFP taken at 20 s intervals. (C) 3D reconstruction of PC12 cell co-expressing ANF-EmGFP and β-actin -mCherry. (D) Extended focus view of cell co-expressing ANF-EmGFP and β-actin -mCherry.

Jie Wang, et al. PLoS One. 2011;6(12):e29162.
2.
Figure 1

Figure 1. Comparison of staining with phalloidin, GFP-utrophin, and β-actin mCherry.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

(A) Fixed cell stained with alexa-fluor phalloidin. Note prominent staining of thick actin filamenents, while thin filaments are lightly stained. (B) Live cell, co-expressing Utrophin-GFP and β-actin -mCherry. Utrophin which only binds to fully stable actin filaments shows similar staining to that seen with phalloidin. β-actin mCherry labels all actin (monomer and filamentous, stable and dynamic). Area marked by white rectangle is shown in (C) at higher magnification, with the channels shown separated and overlaid.

Jie Wang, et al. PLoS One. 2011;6(12):e29162.
3.
Figure 5

Figure 5. Actin facilitates expulsion of vesicular contents.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

(A) Example of individual exocytic event for vesicles labeled with either ANF-EmGFP, or Mepacrine, with the falling phase highlighted by the dashed box. (B) 50 events from 5 cells before and after stimulation were randomly chosen, based only on the criterion that they be clearly distinguished and separate from neighboring events (which tends to select for larger amplitude events also). The decay phase was aligned and averaged and is plotted here. Stimulation caused a marked enhancement of the rate of content expulsion. In latrunculin treated cells (C), when the same analysis was performed, the rate of content release was faster in unstimulated cells. (D) After jasplakinolide treatment the difference between unstimulated and stimulated rates of content expulsion disappeared.

Jie Wang, et al. PLoS One. 2011;6(12):e29162.
4.
Figure 7

Figure 7. Models for the role of actin in secretion.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

(A) Role of actin in regulating availability of vesicle for secretion. At rest (top left) vesicles are in three possible pools; arrested by the cortical actin cytomatrix (“docked and blocked”) associated with actin filaments communicating with sites of secretion, and freely diffusing in the cytoplasm. On stimulation (top right) vesicles held by the cytomatrix become available for secretion, as do vesicles delivered by actin fibers. Latrunculin treatment (lower panel) disassembles much of the cytomatrix, allowing vesicles to spontaneously fuse at a higher rate, but impairing the ability of new vesicles to arrive at sites of secretion, due to loss of connecting actin filaments, leading to a reduced ability to sustain secretion following stimulation. (B) At the site of fusion itself, actin appears to slow the release of vesicular contents; this action is overcome by either latrunculin treatment or stimulation.

Jie Wang, et al. PLoS One. 2011;6(12):e29162.
5.
Figure 4

Figure 4. Effect of latrunculin and jasplakinolide on vesicle recruitment and secretion.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

(A) Visualized ANF-EmGFP vesicles can be observed arriving and fusing at the plasma membrane. (B) To the left, example ROI traces are shown before and after depolarization with 30 mM K+. To the right, release from 6 cells was analyzed before and after stimulation. Release was normalized as events per 10 µm2, per 10 s. (C) 6 cells were treated with latrunculin A; example ROI traces are shown to the left, and pooled data to the right. Latrunculin treatment caused an increase in spontaneous vesicular secretion; stimulation increased this further, however, this additional release fatigued rapidly (right). (D) 7 cells were treated with Jasplakinolide (10 µM), again, example ROI traces are shown on the left, and time binned averages on the right. Jasplakinolide treatment slightly reduced spontaneous rates of exocytosis, and prevented any significant increase following stimulation. Scale bar shows 10 s/10 arbitrary fluorescence units (directly comparable between conditions).

Jie Wang, et al. PLoS One. 2011;6(12):e29162.
6.
Figure 6

Figure 6. Separation between the actin cytoskeleton and exocytic site.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

(A) TIRFM image of β-actin -mCherry (red) overlaid with a summed intensity projection of the ANF-EmGFP image series. Note that green spots, corresponding to sites of secretion, do not align with regions of actin enrichment at the membrane. (B) Plot of summed GFP intensity against mCherry intensity, illustrating the negative relationship. (C) A third method of assessing the relationship between β-actin and site of secretion is to segment β-actin images into regions above and below 50% intensity, and count the number of events within each. Data are expressed as % of total events per cell, and are the average of 6 experiments. *** indicates p<0.001. (D) TIRFM image of a cell expressing β-actin -mCherry. Box indicates region of cell highlighted in B–F. (E) Wide field image of part of cell before and (F) after treatment with latrunculin, with equal image scaling. (G) TIRFM image of β-actin with summed GFP TIRF image, before and (H) after latrunculin treatment. (I) If the pre-latrunculin β-actin image is combined with the post-treatment image of secretion (summed ANF-EmGFP), it can be seen that secretion occurs at sites that were previously unavailable. (J) Pre-latrunculin intensity of β-actin labeling within ROIs used for secretory analysis, plotted against the number of events seen within those regions, showing a negative correlation. (K) this relationship disappears after latrunculin treatment. (L) Relative change in β-actin intensity before and after latrunculin treatment is plotted against change in number of events for the same regions.

Jie Wang, et al. PLoS One. 2011;6(12):e29162.
7.
Figure 3

Figure 3. Effect of Latrunculin A on secretion from PC12 cells (A) PC12 cell co-expressing ANF-EmGFP and β-actin -mCherry.. From: Spatial Regulation of Exocytic Site and Vesicle Mobilization by the Actin Cytoskeleton.

Times indicated reflect time after latrunculin addition. (B) Pooled data from 8 cells, measuring fluorescence intensity within a region corresponding to the outer 25% of the cell, in the presence (red) and absence (black)of Latrunculin A. Data are mean ± sem. (C) Rate of fluorescence loss, corresponding to secretion of ANF-EmGFP, in cells with (solid symbol) and without (open symbol) stimulation. In control cells (black) showed a biphasic release curve that could be fitted to a double exponential. Treatment with latrunculin A (red) caused opposite effects on the two phases of release; speeding the initial component, and slowing the second. Data are from 12 cells, mean ± sem. (D) If unstimulated curves were subtracted from the data corrected curves can be obtained. If these are then fitted, tau values for exponential fits are as shown. Control cells do not fit well to a single exponential (red), but are well described by a double exponential (blue). Latrunculin treated cells (lower) on the other hand are well fit by a single exponential (red). Unsubtracted numbers are Con; τ1 = 2.8 s, τ2 = 38.1 s; Lat A τ1 = 2.2 s, τ2 = 168.8 s. (E) Amplitude of fluorescence loss was calculated for 8 cells which were imaged twice; once before, and again after 2 min of stimulation. Latrunculin A caused a significant reduction (p<0.01) in the amount of fluorescence loss due to stimulation.

Jie Wang, et al. PLoS One. 2011;6(12):e29162.

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