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First published online June 23, 2006; 10.1104/pp.106.080168 Plant Physiology 141:1591-1603 (2006) © 2006 American Society of Plant Biologists Imaging of Dynamic Secretory Vesicles in Living Pollen Tubes of Picea meyeri Using Evanescent Wave Microscopy1,[W] amaj ek Baluka
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China (X.W., Q.W., X.L., X.S., M.Z., J.L.); Graduate School of the Chinese Academy of Sciences, Beijing 100049, China (X.W., Q.W., X.L., M.Z.); Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China (Y.T.); Institute of Cellular and Molecular Botany, Department of Plant Cell Biology, Rheinische Friedrich-Wilhelms-University Bonn, D–53115 Bonn, Germany (J.
Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.
Vesicle trafficking is fundamental to numerous activities in eukaryotic organisms and underlies many of the basic processes involved in cell growth and differentiation. The vesicle trafficking network is responsible for exocytosis and endocytosis (Rothman, 1994  ; Bretscher and Aguado-Velasco, 1998; Hepler et al., 2001; Zefirov et al., 2004; amaj et al., 2005). Exocytosis is the process by which secretory vesicles derived from the trans-Golgi network (TGN) attach and/or fuse with the plasma membrane, releasing their contents into the extracellular space. In contrast, endocytosis is responsible for the uptake of compounds from the extracellular milieu to be used in cellular metabolism, the uptake of signal molecules, and the uptake of membrane proteins and lipids to be recycled (amaj et al., 2005). Vesicle transport of cargo is carried out by coated vesicles from a donor compartment. These vesicles move on to the Golgi apparatus, where further posttranslational modifications are made. Uncoating and subsequent docking and fusion of the vesicles with a target compartment membrane then occur. Three major types of vesicle coating proteins, COPI, COPII, and clathrin, are involved in exocytotic and endocytotic vesicle trafficking. Plant cells grow when secretory vesicles derived from the TGN and endosomes fuse with the plasma membrane, into which they insert cellulose synthases. At the same time, they deliver cell wall matrix into the cell wall (Kimura et al., 1999; Emons and Mulder, 2000).
The last few decades have witnessed many studies of vesicle trafficking, but most studies have focused on animal cells (Rutter and Tsuboi, 2004
With the introduction of evanescent wave microscopy (EWM), direct monitoring of an individual vesicle during secretion has become possible (Tsuboi and Rutter, 2003
In this study, we visualized secretory vesicle motions in living pollen tubes after labeling the vesicles with the endocytotic/exocytotic tracer FM4-64. This amphiphilic styryl dye has been used to investigate endocytosis and visualize the diverse membrane compartments in living fungal hyphae (Fischer-Parton et al., 2000
Visualization of Secretory Vesicles Close to the Plasma Membrane in Living Pollen Tubes Pollen tubes labeled with FM4-64 exhibited a distinct peripheral, bright staining pattern (Fig. 1A ). Confocal microscopic analysis of labeled pollen tubes demonstrated that the peripheral staining was plasma membrane associated, not cell wall associated, as indicated by plasmolysis with 100 µM sorbitol in the presence of FM4-64 (Fig. 1C). Pretreatment of pollen tubes with 500 µM sodium azide impaired dye uptake; therefore, FM4-64 fluorescence could be observed only at the plasma membrane (Fig. 1E), but still allowed staining of the plasma membrane of the apical and subapical regions. Because FM4-64 is a membrane-selective fluorescent dye and does not label the cytoplasm or cell wall (Fig. 1C), it was used to detect vesicle trafficking in living pollen tubes.
Under EWM, apical region versus subapical region defined by de Win et al. (1996) and Wang et al. (2005) can be mostly distinguished because the apical region in Picea meyeri tubes with FM4-64 staining appeared crescent, which was different from inverted cone-shaped (V-shaped) clear zone in angiosperm pollen tubes. Fluorescent spots were distributed plentifully over the apical and subapical regions beneath the plasma membrane of the pollen tube proximal to the coverslip. The spot morphologies of the apical and subapical spots were similar. The density of spots, however, was much higher in the apical region than in the subapical region (Fig. 1G). The spots appeared dim because of the scattering effect induced by out-of-focus vesicles or organelles, such as the Golgi apparatus and endoplasmic reticulum deeper within the pollen tube cytoplasm (Fig. 1H). After processing with flattening and high-pass filters, the spots became clear and could be seen to have similar sizes (Fig. 1I). Spots were identified as vesicles when the average intensity in a 3- x 3-pixel region was 20% greater than the surrounding background gray value in three consecutive frames, the central intensity was a local maximum, and the spot was present in more than three consecutive images. Because the diameter of vesicles in P. meyeri ranged from 100 to 300 nm (Wang et al., 2005), twice the size as those in Lilium and Arabidopsis under the TEM, only those spots with diameter less than 400 nm were considered as secretory vesicles (TGN vesicles) for analysis; other larger fluorescent spots are considered not to be vesicles or organelles, which were excluded from analysis in this article.
To explore vesicle motions, a series of images of growing pollen tubes labeled with FM4-64 was taken under EWM. Movies compiled from a large number of images showed that the vesicles moved around a resting position in the apical and subapical regions of the pollen tubes (Supplemental Movies 1 and 2). These bright fluorescent spots showed short, nonlinear motions in various directions in living pollen tubes. Furthermore, two types of secretory vesicle mobility were observed along the pollen tubes in terms of running length and velocity: short-distance motion (Fig. 2A ) and long-distance motion (Fig. 2B). Long-distance motions were defined as motions of >1 µm in distance and with a maximum velocity of >2 µm/s. All other motions were considered short-distance motions. Approximately 10% of vesicles within the evanescent field could be classified as undergoing long-distance motion, whereas the majority (>80%) of vesicles proximal to the plasma membrane underwent short-distance motion.
Short-distance motions often involved rapid changes or reversals in direction and velocity between consecutive runs, whereas the long-distance motions were directed to the apical region, as though these vesicles were guided to their targets. Moreover, the two types of motion differed in their velocities. The average velocity during short-distance motions was 1.09 ± 0.02 µm/s (n = 30 vesicles), with a maximum velocity of 3.5 µm/s. The run length averaged 75 ± 0.8 µm (n = 30 vesicles), with a duration of 100 s. In contrast, the average velocity during long-distance motions was 1.93 ± 0.05 µm/s (n = 30 vesicles), and the maximum velocity was 5.85 µm/s (Table I ). In addition, vesicle motion could be estimated by the mean squared displacement (MSD) and the diffusion coefficient. The diffusion coefficient in two dimensions, D2, was derived from the slope of a plot of the MSD versus time for  t  0 (Fig. 2, C and D). For long-distance motions, D2 ranged from 2.1 x 10–2 µm2/s to 0.62 µm2/s, with an average value of 0.11 ± 0.07 µm2/s (n = 30 vesicles). For short-distance motions, D2 ranged from 8 x 10–3 µm2/s to 0.12 µm2/s, with an average value of 5.3 x 10–2 µm2/s (n = 30 vesicles).
Quantifying Individual Secretory Vesicle Motions in Living Pollen Tubes
Much variation in velocity was observed during the recording time. In the pollen tubes observed for long periods, single secretory vesicles occasionally traveled distances of 100 µm at a top speed of 6.87 µm/s (in one instance), but such cases were rare (less than one vesicle per cell per minute) and were therefore excluded from further analysis. During pauses between directed runs, complex, oscillatory behaviors were observed. As shown in Figure 3A
, oscillations occurred preferentially during pauses and before direction reversals. The average oscillation frequency was
Exocytosis, the release material from secretory vesicles, was identifiable as a sudden spread of fluorescence. Exocytosis of a vesicle was preceded by a transient brightening of the vesicle followed by a decay of its fluorescence intensity (Fig. 3B), with a characteristic time of 13.7 ± 1.3 s (n = 20 pollen tubes). Fusion of vesicles appeared as a fluorescent spot spreading away from the site of fusion (Fig. 3C). The trace showed apparently random motion, superimposed with a slow drift. The trajectories were constrained in an irregular-shaped value of 1 µm in the x to y direction (Fig. 3D). For analysis, we measured the square of the distance traveled by the vesicles and plotted the MSD against the time interval (Fig. 3E). Table II summarizes the tracking parameters and other findings, as well as the ratios of vesicles with different motions in relation to all vesicles.
Effect of Actin Cytoskeleton Disruption on Secretory Vesicle Mobility Disruption of the actin cytoskeleton by treatment with latrunculin B (LATB) or cytochalasin D (CD) caused significant alterations in secretory vesicle motions (Table III ). The average vesicle path lengths in pollen tubes treated with LATB or CD was reduced to 7.34 ± 1.51 or 9.16 ± 1.37 µm of displacement per 10 s, respectively. These values represent significantly shorter path lengths than those measured in control cells (Fig. 4A ). LATB treatment dramatically reduced the vesicle velocity as well; the velocity ranged from 0.20 to 0.96 µm/s, with an average velocity of 0.62 ± 0.21 µm/s, which is about one-fourth that observed in the control pollen tubes. CD showed similar but less significant effect on the velocity, reducing it to 0.65 to 1.04 µm/s, with an average value of 0.81 ± 0.09 µm/s (Fig. 4B). The alterations in vesicle trajectories caused by LATB and CD treatment were characterized by plotting the x-y coordinates of vesicles in the two experimental conditions. The results showed that long-distance motion occurred only in the untreated cells that had intact actin cytoskeletons (Supplemental Fig. 1A). Vesicles were much less mobile in the presence of both inhibitors (Supplemental Fig. 1, B and C), and their motions became random and were mostly confined to the restricted regions (Supplemental Fig. 1, G and H). Complex dynamics, such as the oscillations, regularly visualized in the control pollen tubes, were not observed in the LATB- and CD-treated pollen tubes (Fig. 4, C and D). Furthermore, the MSD curves (Fig. 4E) revealed that individual vesicle kinetics was clearly affected by these inhibitors, which reduced D2 by 70% (Fig. 4F).
Effects of Microtubule Disruption on Secretory Vesicle Mobility Microtubule disruption by treatment with the inhibitors oryzalin and colchicine led to more moderate alterations in secretory vesicle motion than did by actin cytoskeleton disruption. Incubation for 10 min with 100 µM oryzalin and 5 mM colchicine induced a 10% to 20% reduction in vesicle mobility (Supplemental Fig. 1, D and E). Vesicle tracking showed a clear reduction in vesicle mobility as detected in the x-y plane; the average path lengths were 9.91 and 10.6 µm in 10 s for oryzalin and colchicines, respectively, which represent approximately 80% of the mobility found near the plasma membrane in control cells (Fig. 4A). When traces of individual vesicle motions in control pollen tubes were compared to those in oryzalin- and colchicine-treated cells, a marked difference in velocity was noted (Fig. 4B). In addition, both oryzalin and colchicine treatments had a significant impact on vesicle trajectories, which were much more confined in the lateral directions than those of the control cells (Supplemental Fig. 1, I and J). Plots of MSD versus time revealed that vesicle motions in treated pollen tubes had a number of different, distinctive features, which deviated from the regulated motions observed in control pollen tubes. The averaged MSD plots are shown in Figure 4E. When pollen tubes were exposed to colchicine and oryzalin for 10 min, D2 of mobile vesicles was reduced by only 25% to 40% (Fig. 4F). Tracking parameters of secretory vesicles in treated pollen tubes are summarized in Table IV .
Effects of Brefeldin A on Secretory Vesicle Trafficking We also characterized the effect of brefeldin A (BFA) on the mobility of secretory vesicle in living pollen tubes. When the pollen tubes were observed after a 10-min incubation with BFA, the average path lengths in apical and subapical regions were found to be slightly shorter than the average path lengths measured in control cells. The average path lengths for the apical and subapical regions in BFA-treated pollen tubes were 11.76 ± 2.35 µm and 9.42 ± 1.17 µm of displacement per 10 s, respectively (Fig. 5A ). The velocities of the mobile vesicles varied, but they were generally comparable to those of vesicles in the control pollen tubes. The average velocity of vesicles in the subapical region was 1.87 ± 0.15 µm/s, and the maximum velocity was 3.26 µm/s. The velocity of vesicles in the apical region often decreased to an average value of 0.95 ± 0.34 µm/s (Fig. 5B) when they were moving closer toward the plasma membrane. The x-y coordinates of vesicles in the treated pollen tube are plotted (Supplemental Fig. 1F), which illustrates that BFA treatment had a minimal effect on vesicle trajectories. Nevertheless, oscillations of secretory vesicles in pollen tubes did not disappear upon treatment with BFA (Fig. 5C). As shown in Figure 5D, the MSD curve of individual vesicles in the pollen tubes after BFA treatment was not significantly influenced. D2 values deduced from the MSD curves were reduced by nearly 10% by BFA treatment (Fig. 5E). In BFA-treated pollen tubes, about 30 to 40 vesicles were visible in the observation field, corresponding to average densities of 1.4 x 10–2 ± 0.003 µm–2 and 2.3 x 10–2 ± 0.002 µm–2 in the subapical and apical regions, respectively (Fig. 5F). The total number of visible secretory vesicles decreased after BFA treatment, suggesting that the supply of new secretory vesicles to the observed regions was disrupted.
Imaging techniques are powerful tools for detecting vesicle trafficking in living cells, and they can provide information concerning the mechanisms of vesicle trafficking (Betz and Angleson, 1998 ; Fletcher et al., 2000; Baumann et al., 2005). Because confocal microscopy provides excellent optical sectioning, it has been widely used in the investigation of endocytosis and exocytosis in living cells (Ohara-Imaizumi et al., 2002; Bolte et al., 2004; Ma et al., 2004; amaj, 2006). However, vesicle diameters in pollen tubes range from 75 to 200 nm (Derksen et al., 1995), and, thus, vesicles are beyond the resolution limits of a conventional confocal microscope. In this study, we used a high numerical aperture objective and varied the angle of incidence of the laser beam to generate a penetration depth of the evanescent field of 70 to 400 nm proximal to the plasma membrane with cells adhered to a glass coverslip. EWM has provided valuable and complementary information about vesicle trafficking, especially in living cells, under lengthy observation periods (Watkins and Robertson, 1977; Oheim et al., 1999). In terms of depth discrimination, an evanescent wave microscope is up to 10-fold better than a confocal microscope (Steyer and Almers, 2001). Combined with a high-resolution charge-coupled device, which has extremely high temporal sensitivity, EWM is very useful in tracking 2-D motions of small particles near the coverslip under physiological conditions (Steyer et al., 1997; Sund et al., 1999; Sund and Axelrod, 2000; Wiegand et al., 2002; Mashanov et al., 2003; Ohara-Imaizumi et al., 2004; Xia et al., 2004; Zenisek et al., 2004). In this report, we examined the dynamics and motions of FM4-64-labeled vesicles proximal to the plasma membrane in living pollen tubes of P. meyeri. To our knowledge, this report is the first description of visualization of secretory vesicle tracking in living pollen tubes using EWM. This technique greatly facilitates the quantification of vesicle trafficking in plant cells.
Pollen tubes have been used as in vivo model systems for studying vesicles and organelle tracking because they are highly polarized, tip growing, and easy to manipulate experimentally. Angiosperm pollen tubes have been chosen as the subjects of most previous investigations because they grow quickly. For example, the growth rate of Lilium longiflorum was reported to be about 5 to 25 µm/min (Parton et al., 2001
In previous studies, some organelles moved linearly, in a mostly circulatory pattern (Cai et al., 2001
In our investigations, we observed that exocytotic vesicles entered the evanescent field and then became brighter, losing their lateral mobility while they reached a stable maximum intensity, and then their intensity declined as the dye diffused away. From the spread of fluorescence of secretory vesicles, we deduced that exocytosis is a "full" fusion event involving collapse of the vesicles into the membrane as they release of their internal components. The fusion event in living pollen tubes was not consistent with the "kiss-and-run" model recently reported in synapses (Gandhi and Stevens, 2003
One of the most intriguing aspects of pollen tube growth is the phenomenon of periodicity or oscillation in growth rate (Pierson et al., 1996
Animal cell studies have largely focused on the role of the actin cytoskeleton in vesicle trafficking (Merrifield et al., 1999
The fungal metabolite BFA has been widely used as a useful tool in studies of the relationship between membrane trafficking and secretion. It has been reported as inhibiting vesicle coat formation, blocking endoplasmic reticulum-to-Golgi trafficking, and disrupting the Golgi apparatus (Donaldson et al., 1992 In summary, a fluorescence excitation technique using variable penetration depths of evanescent waves was modified and applied to investigations of secretory vesicles trafficking in living P. meyeri pollen tubes. The secretory vesicles were labeled with the membrane-specific dye FM4-64. Using EWM, we observed exocytosis of secretory vesicles via a "full" fusion event, and we found that there were two types of vesicle motions showing different oscillation frequencies. Contrary to earlier investigations using confocal microscopy, our results indicate that secretory vesicle motions in living pollen tubes are not Brownian, and they are dependent on the actin cytoskeleton. In addition, BFA prominently inhibits vesicle formation, but not vesicle mobility, in living pollen tubes. EWM can be used not only as an independent assay for vesicle trafficking, but also as a new, powerful tool for studies on the dynamics of vesicles, organelles, and single molecules of plant cells, which may now be undertaken in much more detail than ever before.
Plant Materials Cones with mature pollen were collected from Picea meyeri Rehd. et Wils growing in the Botanical Garden of the Institute of Botany, Chinese Academy of Sciences, prior to the beginning of the pollination season in mid-April 2004. Cones were dried overnight at room temperature, and the dry pollen was stored at –20°C until further use.
Stored pollen was equilibrated at room temperature for 30 min and then carefully suspended in a culture medium (2 mg of pollen per mL medium) that contained 12% (w/v) Suc, 0.03% calcium chloride, and 0.01% boric acid in a shaker at 100 rpm. The pH of the medium was adjusted to 6.4 with phosphate buffered saline. Germination took place in the dark at 25°C.
FM4-64 dye was purchased from Sigma. In this experiment, loading of cells with FM4-64 dye was generally achieved by application at a concentration of 2 µM during the imbibition of pollen tubes by direct addition of dye solutions in the culture medium. After 5 min of loading with FM4-64, the medium containing dye was replaced with fresh medium lacking the dye.
Inhibitor stock solutions were made up in 100% ethanol (2 mM CD, 20 mM oryzalin, 50 mM colchicine, and 5 mg/mL BFA), except for LATB, which was a 1 mM stock in dimethyl sulfoxide. All of the inhibitors were purchased from Sigma. The final inhibitor concentrations in the germination media were up to 10 µM for CD, 10 nM for LATB, 100 µM for oryzalin, 5 mM for colchicine, and 5 µg/mL for BFA (Supplemental Fig. 2 shows the appropriate concentrations we selected). Actin cytoskeletons were disrupted using 10 nM LATB or 10 µM CD for 10 min after labeling with FM4-64 at 25°C, respectively. The microtubule was disrupted by incubating pollen tubes with 100 µM oryzalin or 5 mM colchicine for 10 min after labeling with FM4-64 at 25°C. BFA was applied together with FM dyes at a concentration of 5 µg/mL.
Sorbitol (100 µM) and sodium azide (500 µM) were applied directly to the medium. The pollen tubes were examined and digital images were acquired using a laser scanning confocal system installed on an inverted microscope (IX81; Olympus). The samples were excited at 514 nm with a 30-mW argon ion laser operated at full power at an intensity of 3%, achieved by means of neutral-density filters, with a nearly closed pinhole and the gain adjusted to below 7.00. Emission was detected with a 530- to 600-nm band-path filter (Zeiss R510; Carl Zeiss). Images were collected and processed using Adobe Photoshop 7.0 (Adobe Systems).
The total internal reflection system was constructed based on an inverted microscope (IX81; Olympus). Light from a multichannel argon laser (458, 488, 515 nm; 30 mW) was introduced to the microscope through a single-mode fiber and three illumination lenses. The light was focused at the back focal plane of a high aperture objective lens (Apo 100x OHR; NA 1.65; Olympus). This allowed us to generate evanescent waves with penetration depths from approximately 60 to 400 nm in aqueous solution (n = 1.33) at an excitation wavelength of 515 nm laser. FM4-64-labeled cells were visualized by excitation with an argon laser set to 514 nm. Emission was detected with a 530- to 600-nm band-path filter (Zeiss R510; Carl Zeiss).
Fluorescence was gathered through a 100x Apo OHR objective (NA 1.65; Olympus), and the evanescent wave microscope images were captured through the right lateral port of the inverted microscope onto a cooled charge-coupled device camera (Micromax, MMX-512-BFT; Princeton Instruments), which was operated with Metamorph 6.0 (Universal Imaging). Immersion oil with a high refractive index (n = 1.65) was used to bridge the optical contact between the objective and the cover glass, and the incident light for evanescent illumination was introduced from the objective lens. Time-lapse images were acquired every 200 ms and sampled into the computer through a frame grabber with genuine 16 Bit (216; 65,536 gray levels).
Image analysis was performed with Image-Pro Plus 5.1 (Media Cybernetics), Adobe Photoshop 7.0 (Adobe Systems), and ImageJ 1.34e (Wayne Rasband, National Institutes of Health). The raw images were filtered to enhance the visibility of the vesicles. A high-pass fast Fourier transform filter, followed by a 3- x 3-pixel trimmed mean filter, was applied to remove nonuniform background noise, and a flatten filter was used to smooth the background. The pixel size was 0.125 µm with a 1.6x optical zoom, and the image size was typically 326 x 484 pixels. The fluorescence intensity was expressed in the 8-bit value of digitization as described previously (Tsuboi et al., 2000
We thank Dr. Richard Turner, Dr. Mathem Benson, and Prof. Yuxi Hu for valuable discussion at the early stages of these experiments, and Dr. Bai Li for technical assistance with EWM. We also thank Dr. Lingan Kong for his valuable comments on an early draft of this manuscript. Received March 15, 2006; returned for revision June 6, 2006; accepted June 8, 2006.
1 This work was supported by the National Science Fund of China for Distinguished Young Scholars (grant no. 30225005) and a grant for general program (no. 30570100), together with grants from Deutsche Forschungsgemeinschaft (no. SA 1564/2–1 to J. .); from EU Research Training Network TIPNET (project no. HPRN–CT–2002–00265) obtained from Brussels, Belgium; from Slovak grant agency APVV (no. APVT–51–002302; Bratislava, Slovakia); from Slovak grant agency VEGA (no. 2/5085/25; Bratislava, Slovakia); and from Deutsches Zentrum für Luft- und Raumfahrt (Bonn).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jinxing Lin (linjx{at}ibcas.ac.cn).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.080168. * Corresponding author; e-mail linjx{at}ibcas.ac.cn; fax 0086–10–62590833.
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ABSTRACT
RESULTS
osc = 0.34 ± 0.08 per second (n = 12 vesicles). 
