Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching

Abstract

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.

Figure 1.

STICS and FRAP analyses of growing lily pollen tubes labeled with the lipophilic styryl dye FM1-43. A, Fluorescence micrograph showing a single optical section acquired in the LIVE mode of the Zeiss LSM 5. The strong label in the inverted cone at the apex of the tube indicates that vesicles are labeled intensely with the dye. The corresponding time-lapse series is provided as Supplemental Movie S1. Bar = 5 μm. B, Fluorescence micrograph superimposed on a corresponding DIC image of a pollen tube simultaneously labeled with FM1-43 (green) and MitoTracker (red). Mitochondria represent the population of larger organelles that reach farthest into the apical region of growing pollen tubes. The absence of colocalization confirms that in the apical region, the FM1-43 signal corresponds to labeled vesicles. Bar = 5 μm. C, STICS analysis of movements of FM1-43-labeled organelles in the cytoplasm of a lily pollen tube. The vector map overlying the micrograph reveals the direction and speed of particle motion in the pollen tube as analyzed over a 10-s period. The dominant motions occur forward at the periphery of the tube and rearward in the center of the tube. Numbers on color lookup tables are movement rates in μm s⁻¹. Bar = 10 μm. D, Example of the evolution of the correlation function corresponding to the vector marked with a magenta dot in the left panel. The five images at right correspond to the intensity of the two-dimensional correlation function calculated for the lags indicated (numbers in seconds). They reveal the shift in the position of the peak (dark red) as well as a broadening of its waist, resulting from a certain degree of random movement within the assessed region. Note that due to the mathematical calculations used in STICS, the vector indicates a direction opposite to the movement of the peak. Window size, 32 × 32 pixels. Bars = 1 μm. E to G, STICS analysis of vesicle movements in the cytoplasm of a pollen tube apex. Vector maps were obtained using 16 × 16 pixel windows and sequences of 50 (G) or 100 (E and G) frames. Each image shows individual small waves of vesicle motion. E, Strong motion from the shoulder of the apex into its center (arrow). F, Prominent motion within the inverted cone in the direction of its tail (arrow). G, Outward motion from the cone to the periphery of the tube in the subapical region (arrows). Numbers on color lookup tables are movement rates in μm s⁻¹. Bars = 5 μm. H, FRAP analysis of vesicle dynamics in the apex of a growing pollen tube. The time-lapse series shows a pollen tube labeled with FM1-43 after removal of the dye in the medium (first frame) and at various times during recovery after photobleaching. Numbers indicate time in seconds after photobleaching. During recovery from photobleaching, there is an initial accumulation of label in the shoulders of the apex. The complete time-lapse series is available as Supplemental Movie S3. Bar = 5 μm. I, Fluorescence micrograph showing a single optical section acquired in the 510 Meta mode. FM1-43 was not removed prior to imaging, revealing intense label of the plasma membrane. Bar = 5 μm. J, Fluorescence micrographs and corresponding DIC images of a pollen tube changing growth direction during FRAP analysis. The images correspond to the graph shown in Figure 2D. Numbers correspond to time in seconds after photobleaching. Images were acquired with the Zeiss 510 Meta system. Arrows in the DIC images indicate the growth direction. Arrows in the fluorescence images indicate the region of low fluorescence recovery at the extreme apex changing its position relative to the original growth direction. Bar = 10 μm.

Figure 2.

FRAP analysis of the cortical region of a growing pollen tube. A, Subdivision of the cortical and central regions of the pollen tube apex into zones. In this fluorescence micrograph, all pixels that were not completely black were given a value of 255 (red) to illustrate that no background noise was observed outside of the tube after FM1-43 labeling and subsequent removal of the dye from the medium. Inset, Corresponding DIC image. Bars = 10 μm. B to D, Three examples of recovery of fluorescence intensity in the cortical zones identified in A. B, The recovery of fluorescence is time delayed between the different sections, with the central sections being last to initiate recovery. C, Fluorescence intensity in the extreme apex (zones 1/−1) does not recover the same fluorescence intensity as the adjacent zones. D, Example of a pollen tube undergoing a change of growth direction during recovery. The central region of slow and incomplete fluorescence recovery shifts from zones 1/−1 to zones 1/2.

Figure 3.

Determination of turnover time of an inverted vesicle cone using FRAP analysis. A, Recovery of mean fluorescence in three central circular zones identified in Figure 2A. Two sets of arrows (one set pointing upward, the other set pointing downward) indicate peaks of fluorescence intensity seemingly moving through the three circular zones with an average delay of 2 to 3 s. B, Measured and theoretical turnover times for the vesicle population in the bleached apex. The solid line is the theoretical turnover time based on the number of vesicles required for material delivery sustaining pollen tube elongation. The experimental values and the resulting regression curve are the times measured for the inverted cone to reach a plateau in fluorescence intensity after photobleaching.

Figure 4.

Transmission electron micrograph of a median section of a freeze-fixed pollen tube illustrating the cylinder/hemisphere-shaped geometry of the tube and the distribution of vesicles in the shape of an inverted cone. R_i, Radius of the hemisphere-shaped apex and of the cylindrical shank; L_B, length of the cylindrical shank bleached during FRAP analysis. Bar = 3 μm.

Figure 5.

Schematic drawing illustrating the principal directions of vesicle flow in the apical region of a pollen tube. Following delivery into the apical region on the actin filaments forming the cortical fringe, vesicles are released into the apical cytoplasm in an annulus-shaped zone. Some of the vesicles that succeed in contacting the plasma membrane fuse with it and undergo exocytosis, whereas a significant portion might deliver their contents by a kiss-and-run mechanism. Vesicles that do not succeed in contacting the plasma membrane stream rearward within the cone-shaped vesicle pool. Many of these vesicles are recirculated back into the forward stream immediately in the subapical region. At the extreme apex, the principal activity is endocytosis, either by a clathrin-mediated or a clathrin-independent (smooth) mechanism. Clathrin-mediated endocytosis also takes place in a more distal region, based on observations by Derksen et al. (1995), Moscatelli et al. (2007), and Zonia and Munnik (2008). The position of the actin fringe is based on Lovy-Wheeler et al. (2005). Objects are not drawn to scale. For clarity, except for vesicles, no other organelle or the cell wall is drawn. The sketch of the tube at the bottom shows the position of the annulus-shaped release zone.