Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

Abstract

Pollen tubes are one of the fastest growing eukaryotic cells. Rapid anisotropic growth is supported by highly active exocytosis and endocytosis at the plasma membrane, but the subcellular localization of these sites is unknown. To understand molecular processes involved in pollen tube growth, it is crucial to identify the sites of vesicle localization and trafficking. This report presents novel strategies to identify exocytic and endocytic vesicles and to visualize vesicle trafficking dynamics, using pulse-chase labelling with styryl FM dyes and refraction-free high-resolution time-lapse differential interference contrast microscopy. These experiments reveal that the apex is the site of endocytosis and membrane retrieval, while exocytosis occurs in the zone adjacent to the apical dome. Larger vesicles are internalized along the distal pollen tube. Discretely sized vesicles that differentially incorporate FM dyes accumulate in the apical, subapical, and distal regions. Previous work established that pollen tube growth is strongly correlated with hydrodynamic flux and cell volume status. In this report, it is shown that hydrodynamic flux can selectively increase exocytosis or endocytosis. Hypotonic treatment and cell swelling stimulated exocytosis and attenuated endocytosis, while hypertonic treatment and cell shrinking stimulated endocytosis and inhibited exocytosis. Manipulation of pollen tube apical volume and membrane remodelling enabled fine-mapping of plasma membrane dynamics and defined the boundary of the growth zone, which results from the orchestrated action of endocytosis at the apex and along the distal tube and exocytosis in the subapical region. This report provides crucial spatial and temporal details of vesicle trafficking and anisotropic growth.

Self-regulation in tip-growth: The role of cell wall ageing

“…Fayant et al, 2010) measurements that probe the local properties of the wall with quantitative measurement on the local rate of exocytosis (see e.g. Zonia and Munnik, 2008) may serve to validate some of the ideas presented here.…”

“…the apical region, than areas farther away along the tube. Finally, it should be mentioned that there is strong experimental evidence that in several tip-growing cells the maximum rate of vesicle delivery is not at the apex, but rather at an annular region just downstream from the apex (Bove et al, 2008;Zonia and Munnik, 2008). This could in principle be modelled by a non-monotonic supply factor, with a maximum at an apex distance s max > 0, but we chose to forego these additional complexities here.…”

Pollen Proteins Bind to the C-terminal Domain of Nicotiana alata Pistil Arabinogalactan Proteins

“…For 120K to arrive at a vacuole, it must travel from the ECM through the pollen tube endomembrane system. Recent work has highlighted two distinct endocytic pathways in pollen tubes (54,55). Both clathrin-coated and noncoated vesicle endocytic pathways exist; clathrincoated vesicles traffic to the trans-Golgi, whereas noncoated vesicles travel to the multivescular body and a vacuole.…”

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

“…However, the PME data in tobacco challenge this view by showing that the inverted cone stains much less brightly than the flanks surrounding the cone ; data herein). It is possible that material moves forward along the edge of the cell, being directed by the cortical actin fringe (Lovy-Wheeler et al, 2005), and deposits as an annulus outside of the polar axis (Bove et al, 2008;Zonia and Munnik, 2008), as has been shown for root hairs (Shaw et al, 2000;Dumais et al, 2004). This is an attractive idea, but more work is needed to establish its occurrence in pollen tubes.…”