Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes

Regulated secretory vesicle delivery, vesicle fusion and rapid membrane rec ycling are all contentious issues with respect to tip growth in plant, fung al and animal cells. To examine the organisation and dynamics of membrane m ovements at the growing pollen tube apex and address the question of their relationship to growth, we have used the membrane stain FM4-64 both as a st ructural marker and as a quantitative assay. Labelling of living Lilium Lon giflorum pollen tubes by FM4-64 resulted in a distinct staining pattern in the tube apex, which corresponds spatially to the previously identified con e-shaped 'apical clear zone' containing secretory vesicles. Dye uptake coul d be inhibited by sodium azide and followed a strict temporal sequence from the plasma membrane to a population of small (1-2 mum diameter) discrete i nternal structures, with subsequent appearance of dye in the apical region and ultimately in vacuolar membranes. Washout of the dye rapidly removed th e plasma membrane staining, which was followed by a gradual decline in the apical fluorescence over more than an hour. Injected aqueous FM4-64 solutio n showed a relatively even distribution within the pollen tube. Association of FM4-64 with apical secretory vesicles was supported by the effects of t he inhibitors Brefeldin-A and Cytochalasin-D, which are known to affect the localisation and number of such vesicles, on the FM4-64 staining pattern. Examination of the dynamics of FM4-64 labelling in the pollen tube tip by t ime-lapse observation, supported by fluoreseence-recovery-after-photobleach ing (FRAP) analysis, suggested the possibility of distinct pathways of bulk membrane movement both towards and, significantly, away from the apex. Qua ntitative analysis of FM4-64 distribution in the apex revealed that fluctua tions in fluorescence 5 to 10 mum subapically, and to a lesser extent the a pical 3 mum, could be related to the periodic oscillation in pollen tube gr owth rate. This data reveals a quantitative relationship between FM4-64 sta ining and growth rate within an individual tube.