Rm. Parton et al., Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes, J CELL SCI, 114(14), 2001, pp. 2685-2695
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.