Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain
* Author for correspondence (e-mail: Luisguti{at}umh.es)
Accepted 5 April 2005
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Summary |
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Key words: Exocytosis, Confocal microscopy, Adrenomedullary cells, Cytoskeleton, Vesicle transport
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Introduction |
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In this study we address cytoskeleton dynamics during secretion using both transmitted light scanning microscopy and confocal fluorescence. The former is used as a new tool allowing the direct visualisation of complex cytoplasmic structures inside cell cytosol. These structures, with differential density to the passage of visible light, are strictly sensitive to actin and myosin inhibitors and present a distribution characteristic of the F-actin network. More interestingly, there is a clear association between the dynamics of both chromaffin granule movements and these cytoplasmic structures, which appears to suffer complex reorganisations in the subcortical and internal cytoplasmic regions during the secretory cycle of chromaffin cells. Taken together, these studies and the data gathered using fluorescent-tagged ß-actin support an active dual role of F-actin as a barrier and carrier system during secretion of neuroendocrine cells.
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Materials and Methods |
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Dynamic confocal microscopy
Cells were loaded with quinacrine using a 4 µM concentration in culture medium for 10 minutes as described (Ñeco et al., 2002; Ñeco et al., 2003
; Ñeco et al., 2004
). Fluorescent emission from quinacrine trapped in the acidic vesicles was investigated using an Olympus Fluoview FV300 confocal laser system mounted in a BX-50 WI upright microscope incorporating a 100 x LUMPlan FI water-immersion objective. Excitation was performed using Ar and HeNe visible light lasers. This system allows for Z-axis reconstruction (0.5-0.55 µm theoretical Z slice) and time-lapse dynamic studies with time resolutions ranging from 0.1 second for 200 x150 pixels image acquisition (adequate for region studies) to about 0.6 second for images of 400 x300 pixels (for visualisation of the entire cell). Simultaneous acquisition of transmitted light images was obtained using the channel implemented in the confocal microscope and using bright field optics (theoretical depth of field is 0.55-0.65 µm). The intensity of this channel was adjusted to avoid saturation of the subcortical region and visualise the lighter cytoplasmic structures. Distribution of the F-actin network was studied using rhodamine coupled to phalloidin in intact or permeabilised cells. As this chemical presents low permeability throughout the plasmalemma, intact cells in culture media were incubated with 1 µM rhodamine-phalloidin for 30 minutes at 37°C, in this way a low percentage (5-10%) of the cells, were labelled enabling Z-axis confocal reconstructions. Microtubules were visualised in intact cells using 1 µM taxol conjugated with Bodipy 564/570 (Molecular Probes, Eugene, OR). Incubation of this compound for 15-20 minutes at 37°C allowed efficient labelling of the total population of cultured chromaffin cells. Other actin and myosin affecting chemicals were used as described (Gil et al., 2000
; Ñeco et al., 2002
; Ñeco et al., 2003
; Ñeco et al., 2004
).
Production of GFP-actin and mutant and wild-type forms of RLC-GFP constructs
A GFP-actin construct was obtained by NheI/MunI digestion of pEGFP-actin (Clontech, Palo Alto, CA) and inserted into an XbaI/EcoRI-digested pHSVpuc amplicon, upstream the IE 4/5 promoter. Packaging and amplification of this vector were performed using standard procedures (Lim et al., 1996). A plasmid encoding chicken gizzard smooth muscle myosin II regulatory light chain (RLC; a gift of Dr Kendrick-Jones, MRC, Cambridge, UK) was used for site-directed PCR cassette mutagenesis. The modifications were confirmed by sequencing in both directions. pRLC-wt-GFP and pRLC-T18A/S19A-GFP were generated using pEGFP-N1. The final construct was inserted in pHSVpuc and amplicons produced as indicated above. Primary cultures of chromaffin cells were infected with Herpes simplex virus (HSV-1) amplicon containing the constructs described above as described elsewhere (Gíl et al., 2002
). Efficiency of virus infection was determined by fluorescent microscopy, using serial dilutions of purified virus. The dilution chosen for further experiments (20-30 µl virus per 1 ml of medium) produced 10-15% infection efficiency.
Image analysis and particle tracking
Analysis of frames was performed using the program of public domain ImageJ with plug-ins for ROI measurement, image average and comparison of multiple channel images and 3D reconstruction (obtained from http://rsb.info.nih.gov/ij/). A multi-tracker plug-in was used to determine particle centroid in time-lapse studies as described earlier (Ñeco et al., 2003; Ñeco et al., 2004
). The x-y coordinates determined for vesicles or threshold visible images (structure particles obtained after threshold of the transmitted light channel images) were transferred to Igor Pro, where specialised macros were used to calculate total lateral displacement and the mean square displacement (MSD) for any given time interval based in the equations defined by Qian et al. (Qian et al., 1991
),
![]() | (1) |
This equation is a good description of vesicle movement when subjected to a single coefficient of diffusion; the downward curvature of the experimental data indicated the diffusion of vesicles in a cage, which can be described by the approximate equation (Saxton and Jacobson, 1997),
![]() | (2) |
The Student's t-test for paired samples or the two-way ANOVA test was used to establish statistical significance among the different experimental data (samples were considered significantly different when P<0.05). All data were expressed as mean±s.e.m. of experiments performed in a number (n) of individual cells. The data presented represents experiments performed with cells from at least three different cultures.
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Results |
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Interestingly, the MSD versus time plot for control granules showed a downward curvature and fitted better to an equation representing vesicle movements inside a cage (see Equation 2). The best fit for these data (R2 = 0.96 compared to 0.91 for lineal regression) gave a value of the theoretical radius of the cage confining the vesicles (rc) of 0.86 µm. In order to estimate whether the dimensions of the polygonal structures visualised with transmitted light scanning microscopy could be limiting such vesicle movements, we measured the wall to wall maximal X and Y distances for an high number of such cages (n=272 measurements in five cells) and obtained an average value of 1.5±0.7 µm (mean±s.d.), that closely matches the diameter of 1.72 µm for the theoretical circular cage imprisoning vesicle movement.
Chemicals affecting F-actin alter the structure and dynamics of the cytoplasmic structures visualised with transmitted light
What is the molecular nature of these visible structures? Based on its distribution accumulating in the cell periphery, our first guess would be the cytoskeletal network of F-actin forming a subplasmalemmal barrier in chromaffin cells (Trifaró et al, 1984; Perrin and Aunis, 1985
). In consequence, we treated chromaffin cultures with specific agents affecting such filamentous protein. Treatment of cultured cells with 1 µM latrunculin A, a chemical stabilizing monomeric actin (Spector et al., 1983
), for 15 minutes resulted in drastic changes in the characteristics of these visible structures. First, there was an obvious alteration of the cell shape that was accompanied by a reduction of the intensity of the peripheral cortical network (Fig. 3). Quantification by measurement of the density of the cell cortex showed a 60% decrease in the average optical intensity of this cortical structure in measurements performed in nine cells before and after latrunculin treatment (Fig. 3C). Moreover, the incubation of chromaffin cells with latrunculin A also affected the internal network of polygonal cages (Fig. 3D), which continuously decreased its optical density during incubation with this chemical (Fig. 3E).
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GFP-ß-actin and the regulatory light chain of myosin II colocalise with the transmitted light network within the cortical region
We have shown in a recent publication that myosin II is associated with the F-actin network in the cytoplasm and periphery of chromaffin cells (Ñeco et al., 2002; Ñeco et al., 2004
). Based on this, we conceived experiments expressing both the ß-actin and regulatory light chain (RLC) of myosin II linked to enhanced green florescence protein (GFP) by their N-terminal and C-terminal domains respectively, using an amplicon-based strategy that has proven successful for the expression of other proteins in chromaffin cells in culture (Gíl et al., 2002
; Ñeco et al., 2004
). GFP-ß-actin was heavily expressed in the cytoplasm and the cortical region of chromaffin cells (Fig. 4F), and colocalised with the cortical density visualised with transmitted light (Fig. 4G,H), whereas in the cytoplasm there was a partial alignment. In addition, when we expressed RLC-GFP and specially an inactive unphosphorylatable form, T18A/S19A RLC-GFP (Komatsu et al., 2000
; Ñeco et al., 2004
) in chromaffin cells, it could be observed in the cytoplasm forming a network that matched that visualised with transmitted light (Fig. 4I,K). In addition, if granules were labelled with quinacrine, its brilliant fluorescence could be observed in association with both the transmitted light structures and the GFP fluorescence associated with T18A/S19A RLC-GFP (Fig. 4K). Interestingly, the expression of this inactive form of RLC yielded a static network of transmitted light structures whereas the wild-type form of RLC sustained both vesicle (Ñeco et al., 2004
) and structure mobility (D.G., unpublished observation).
Structure mobility was affected by chemicals affecting actin-myosin but not by microtubule inhibitors
If these structures visualised by transmitted light are composed by F-actin-myosin II, their dynamics should also be altered by 2,3-butanodione monoxime (BDM), which affects myosin ATPase activity (Herrmann et al., 1992); or wortmannin, which alters the activity of myosin light chain kinase (MLCK) (Nakanishi et al., 1992
). Incubation of chromaffin cells with concentrations of 10 mM BDM, or 1 µM wortmannin for 15 minutes, clearly affects the dynamics of the F-actin visible light structures, producing stabilisation similar to that caused by 1 µM phalloidin or 10 µM jasplakinolide. Under these conditions, transmitted light particle tracking measurements indicated a 50-65% reduction in the mobility of these structures using chemicals affecting actin-myosin in numerous measurements performed in more than 40 threshold density particles in 5-20 cells under each experimental condition (Fig. S2 in supplementary material).
Similar experiments were conceived to test whether or not microtubules control the activity and dynamics of this network. Cells were incubated for 15 minutes with 10 µM vinblastine or 1 µM taxol labelled with Bodipy 564/570. None of these compounds were able to modify the dynamics of this network observed in the transmitted light channel (Fig. S2 in supplementary material).
Secretagogues induce changes in the density and distribution of the visualised transmitted light structures
The cellular distribution, colocalisation with phalloidin, GFP-ß-actin and RLC-GFP and its sensitivity to chemicals affecting actin-myosin strongly suggest that the structure visualised by transmitted light scanning microscopy might be composed of the chromaffin cell cytoskeleton complex of F-actin modulated by the activity of structural myosin II (Kumakura et al., 1994; Ñeco et al., 2002
; Ñeco et al., 2004
). Thus, transmitted light scanning microscopy could be used to study the cytoskeletal dynamics that accompanies secretion, obtaining a dynamic picture of the complex rearrangements of cytoskeletal elements occurring during the secretory cycle. The stimulation of the cells with 10 µM acetylcholine resulted in a 2-4% increase in the cell perimeter as a consequence of exocytotic membrane incorporation and the simultaneous formation of evident discontinuities in the subcortical structures (Fig. 5A and Movie 3 in supplementary material). Measurements of optical density in a ROI located over one of the detected disruptions, such as those depicted in Fig. 5B at 2-second intervals, showed that the formation of these subcortical patches within a few seconds was fast enough to encompass secretion (Fig. 5D). In contrast with these rapid changes occurring in the cell periphery, we also observed the reorganisation of intracellular structures forming polygons, increasing the open space in their interior (Fig. 5C). This process, however, takes tens of seconds to fully develop as shown in the images of this panel taken at 10-second intervals. These changes were observed in dozens of cells stimulated with either physiological agonist or by sustained superfusion with high potassium stimulatory solution and they were strictly dependent of the presence of calcium in the stimulatory medium. In similar experiments, stimulation for 1 minute with acetylcholine in a medium lacking Ca2+ (and in the presence of 1 mM EGTA) produced no apparent changes in the cytoskeletal peripheral integrity compared with resting conditions (Fig. S3 in supplementary material), whereas the posterior perfusion of this cell with this secretagogue in a medium containing 2 mM CaCl2 induced rapid and drastic changes in the cytoskeletal structure.
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In another series of experiments, cells were depolarised briefly with a 10-second superfusion with 59 mM KCl. Under these conditions cells secrete catecholamines for 10-12 seconds as measured using single cell amperometry (Gil et al., 1998; Ñeco et al., 2003
), and the cortical cytoskeleton disassembles in parallel (Fig. 6A,B and compressed Movie 4 in supplementary material), after that, the cortical barrier slowly increased its optical density to recover the initial value 80-100 seconds after the initiation of the transient stimulus (Fig. 6B). Interestingly, disruption of the cytoskeletal F-actin structure was a process involving transfer of material from the subcortical area to the adjacent cytoplasmic regions, as ROI measurements within this zone showed that, with a few seconds delay, increases were detected in the optical density encompassing the parallel decrease of the peripheral barrier (Fig. 6A,B, comparison of the optical density in cortical and interior ROIs). The concept of cortical F-actin transfer rather than its destruction was also supported when the average transmitted light intensity of the overall region studied in Fig. 6 did not change significantly during secretion (Fig. 6B). We observed in many stimulated cells, that the reorganisation of F-actin implied the formation of channel-like structures perpendicular to the membrane plane (see second and third frame of Fig. 6A). A detailed observation of the granule positions during secretion showed that quinacrine-loaded vesicles accessed the cortical region through the newly opened disruptions and they were frequently found in the narrow space left by the channel-forming structures. Finally, they increased their presence in the subplasmalemmal regions 10-15 seconds after disruption of the cortical barrier as detected by measuring quinacrine fluorescence within a ROI located in the exterior of the cortical structure (Fig. 6C).
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Vesicles moved along F-actin during stimulus-dependent cytoskeletal reorganisations
During cell stimulation, F-actin reorganised and formed disruptions and the open cytoplasmic spaces as described above, but could the vesicles use such spaces to diffuse and access the subplasmalemmal area? A simple, yet accurate way to observe vesicle positions is to obtain the cumulative image of vesicles in relation to cytoskeletal structures during secretion or even under resting conditions. Cumulative images obtained over 15 seconds in non-stimulated cells show that vesicles moved in the internal face of the cytoskeletal barrier and appeared to avoid the dark area located in the centre of polygons formed by the F-actin network (Fig. 6D, arrows). After stimulation, most of the vesicles either were released or moved to other cell areas but the vesicles observed moved in contact with the cytoskeletal network and again avoided the use of open spaces (arrows in Fig. 6E). These observations were made in dozens of stimulated cells and indicated that during rest conditions or stimulatory activity, chromaffin granules moved preferentially along F-actin structures avoiding access to bare cytoplasmic zones.
Changes in the fluorescence of GFP-ß-actin-expressing cells support the F-actin dynamics observed using transmitted light scanning microscopy
In order to confirm the observations made with transmitted light scanning microscopy, we studied the cytoskeletal dynamics in chromaffin cells expressing GFP-ß-actin construct, where fluorescence is distributed in the cell periphery and also in cytoplasmic patches produced by overexpression (Fig. 7A). These patches corresponded to F-actin aggregates as they were labelled by phalloidin (Fig. 7B,C), which also stained the peripheral ring corresponding to the cortical F-actin barrier. Additional proof of the accumulation of GFP-ß-actin in the F-actin cortical network, was obtained by treatment with 1 µM latrunculin A. This substance affected the integrity of the peripheral fluorescence that decreased in the cortical area and continuously increased in parallel in the internal cytoplasm as shown in the ROI determinations (Fig. 7D,E), thus validating the use of this fluorescent construct to study of cortical F-actin dynamics in chromaffin cells.
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Interestingly, cells overexpressing GFP-ß-actin manifested fluorescent changes encompassing those occurring in the transmitted light channel upon cell stimulation by depolarisation. In these experiments a temporal decrease of fluorescence integrated in the cortical zone and the parallel increase in the fluorescence of the cytoplasmic subcortical area was observed and similar results were obtained by analysing the transmitted light images. Furthermore, the formation of cortical disruptions during stimulation and F-actin transfer has also been observed in cells expressing the GFP-ß-actin construct (Fig. 8) for a depolarised cell. These experiments proved simultaneous detection of these disruptions in the confocal images of GFP-ß-actin fluorescence (Fig. 8A) and in the transmitted light channel images (Fig. 8B). Determination of fluorescence intensity at different distances from the cell limits demonstrated GFP-ß-actin transfer from the external to the internal zones during the formation of such disruptions (Fig. 8C). Taking these observations from both transmitted light and fluorescence GFP-ß-actin images together, it is clear that during stimulation F-actin redistributed to leave open space in the peripheral barrier and to form the trails for vesicle movement.
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Discussion |
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A model to understand the double role of F-actin as both a barrier and a support for vesicle access to the subplasmalemmal area in chromaffin cells
Even if the combination of pharmacological tools and the observed distribution and colocalisation of the transmitted light structures with phalloidin, GFP-ß-actin, and myosin II RLC labelling may constitute convincing evidence of its F-actin identity, the complexity of the changes occurring in these structures during cell stimulation is additional and important proof of its nature. As shown in Figs 5, 6, 7, 8, using both transmitted light and confocal observation of GFP-ß-actin, three types of major change were characterised in the cytoplasmic F-actin distribution after secretory stimulation: fast and discrete disruptions in the cortical peripheral barrier, formation of channel-like structures perpendicular to the membrane plane, and slower changes in the interior of the cytoplasm with the appearance of the formation of wider open spaces devoid of F-actin. The formation of cortical disruptions in response to different secretagogues was the key element supporting the role of peripheral F-actin as an effective barrier avoiding vesicle access to secretory active sites (Aunis and Bader, 1988; Vitale et al., 1995
). Our experiments are the first to show the real-time dynamics of this process, in resting conditions, vesicles tend to locate within 200-400 nm of the membrane, following secretagogue superfusion and within 5-10 seconds, F-actin reorganises itself forming cortical disruptions of about 0.5-2 µm and the vesicles penetrate to previously forbidden areas in the cell limits. After 10-20 seconds of stimulation the general disposition of F-actin, that was parallel to the membrane, changed to a perpendicular alignment increasing its concentration in deeper subcortical regions, thus forming frequently channel-like open spaces of about 300-400 nm used by granules to access the membrane. A static view of these structures was previously shown when the cortical region of rhodamine-phalloidin-labelled chromaffin cells was studied by z-reconstruction of confocal images (Ñeco et al., 2003
). Vesicles using these channel-like structures will be accessing docking sites 10-20 seconds after initiation of secretion and therefore will refill the pools of docked vesicles released during sustained stimulation. Finally, after 60-100 seconds, F-actin reorganisation in response to transient stimuli recovered the original distribution of parallel to the plasma membrane and again vesicles were restricted in their access to active sites. This reorganisation of F-actin is of extreme importance as it provides the basis to understanding the double nature of the cytoskeletal network of F-actin as a dense barrier that does not allow vesicle access to the docking sites and also, after stimulation and reorganisation, as providing the system to transport vesicles from the cytoplasmic pool to specific points of the peripheral area. In contrast to these rapid changes, continuous cell stimulation also induced the slow formation of intracellular spaces lacking F-actin with the aspect of empty polygons. We do not know the functional relevance of this process as vesicles do not seem to access the interior of these zones as they only move only along the F-actin walls. The formation of these polygons during secretion is supported by electron microscopy studies in fixed chromaffin cells (Tchakarov et al., 1998
).
Taking these observations together, an active role as both a barrier and a carrier for F-actin-myosin is suggested in this neuroendocrine model, but our studies do not exclude another scaffold function as has been proposed in presynaptic terminals (Sankaranarayanan et al., 2003). We observed that these transmitted light structures are also easily visible and dynamic in the cell body of motoneurons and hippocampus neurons, therefore the possible application of transmitted light scanning microscopy to the study of secretion in neuronal and other cells could open new alternatives for the study of transport and secretion processes in living cells.
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Acknowledgments |
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Footnotes |
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References |
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