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First published online 19 August 2003
doi: 10.1242/jcs.00672

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Interaction of the actin cytoskeleton with microtubules regulates secretory organelle movement near the plasma membrane in human endothelial cells

¹ National Institute for Medical Research, London NW7 1AA, UK
² MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT, UK
³ Department of Neuroscience, University of Edinburgh, Edinburgh EH8 9YL, UK
⁴ Department of Pharmacology, UCL, London WC1E 6BT, UK
⁵ Department of Biomedical Sciences, Imperial College, London SW7 2AZ, UK

View larger version (25K): [in a new window]   Fig. 3. Long-range movements of WPb in HUVECs. (A) Long-range directed motions visualized by thresholding and accumulating frames from a sequence of 120 images. Bar, 10 µm. (B) Example of long-range directed motion. The three-dimensional trajectory is shown in the left-hand panels. Note the difference in scale between the z-axis and the x-y axes. The initial point (t=0) is indicated by an asterisk. The right-hand panel shows plots of vertical position z (solid line, left axis in µm) and velocity (dotted line, right axis in µm/second) as a function of time, with a rolling average of three frames. The WPb slows down as it approaches the plasma membrane (arrows) and accelerates as it moves away from the membrane (arrowheads). (C) WPb rotation. Plot of the angle of a rotating WPb as a function of time (anticlockwise rotation, frequency rot=0.40 per second). Top panels show the corresponding TIRF images (Bar, 1 µm). (D) WPb oscillations. Plot of the x-y velocity of an oscillating WPb as a function of time (oscillation frequency osc=0.14 per second).

View larger version (31K): [in a new window]   Fig. 4. Short-range motions of WPb in HUVECs. (A) Short-range diffusive motions visualized by averaging the same sequence as in Fig. 3A. Examples are shown of simple diffusion (WPb 1), directed diffusion (WPb 2) and restricted diffusion (WPb 3). The WPb x-y trajectories are given in the centre panels (x and y in µm). Three-dimensional MSD plots (right-hand panels) were fitted according to Eqs 1-3 (see Materials and Methods). The parameters deduced from the fits are: WPb 1, D=1.5 10-4 µm2/second; WPb 2, D=1.05x10-3 µm2/second, v=1.07x10-2 µm/second; WPb 3, D=1.0x10-4 µm2/second, Dcage=3.6x10-5 µm2/second, Rcage=49 nm. Bar, 10 µm. (B) Averages of simple diffusion (n=21), directed diffusion (n=11), and restricted diffusion (n=16) 3D MSD plots.

View larger version (80K): [in a new window]   Fig. 1. Visualization of secretory organelles in living HUVECs by TIRF microscopy. (A,B) Epifluorescence (EPI) and corresponding TIRF (TIRF) images of tPAGFP 4 hours after microinjection (A) and Rab27a-GFP 48 hours after microinjection (B). Details of the TIRF image show small diameter tPA-GFP vesicles (A) and tubular rod-shaped Rab27a-GFP organelles (B). Arrowheads indicate the Golgi region in epifluorescence images. (C) Rab27a-GFP colocalizes with vWF on Weibel-Palade bodies (TIRF images; green, Rab27a-GFP; red, vWF). Bars, 10 µm.

View larger version (54K): [in a new window]   Fig. 2. Exocytosis of endothelial secretory organelles. (A,B) Individual fusion events of a Rab27a-GFP-positive organelle (A) and a tPA-GFP vesicle (B) stimulated by 100 µM histamine. Rab27a-GFP diffuses in the plasma membrane, whereas tPA-GFP remains at the fusion site after exocytosis. Numbers indicate time (in seconds) relative to the moment of fusion. Lower panels show three-dimensional luminance plots of four successive frames starting one frame before fusion. Bars, 1 µm. (C) Plot of the half-width Rfluo2(t) obtained by a Gaussian fit of the distribution of fluorescence intensities from the images shown in A. A linear fit (grey line) yields the diffusion coefficient of Rab27a-GFP in the membrane Dfluo=0.12±0.01 µm2/second. (D) Time course of the fluorescence intensity of the tPA-GFP vesicle shown in B. The characteristic decay time of the fluorescence is given by an exponential fit (grey line): =13.7±0.9 s.

View larger version (50K): [in a new window]   Fig. 5. Visualization of cytoskeletal elements in HUVECs by TIRF microscopy. (A) TIRF image of the actin cytoskeleton labelled with rhodaminephalloidin. Arrowheads point to focal adhesions. The diffuse staining is probably due to the actin cortex. (B) TIRF images of the microtubule cytoskeleton visualized by immunostaining of -tubulin. Left-hand panel: cell with radially oriented microtubules; arrowheads show bright peripheral microtubules. Right-hand panel: detail of the cell periphery in another cell. (C) WPb align with microtubules. Arrowheads indicate WPb colocalizing with microtubules in two different cells. WPb preferentially accumulate at microtubule plus-ends (green, Rab27a-GFP; red, -tubulin). Bars, 10 µm.

View larger version (25K): [in a new window]   Fig. 6. Effects of cytoskeleton disruption on long-range movements of WPb. (A) Trajectories of the ten most mobile WPb in a nontreated cell (NT), a nocodazole-treated cell (noco) and a latrunculin B-treated cell (latB). Bars, 10 µm. (B) Analysis of long-range motions in nontreated cells (NT, n=54 from three cells), nocodazole-treated cells (noco, n=2 from three cells), cells treated with the kinesin ATPase inhibitor ATA (n=16 from three cells), latrunculin B-treated cells (n=33 from three cells) and cells treated with the myosin ATPase inhibitor BDM (n=44 from three cells). Parameters derived from three-dimensional tracking are: frequency of long-range motions (in s-1), defined as the number of WPb undergoing long-range motions per total number of WPb per unit time, average and maximum velocities (in µm/second) and total run length (in µm).

View larger version (28K): [in a new window]   Fig. 7. Effects of cytoskeleton disruption on short-range motions of WPb. (A) Data from all classes of short-range diffusive behaviours (simple, directed and restricted diffusion) were pooled to calculate the average diffusion coefficient D (left panel) and to plot the averaged three-dimensional MSD (right panel) in nontreated cells (circles or NT, n=48 from three cells), nocodazole-treated cells (diamonds or noco, n=51 from three cells), cells treated with the kinesin inhibitor ATA (n=48 from three cells), latrunculin B-treated cells (triangles or latB, n=60 from three cells) and cells treated with the myosin inhibitor BDM (n=59 from three cells). A numerical constant was added to the averaged MSD data so that all three plots coincide on their first data point. (B) Same analysis as in A for vertical motions (in the z direction) in nontreated cells (n=47 from three cells), nocodazole-treated cells (n=46 from three cells), cells treated with the kinesin inhibitor ATA (n=41 from three cells), latrunculin B-treated cells (n=53 from three cells) and cells treated with the myosin inhibitor BDM (n=53 from three cells). The left-hand panel shows the z-direction diffusion coefficient Dz. Averaged one-dimensional MSDz are plotted on the right panel. A numerical constant was added to the averaged MSD data so that all three plots coincide on their first data point.

View larger version (48K): [in a new window]   Fig. 8. Effects of cytoskeleton disruption on simple, directed and restricted diffusive behaviours of WPb. A percentage of simple (S), directed (D) and restricted (R) diffusion in nontreated cells (NT), nocodazole-treated cells (noco) and latrunculin-treated cells (latB). (B) Restricted diffusion. Diffusion coefficient of the cage (Dcage in µm2/second) and radius of the cage (Rcage in µm) in nontreated cells (n=16), nocodazole-treated cells (n=1) and latrunculin-treated cells (n=11).