Received April 1, 2008
Accepted May 19, 2008
Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy (STICS) and fluorescence recovery after photobleaching (FRAP)
Jerome Bove , Benoit Vaillancourt , Jens Kroeger , Peter K. Hepler , Paul W. Wiseman , and Anja Geitmann *
Institut de Recherche en Biologie Vegetale, Universite de Montreal, 4101 rue Sherbrooke est, Montreal, Quebec, H1X 2B2, Canada; Department of Physics, McGill University, 3600 rue University, Montreal, Quebec, H3A 2T8, Canada; Biology Departement, University of Massachusetts, Amherst, MA 01003, USA; Department of Chemistry, McGill University, 801 rue Sherbrooke ouest, Montreal, Quebec H3A 2K6, Canada
* Corresponding author; email: anja.geitmann{at}umontreal.ca.
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 FM 1-43, we applied spatio-temporal correlation spectroscopy (STICS) on time lapse series obtained with high speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate towards the apex in the cell cortex, they accumulate in an annulus shaped region adjacent to the extreme tip and then turn back to flow rearwards in the center of the tube. Fluorescence recovery after photobleaching (FRAP) 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. FRAP 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. We therefore 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.
