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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02419

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Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells

Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain

View larger version (129K): [in a new window]   Fig. 1. Simultaneous visualisation of granules and cytoplasmic structures by confocal scanning microscopy. Granules were detected in quinacrine-treated chromaffin cells by epifluorescence whereas cytoplasmic structures were evident in the transmitted light channel implemented in an Olympus Fluoview FV300 microscope incorporating a 100 x LUMPlan FI water-immersion objective. (A-D) Images of four planes separated by 1.0 µm along the Z axis. Structure density increased in the region beneath the plasmalemma forming a peripheral ring and extending into the cytoplasm with the appearance of polygonal cages. The nucleus presents a less defined blurry organisation. (E) Vesicle fluorescence and optical density of the transmitted light image across the line depicted in C. Vesicles accumulate in the proximity of the peripheral ring visualised by transmitted light scanning microscopy. (F) Cross-sectional images of transmitted light structures in a chromaffin cell in the conventional X-Y plane and two X-Z and Y-Z cytoplasmic planes (indicated by coloured lines in the X-Y section). (G) 3D reconstruction of a portion of the cytoplasmic structures showing an intricate network. Black surfaces represent solid structure. Bar, 10 µm (A-D); 2 µm (F); 1 µm (G).

View larger version (51K): [in a new window]   Fig. 2. Cytoplasmic structures visualised by transmitted light are dynamic but move at a lower speed than chromaffin granules. Time-lapse high magnification images of chromaffin granule fluorescence (B) and transmitted light structures (A) acquired at 1 Hz over a 1-minute period in basal medium. Arrows depict disruptions in the peripheral ring and vesicles accessing this region. (C) Dynamics were analysed by measuring the time-dependent variations of averaged intensity in a region of interest (ROI, see boxes in A,B). Vesicles present short cycle oscillations in fluorescence (2-5 seconds), whereas visible light intensity changed in cycles of time ranging from 10-20 seconds. (D) Distribution of X-Y displacements for vesicles and particle structures obtained after threshold of the transmitted light images. The average displacements were 0.79±0.09 µm over 20 seconds (for 60 threshold particles in 16 cells) for the visible light structures and 3.75±0.50 µm/20 seconds (n=135 vesicles corresponding to 11 cells) for the vesicle displacement. (E) Mean square displacement (MSD) as a function of time obtained for vesicles (n=27) and particle structures (n=18). Also plotted were the best linear fits for the different data, used to estimate the diffusion coefficient according to Qian et al. (Qian et al., 1991) (continuous lines), and the best fit to the equation MSD(t) = rc2 [1 – A1 exp (–4 A2 D t /rc2)], defining movement restricted by a cage (dashed line).

View larger version (94K): [in a new window]   Fig. 3. Latrunculin A affects the distribution and density of transmitted light structures. Chromaffin cell treated with latrunculin A (1 µM, 15 minutes) affects the integrity of the peripheral ring visualised with transmitted light scanning microscopy. Representative cell depicted before (A) and after (B) latrunculin A incubation. (C) Estimation of the effect by averaging the value of optical density in the cortical area for nine cells. (D,E) Time-course of the effect of latrunculin A on the internal organisation of transmitted light structures. Images were taken at 3-minute intervals (D) and mean image intensity (±s.e.m.) taken for each frame acquired. Bar, 10 µm (A,B); 1 µm (D).

View larger version (110K): [in a new window]   Fig. 4. Phalloidin, ß-actin and myosin II RLC-GFP colocalise with the structures visualised by transmitted light in the cortical region. (A-C) Rhodamine-phalloidin (1 µM, 30 minutes) labelling of the cortical region overlapped with the peripheral ring visualised with transmitted light scanning microscopy. Images obtained in the transmitted light channel using a green LUT table (A), epifluorescence channel for rhodamine (B) and merged image (C). (D) Chromaffin cell labelled with quinacrine and treated with phalloidin showing static vesicles located over the immobilised cytoplasmic structures observed by transmitted light. (E) Cytoplasmic zone magnified. (F-H) GFP-ß-actin and transmitted light colocalised in the cortical region. Depicted is a cell expressing GFP-ß-actin (F) visualised by confocal microscopy and the corresponding transmitted light image using a red-LUT (G) with the merged image (H). (I-K) Cells overexpressing the double mutant T18A/S19A RLC-GFP were studied by laser confocal microscopy. Depicted is a high magnification field of a cell expressing this inactive form of myosin II, where vesicles are also observed by quinacrine labelling (I). (J) Simultaneous acquisition of transmitted light image corresponding to the same field using a red LUT. (K) Merged fluorescent and transmitted light images. Bar, 10 µm (A-C,D,F-H); 1 µm (E,I-K).

View larger version (85K): [in a new window]   Fig. 5. Secretagogues induced fast reorganisations in F-actin cytoskeleton in chromaffin cells. Cells were stimulated by addition of acetylcholine (10 µM) to the basal bathing medium and the changes in the F-actin cytoskeleton studied by acquiring transmitted light images of the entire cell at 1 Hz. (A) Frames separated by 10-second intervals after secretagogue addition. The formation of clear discontinuities in the subcortical structures is visible. (B) Measurements of optical density in a ROI located over one of the detected disruptions for 2-second intervals, showed that the formation of these subcortical patches within a few seconds was fast enough to encompass secretion measured by the increase in the cell perimeter (D). (C) In contrast with these rapid changes occurring in the cell periphery, reorganisation of intracellular structures forming polygons increased the open space in their interior. This process takes tens of seconds to fully develop as shown in the images taken at 10-second intervals. Bar, 10 µm (A); 1 µm (B,C).

View larger version (49K): [in a new window]   Fig. 6. Cytoskeletal changes during secretion involved F-actin transfer in the sub-cortical zone. (A,B) High-magnification frames acquired at 1 Hz of a cortical region in a chromaffin cell stimulated by fast and transient superfusion (10 seconds) with 59 mM KCl depolarising solution. The cortical cytoskeleton disassembles. After that the cortical barrier slowly increases its optical density to recover the initial value 80 to 100 seconds after the initiation of the transient stimulus (B). Interestingly, the disruption of the cytoskeletal F-actin structure was a process involving transfer of material from the subcortical area to the adjacent cytoplasmic regions (boxed areas in A), as ROI measurements within this zone detected increases in optical density encompassing the parallel decrease of the peripheral barrier (A,B). Quinacrine-loaded vesicles access the cortical region through the newly opened disruptions and were frequently found in the narrow space left by the channel-like structures. (C) Vesicles increased their presence in the subplasmalemmal regions 10-15 seconds after the disruption of the cortical barrier as detected by measuring quinacrine fluorescence in a ROI located in the exterior of the cortical structure. (D,E) Representative images obtained from 10 frames after and before the cell was stimulated by transient depolarisation over 10 seconds. Arrows indicate the positions of open areas lacking F-actin structures. Cytoskeletal structures are depicted in red LUT. Bar, 1 µm (A,D,E).

View larger version (60K): [in a new window]   Fig. 7. GFP-ß-actin can be used to study cortical F-actin dynamics in chromaffin cells. Cells infected with amplicons containing the GFP-ß-actin expressing vector were visualised by confocal fluorescence microscopy. (A) Cells expressing GFP-ß-actin were fixed, permeabilised and incubated with phalloidin-rhodamine as indicated in methods. Depicted are confocal images of GFP-ß-actin (A), phalloidin-rhodamine (B) and both channels showing evident overlapping (C). (D) GFP-ß-actin-expressing cell treated with 1 µM latrunculin A for 30 minutes. Images were acquired at 1 Hz, and frames were separated by 5-minute intervals. (E) Fluorescence in the interior or cortical region was determined and plotted against time after latrunculin A addition. Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 8. Simultaneous observation of peripheral F-actin reorganisation by GFP-ß-actin fluorescence and transmitted light scanning microscopy. Cells were infected with amplicons containing the GFP-ß-actin construct as indicated in Materials and Methods. GFP fluorescence (A) and transmitted light images (B) of a cortical region were acquired at 1 Hz. Time-lapse high magnification images separated by 10 seconds before (first frame) and after stimulation with 10 µM acetylcholine are shown. Arrows indicate the positions of F-actin disruptions visualised in both channels. (C) Fluorescence determination in three different regions of interest (ROI) located at different distances from the cell limit (boxed areas in A). F-actin transfer to the cell interior follows fragmentation of the peripheral cortex. Bar, 1 µm (A,B).

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