Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K⁺ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.     

Apical epidermal growth factor receptor signaling: regulation of stretch-dependent exocytosis in bladder umbrella cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.     

Analysis of hydrostatic pressure-induced changes in umbrella cell surface area

All cells experience and respond to external mechanical stimuli including shear stress, compression, and hydrostatic pressure. Cellular responses can include changes in exocytic and endocytic traffic. An excellent system to study how extracellular forces govern membrane trafficking events is the bladder umbrella cell, which lines the inner surface of the mammalian urinary bladder. It is hypothesized that umbrella cells modulate their apical plasma membrane surface area in response to hydrostatic pressure. Understanding the mechanics of this process is hampered by the lack of a suitable model system. We describe a pressure chamber that allows one to increase hydrostatic pressure in a physiological manner while using capacitance to monitor real-time changes in the apical surface area of the umbrella cell. It is demonstrated that application of hydrostatic pressure results in an increase in umbrella cell apical surface area and a change in the morphology of umbrella cells from roughly cuboidal to squamous. This process is dependent on increases in cytoplasmic Ca(2+). This system will be useful in further dissecting the mechanotransduction pathways involved in cell shape change and regulation of exocytic and endocytic traffic in umbrella cells.     

Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition

The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane lipid composition on sorting of the endocytosed material. This suggests that changes in the membrane organization can be one of the underlying mechanisms for regulating the endocytosis/exocytosis processes and membrane intracellular trafficking.     

Membrane lipids and proteins as modulators of urothelial endocytic vesicles pathways

The increased studies on urinary bladder umbrella cells as an important factor for maintaining the permeability barrier have suggested new pathways for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. The biological role of these vesicles was defined, for many years, as a membrane reservoir for the umbrella cell apical plasma membrane which are subject to an increased tension during the filling phase of the micturition cycle and, therefore, the vesicles are fused with the apical membrane. Upon voiding, the added membrane is reinserted via a non-clathrin or caveolin-dependant endocytosis thereby restoring the vesicle cytoplasmic pool. However, in the last decade, new evidence appeared indicating alternative pathways of the endocytic vesicles different than the cycling process of exocytosis/endocytosis. The purpose of this review is to analyze the molecular modulators, such as membrane lipids and proteins, in the permeability of endocytic vesicles, the sorting of endocytosed material to lysosomal degradation pathway and recycling of both membrane and fluid phases.     

Endocytosis and exocytosis in hyphal growth

Two ancient processes, endocytosis and exocytosis, are employed by eukaryotic cells to shape their plasma membrane and interact with their environment. Filamentous fungi have adapted them to roles compatible with their unique ecological niche and morphology. These organisms are optimal systems in which to address questions such as how endocytosis is localized, how endocytosis and exocytosis interact, and how large molecules traverse eukaryotic cell walls. In the tips of filamentous (hyphal) cells, a ring of endocytosis encircles an apical crescent of exocytosis, suggesting that this area is able to support an endocytic recycling route, although both processes can occur in subapical regions as well. Endocytosis and exocytosis underlie growth, but also facilitate disease progression and secretion of industrially relevant compounds in these organisms. Here we highlight recent work on endocytosis and exocytosis in filamentous fungi.     

Apical membrane area of rabbit urinary bladder increases by fusion of intracellular vesicles: An electrophysiological study

Summary Mammalian urinary bladder undergoes, in a 24-hour period, a series of slow fillings and rapid emptying. In part the bladder epithelium accommodates volume increase by stretching the cells so as to eliminate microscopic folds. In this paper we present evidence that once the cells have achieved a smooth apical surface, further cell stretching causes an insertion of cytoplasmic vesicles resulting in an even greater apical surface area per cell and an enhanced storage capacity for the bladder. Vesicle insertion was stimulated by application of a hydrostatic pressure gradient which caused the epithelium to bow into the serosal solution. Using capacitance as a direct and nondestructive measure of area we found that stretching caused a 22% increase in area. Removal of the stretch caused area to return to within 8% of control. An alternate method for vesicle insertion was swelling the cells by reducing mucosal and serosal osmolarity. This perturbation resulted in a 74% increase in area over a 70-min period. Returning to control solutions caused area to decrease as a single exponential with an 11-min time constant. A microtubule blocking agent (colchicine) dit not inhibit the capacitance increase induced by hypoosmotic solutions, but did cause an increase in capacitance in the absence of a decreased osmolarity. Microfilament disrupting agent (cytochalasin B, C, B.) inhibited any significant change in capacitance after osmotic challenge. Treatment of bladders during swelling with C.B. and subsequent return, to control solutions increased the time constant of the recovery to control values (22 min). The Na⁺-transporting ability of the vesicles was determined and found to be greater than that of the apical membrane. Aldosterone increased the transport ability of the vesicles. We conclude that some constituent of urine causes a loss of apical membrane permeability. Using electrophysiological methods we estimated that the area of cytoplasmic vesicles is some 3.3 times that of the apical membrane area. We discuss these results in a general model for vesicle translocation in mammalian urinary bladder.     

Endocytosis and exocytosis protect cells against severe membrane tension variations

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.     

Compensatory endocytosis in bladder umbrella cells occurs through an integrin‐regulated and RhoA‐ and dynamin‐dependent pathway

Compensatory endocytosis (CE) ensures recycling of membrane components and maintenance of plasma membrane size; however, the mechanisms, regulation, and physiological functions of clathrin‐independent modes of CE are poorly understood. CE was studied in umbrella cells, which undergo regulated exocytosis of subapical discoidal/fusiform vesicles (DFV) during bladder filling, and may then replenish the pool of DFV by internalizing apical membrane during voiding. We found that voiding‐stimulated CE, which depended on β₁ integrin‐associated signalling pathways, occurred by a dynamin‐, actin‐, and RhoA‐regulated mechanism and was independent of caveolins, clathrin, and flotillin. Internalized apical membrane and fluid were initially found in ZO‐1‐positive vesicles, which were distinct from DFV, classical early endosomes, or the Golgi, and subsequently in lysosomes. We conclude that clathrin‐independent CE in umbrella cells functions to recover membrane during voiding, is integrin regulated, occurs by a RhoA‐ and dynamin‐dependent pathway, and terminates in degradation and not recapture of membrane in DFV.     

The uroepithelium: not just a passive barrier

The uroepithelium lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, where it forms a tight barrier that allows for retention of urine, while preventing the unregulated movement of ions, solutes, and toxic metabolites across the epithelial barrier. In the case of the bladder, the permeability barrier must be maintained even as the organ undergoes cyclical changes in pressure as it fills and empties. Beyond furthering our understanding of barrier function, new analysis of the uroepithelium is providing information about how detergent-insoluble membrane/protein domains called plaques are formed at the apical plasma membrane of the surface umbrella cells, how mechanical stimuli such as pressure alter exocytic and endocytic traffic in epithelial cells such as umbrella cells, and how changes in pressure are communicated to the underlying nervous system.     

Adenosine receptor expression and function in bladder uroepithelium

The uroepithelium of the bladder forms an impermeable barrier that is maintained in part by regulated membrane turnover in the outermost umbrella cell layer. Other than bladder filling, few physiological regulators of this process are known. Western blot analysis established that all four adenosine receptors (A₁, A_2a, A_2b, and A₃) are expressed in the uroepithelium. A₁ receptors were prominently localized to the apical membrane of the umbrella cell layer, whereas A_2a, A_2b, and A₃ receptors were localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Adenosine was released from the uroepithelium, which was potentiated 10-fold by stretching the tissue. Administration of adenosine to the serosal or mucosal surface of the uroepithelium led to increases in membrane capacitance (where 1 µF 1 cm² tissue area) of 30% or 24%, respectively, after 5 h. Although A₁, A_2a, and A₃ selective agonists all stimulated membrane capacitance after being administrated serosally, only the A₁ agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells. capacitance; exocytosis     

Stretch induces granule exocytosis in toad urinary bladder

Evidence from thin sections and freeze-fracture is presented showing that stretch induces a burst of exocytosis in granular cells of the toad urinary bladder epithelium. Since the role of granule exocytosis in hormonally-induced permeability changes in this tissue has not yet been clarified, we propose that the stretch factor is an important parameter to consider and standardise in future physiological and morpho-functional studies using this model transporting system.     

Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5–15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder

Summary This study concerns the timing and magnitude of exocytosis and endocytosis in the granular cells of toad bladder during the hydroosmotic response to antidiuretic hormone. Granule exocytosis at the luminal cell surface is extensive within 5 min of the administration of a physiological dose of hormone. Hydroosmosis becomes detectable during this time period. The amount of membrane added to the luminal surface by exocytosis during 60 min of exposure to hormone can be of the same order of magnitude as the extent of the luminal plasma membrane. Endocytosis, demonstrated by peroxidase uptake from the luminal surface, becomes extensive during the period 15–45 min after hormone administration. Thus, maximal endocytic activity occurs later than the period of most extensive exocytosis and seems to correlate with the onset of the decline in water movement. The amount of membrane retrieved from the luminal surface by endocytosis during 60 min of stimulation is at least three quarters of that added by exocytosis. The bulk membrane movement in ADH stimulated preparations does not require the presence of an osmotic gradient. Colchicine inhibits the hydroosmotic response, the exocytosis of granules, and endocytosis at the luminal surface. These results strengthen our view that the bulk circulation of membrane at the cell surface, via exocytosis and endocytosis, is closely related to the permeability changes occuring at the surface.     

The maintenance of the permeability barrier of bladder facet cells requires a continuous fusion of discoid vesicles with the apical plasma membrane

The luminal surface of the bladder epithelium is continuously exposed to urine that differs from blood in its ionic composition and osmolality. The apical plasma membrane of facet or umbrella cells, facing the urine, is covered with rigid-looking plaques consisting of hexagonal uroplakin particles. Together with tight junctions these plaques form a specialized membrane compartment that represents one of the tightest and most impermeable barriers in the body. Plaques also occur in the membrane of cytoplasmic discoid vesicles. Here it is shown shown that synaptobrevin, SNAP23 and syntaxin are perfectly colocalized with uroplakin III at the apical plasma membrane as well as with membranes of discoid vesicles. Such a distribution suggests that discoid vesicles in facet cells may gain access to the apical plasma membrane probably by combination of homotypic and heterotypic fusion events. Furthermore, we detected uroplakin III-containing membranes of different sizes in the urine of healthy humans and rats. Probably facet cells maintain their permeability barrier by a process of continuous membrane regeneration that includes the cutting off of areas of the apical membrane and its replacement by newly fused discoid vesicles.     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.     

A Highlights from MBoC Selection: A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells

Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a–Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.     

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

Pollen tubes are one of the fastest growing eukaryotic cells.Rapid anisotropic growth is supported by highly active exocytosisand endocytosis at the plasma membrane, but the subcellularlocalization of these sites is unknown. To understand molecularprocesses involved in pollen tube growth, it is crucial to identifythe sites of vesicle localization and trafficking. This reportpresents novel strategies to identify exocytic and endocyticvesicles and to visualize vesicle trafficking dynamics, usingpulse-chase labelling with styryl FM dyes and refraction-freehigh-resolution time-lapse differential interference contrastmicroscopy. These experiments reveal that the apex is the siteof endocytosis and membrane retrieval, while exocytosis occursin the zone adjacent to the apical dome. Larger vesicles areinternalized along the distal pollen tube. Discretely sizedvesicles that differentially incorporate FM dyes accumulatein the apical, subapical, and distal regions. Previous workestablished that pollen tube growth is strongly correlated withhydrodynamic flux and cell volume status. In this report, itis shown that hydrodynamic flux can selectively increase exocytosisor endocytosis. Hypotonic treatment and cell swelling stimulatedexocytosis and attenuated endocytosis, while hypertonic treatmentand cell shrinking stimulated endocytosis and inhibited exocytosis.Manipulation of pollen tube apical volume and membrane remodellingenabled fine-mapping of plasma membrane dynamics and definedthe boundary of the growth zone, which results from the orchestratedaction of endocytosis at the apex and along the distal tubeand exocytosis in the subapical region. This report providescrucial spatial and temporal details of vesicle traffickingand anisotropic growth. Key words: Endocytosis; exocytosis, hydrodynamics, lipophilic FM dyes, pollen tube growth, vesicle trafficking Received 14 September 2007; Revised 23 November 2007 Accepted 7 January 2008