Pollen tube extension is an excellent example of polarisedgrowth and provides an ideal model system for understandingthe processes of organisation and regulation involved. Centralto this growth, and to the control of growth rate and orientation,are the processes of localised delivery to and secretion(exocytosis) of cell wall material and membrane at the tubeapex (Messerli et al., 2000; Li et al., 1999; Roy et al., 1999;Holdaway-Clarke et al., 1997; Malhó and Trewavas, 1996;Derksen et al., 1995; Steer and Steer, 1989).
At the pollen tube tip Golgi-derived secretory vesiclescontaining cell wall materials are accumulated in a particularregion of the pollen tube apex (defined in Lancelle and Hepler,1992, as the apical clear zone) prior to fusion with the apicalplasma membrane (Heslop-Harrison, 1987; Picton and Steer,1982; Picton and Steer, 1983; Van Der Woude et al., 1971).Studies to date on pollen tube growth suggest that secretoryvesicle production and delivery to the clear zone are notregulated with respect to the average growth rate (Roy et al.,1998; Picton and Steer, 1981; Picton and Steer, 1982; Pictonand Steer, 1983). Furthermore, it has been estimated thatmembrane incorporation at the apex exceeds the increase inmembrane area required for tip extension and hence a
requirement for active membrane recovery from the tip wasproposed (Steer and Steer, 1989). Membrane uptake in thepollen tube tip (endocytosis) has been supported by thediscovery of clathrin-coated vesicles (Derksen et al., 1995), thelocalisation of clathrin (Blackbourn and Jackson, 1996) anduptake of cell impermeant fluorescent markers (O’Driscoll etal., 1993). Whether membrane uptake is localised at theextreme apex or in a region behind the clear zone is stillundecided.
How the balance between secretory vesicle delivery,membrane recovery and growth rate is organised has yet to befully understood, especially with the discovery that tipextension rate in the pollen tube is not constant but varies inan oscillatory fashion (Feijó et al., 2001). A correlationbetween oscillatory fluctuations in growth rate and oscillatoryfluctuations in the magnitude of the tip-focused calciumgradient has been established; the latter was proposed as themechanism regulating secretory vesicle delivery at the apex(Messerli et al., 2000; Holdaway-Clarke et al., 1997).
Work in a variety of experimental systems has led to a betterunderstanding of the mechanisms of exocytotic and endocyticpathways (Zheng and Yang, 2000; Edwardson, 1998; Pelham,1997). However, understanding the dynamics of membranedelivery to and recycling from the apical plasma membrane in
2685
Regulated secretory vesicle delivery, vesicle fusion andrapid membrane recycling are all contentious issues withrespect to tip growth in plant, fungal and animal cells. Toexamine the organisation and dynamics of membranemovements at the growing pollen tube apex and address thequestion of their relationship to growth, we have used themembrane stain FM4-64 both as a structural markerand as a quantitative assay. Labelling of living LiliumLongiflorum pollen tubes by FM4-64 resulted in a distinctstaining pattern in the tube apex, which correspondsspatially to the previously identified cone-shaped ‘apicalclear zone’ containing secretory vesicles. Dye uptake couldbe inhibited by sodium azide and followed a strict temporalsequence from the plasma membrane to a population ofsmall (1-2 µm diameter) discrete internal structures, withsubsequent appearance of dye in the apical region andultimately in vacuolar membranes. Washout of the dyerapidly removed the plasma membrane staining, which wasfollowed by a gradual decline in the apical fluorescenceover more than an hour. Injected aqueous FM4-64 solution
showed a relatively even distribution within the pollen tube.Association of FM4-64 with apical secretory vesicles wassupported by the effects of the inhibitors Brefeldin-A andCytochalasin-D, which are known to affect the localisationand number of such vesicles, on the FM4-64 stainingpattern. Examination of the dynamics of FM4-64 labellingin the pollen tube tip by time-lapse observation, supportedby fluorescence-recovery-after-photobleaching (FRAP)analysis, suggested the possibility of distinct pathways ofbulk membrane movement both towards and, significantly,away from the apex. Quantitative analysis of FM4-64distribution in the apex revealed that fluctuations influorescence 5 to 10 µm subapically, and to a lesser extentthe apical 3 µm, could be related to the periodic oscillationin pollen tube growth rate. This data reveals a quantitativerelationship between FM4-64 staining and growth ratewithin an individual tube.
Key words: Tip growth, Oscillation, Pollen tube, Polarity, Vesicles,FM4-64
SUMMARY
Dynamics of the apical vesicle accumulation and therate of growth are related in individual pollen tubesR. M. Parton*, S. Fischer-Parton, M. K. Watahiki and A. J. TrewavasInstitute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JH, UK*Author for correspondence (e-mail: [email protected])
actively tip-growing systems has to some extent beenhampered by the lack of an adequate means to visualise andtrack these processes in action. Dyes such as FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl) pyridinium dibromide] promise the possibility ofa new perspective on such dynamic activities at the growingapex. Such amphiphilic fluorescent dyes have been developedwith animal cell systems as tools for the study of membranetrafficking in living cells (Betz et al., 1996). FM4-64 acts as amembrane marker, being only weakly fluorescent in water witha quantum yield that increases dramatically when associatedwith membranes (Betz et al., 1996). This dye has been used inyeast to follow membrane uptake and transport to the vacuole(Vida and Emr, 1995). More recently, the application of FM4-64 to track membrane trafficking in tip growing plant andfungal cells has emerged (Belanger and Quatrano, 2000;Fischer-Parton et al., 2000; Hoffmann and Mendgen, 1998).
A possible problem with the use of FM-dyes is theambiguity in their mode of internalisation by certain cell typesand subsequently their internal localisation (Fischer-Parton etal., 2000; Nishikawa and Sasaki, 1996). We have addressed thisissue in pollen tubes and present evidence of an endocyticuptake mechanism and localisation to membrane vesicles at thepollen tube apex. Analysis of FM4-64 staining in growingpollen tubes reveals a distinct apical staining pattern, dynamicmovements of FM4-64 stained material and a quantitativerelationship with growth rate in individual tubes. Thesignificance of these observations is discussed in relation to therole of the apical vesicle accumulation in pollen tube extension.
MATERIALS AND METHODS
Chemicals and materialsChemicals for culture media were obtained from BDH Chemical Co.(Poole, UK) or Fisher Scientific (Loughborough, UK) unlessotherwise stated. Dyes and inhibitors were obtained from MolecularProbes Europe (Leiden, Netherlands) and were prepared as directedby the manufacturer.
Plant materialPollen of Lilium longiflorumwas collected from freshly cut flowersobtained locally. Anthers were dried at room temperature for two daysthen vortexed vigorously to release the pollen, which was aliquoted,frozen and stored at −80°C.
Pollen culture and handlingPollen was imbibed for 5 minutes in culture medium (modified fromFeijó et al., 1999): 1.6 mM H3BO3, 2.0 mM CaCl2, 1.0 mM KCl, 5%sucrose, 0.05 mM MES before being transferred to thin layers of 0.2%Gellangum (from Wako, Osaka, Japan) solidified culture medium.Thin gel layers (optimised for imaging) were prepared on washed No.1g, 48×64 mm, 0.005% polylysine-coated glass coverslips (ChancePropper, Smethwick, UK) by sandwiching hot medium between thecoverslip and a top plate and subsequently sliding off the latter aftersolidifying the medium at 4°C. Thin gel layers were sown with pollensuspension and incubated in a dark humid environment at 25°C for 3-48 hours.
Confocal microscopyConfocal microscopy was performed with a Leica TCS NT confocalmicroscope (Leica Microsystems Heidelberg GmbH, Germany) fittedwith a 100 mW argon ion laser (488 or 514 nm excitation), a 25 mW
Krypton laser (568 nm excitation) and tunable emission wavelengthcollection. A ×63 Leica water immersion plan apo (NA 1.2) objectivewas used throughout. Bright field imaging was performed with thetransmitted light detector of the TCS NT. For quantitative imaging ofFM4-64, the requirements for image capture and analysis discussedpreviously (Parton and Read, 1999) were adhered to. Axial resolutionwas assessed by imaging 210 nm fluorescent beads (MolecularProbes: excitation 514 nm; emission 580-700 nm) according to Partonand Read (Parton and Read, 1999). The ‘full-width-half-maximum’estimated axial resolution (optical sectioning) was 0.85-0.95 µm;lateral resolution was 0.3-0.35 µm.
Loading cells with FM4-64 dye was generally achieved byapplication (at 4-8 µM) during the imbibition of pollen grains in liquidmedium, or by direct addition of dye (1-3 µM) in 115% liquidmedium, to growing tubes on thin gel layers. Injection of FM4-64 wasperformed using the Nanoject pressure injector (World PrecisionInstruments) with borosilicate glass needles pulled using a Sutter P97microelectrode puller. Viability of cells was assessed by growth rateand appearance. Extensively irradiating dye-loaded cells was found tobe deleterious; therefore, laser dosage was minimised by controllinglaser power and laser dwell time.
Fluorescence-recovery-after-photobleaching (FRAP) experimentswere performed on the Leica confocal using the 514 nm laser line andthe bleach protocol of the TCSNT software.
Time-course analysis was performed using the Leica TCSNTsoftware time-course option (1-4 frames per second). Numbered stillswere converted to AVI file format using Adobe Premiere (version 5.1)software to view as video clips.
Observations were based upon at least five pollen tubes unlessotherwise stated.
RESULTS
FM4-64 displays a distinct staining pattern in L.longiflorum pollen tubesIn median confocal optical section (Fig. 1A), FM4-64consistently produced a distinct bright peripheral staining andbright, V-shaped apical staining. The brightness of theperipheral staining was dependent upon the availability ofextracellular dye and diminished with increasing time after dyeapplication. Peripheral staining was shown to be plasma-membrane-associated, not cell-wall associated, by plasmolysisin the presence of FM4-64 (Fig. 2). Growth of L. longiflorumpollen tubes was unaffected by the presence of FM4-64 at theconcentrations used (continuing at normal growth rates of 5-25 µm/minute) and, although growth rate diminished withtime, it continued for up to 48 hours. Pollen tubes of Nicotianatabacum, N. Plumbaginifolia and Agapanthus umbellatusshowed similar FM4-64 staining patterns to L. longiflorum(data not shown).
Apical FM4-64 staining of L. longiflorum pollen tubescoincided approximately with the area known commonly as the‘apical clear zone’, described previously (Lancelle and Hepler,1992) as a vesicle rich V-shaped region of the apex excludinglarge organelles (Fig. 1; Fig. 3). Careful analysis of manymedian optical section confocal images of FM4-64fluorescence in growing tubes consistently revealed a patternof a sharply defined high signal in the extreme apical 3-5 µmregion. Beyond this region, FM4-64 staining was generallylower and varied considerably in both fluorescence signal anddistribution (Fig. 1; Fig. 3). Examination of FM4-64 stainingin three dimensions by rapid confocal optical sectioning
JOURNAL OF CELL SCIENCE 114 (14)
2687Vesicle accumulation and growth rate in pollen
through different focal planes (Z-series) showed the radialsymmetry of pollen tubes. Rather than a simple cone shape,FM4-64 staining defined a distinct, bright, lens-shaped apicalregion with a dimmer, much less distinct region of stainingmore subapically, often extending considerably away from theapex as a narrow ‘tail’ (Fig. 1; Fig. 3). In many images an area
of reduced staining could be observed that separated theextreme apical region and more subapical regions of staining.
FM4-64 shows time- and energy-dependentinternalisationUptake of FM4-64 into L. longiflorumpollen tubes followed astrict time sequence (Fig. 4; Fig. 5). Immediately after dyeapplication, staining associated with the plasma membrane wasobserved. Within 1-2 minutes, clear dye internalisation couldbe discerned that was associated with small, near-sphericalstructures located behind the apex (Fig. 4B,C). Thesestructures moved towards the tip with the cytoplasmicstreaming but tended to be excluded from the extreme apicalregion; they persisted as long as high concentrations of external
Fig. 1.FM4-64 staining distribution in a growing pollen tube of L.longiflorum. (A) Typical median focal plane confocal optical section;the bright field image at 1/3 size appears as an insert. (B) Pixelvalues along a central transect through the fluorescence image in (A).(C) Representative diagram of FM4-64 staining in the L. longiflorumpollen tube apex. The apical V-shaped ‘clear zone’ of secretoryvesicles defined previously (Lancelle and Hepler, 1992) on the basisof electron microscopy analysis is shown in relation to the different‘zones’ defined by the FM4-64 signal (see text).
Fig. 2.Plasmolysis of an FM4-64-loaded L. longiflorumpollen tube.(A) 5 minutes after applying standard medium with 0.3 M sorbitoland 2 µM FM4-64. The detector sensitivity was increased to showdye fluorescence associated with Hechtian strands at the apex(arrowhead). (B) Corresponding bright field image. Bar, 15 µm.
Fig. 3. (A-E) Sequence of confocal optical sections (Z-series) takenat 1.25 µm stepped focus positions through a growing FM4-64-stained L. longiflorum pollen tube. Half of the ten-image sequence isshown from the periphery to the median focal plane. (The other fiveimages of the sequence (not shown) were very similar reflecting theradial symmetry of the pollen tube.) Pixel intensity values along acentral transect through each image are shown. Bar, 15 µm.
2688
dye were present (indicated by bright peripheral staining).During the following 3-10 minutes a fainter, more diffuse,internal staining could be seen, initially behind the extremeapex but soon spreading to the whole apical 20-25 µm region.Only after 10-15 minutes did the typical FM4-64 staining(described in Fig. 1) become apparent (Fig. 4H-J; Fig. 5A,B).Staining intensity reached a relatively stable state ~30 minutesafter dye application. By this time peripheral staining wasreduced relative to the internal fluorescence and the earlystaining spherical structures were more difficult to distinguish.Further application of dye at this point increased peripheralstaining and resulted in a ‘reappearance’ of the sphericalstructures seen earlier (data not shown). It is likely that the freeexternal FM4-64 concentration is reduced with increasing timeafter application as the dye becomes sequestered or diluted.From 30-60 minutes, the staining pattern was indistinguishablefrom that achieved with L. longiflorum pollen tubes culturedfor 4-7 hours after FM4-64 applied to imbibed spores (seeMaterials and Methods; Fig. 1A) and did not appreciably differover the following 12 hours. By ca. 24 hours a distinct stainingof subapical structures, possibly vacuolar membranes, haddeveloped (Fig. 5C,D). The apical staining pattern, althoughsomewhat fainter, was still visible, whereas the peripheral
(plasma membrane) staining could not be easily distinguishedfrom internal fluorescence levels.
Introduction of FM4-64 into pollen tubes by microinjectionproduced a different staining pattern to that described above(Fig. 6). Injected dye spread slowly from the site of injectionto give a general fluorescence with no distinctive localisation.No bright peripheral staining was seen. Despite the absence ofexternal dye, the internal dye staining persisted, diminishingonly gradually with time (>30 minutes) after injection.
Uptake of FM4-64 was inhibited in a concentration-dependent manner by the metabolic inhibitor sodium azide(Fig. 7). Concentrations of azide that arrested growth but didnot halt cytoplasmic streaming allowed uptake to progress at areduced rate (Fig. 7A), whereas higher concentrations (Fig.7B), which inhibited both growth and streaming, effectivelyhalted uptake.
Removal of FM4-64 from pollen tubes could be achieved bycontinuous perfusion with dye-free medium (Fig. 8). Dye wasrapidly removed from the PM but persisted for >2 hours withinthe apical region, declining slowly with time. Removal of dyefrom different regions of internal staining all showed similarkinetics of decline in fluorescence intensity (Fig. 8B).
JOURNAL OF CELL SCIENCE 114 (14)
Fig. 4.FM4-64-uptake time course in a growing L. longiflorumpollen tube. (A-J) Median confocal fluorescence images at increasingtimes (minutes) after addition of FM4-64 (2 µM in 115% standardmedium). To avoid osmotic perturbation, pollen tubes werepretreated with 115% medium before dye application. Inserts showbright field images at 1/3 size. Bar, 20 µm.
Fig. 5.Median focal plane images of FM4-64-stainedL. longiflorumpollen tubes (A) 15 minutes, (B) 30 minutes, (C) 24 hours and (D) 48hours after staining. Inserts show bright field images at 1/3 size. Barin A,B, 15 µm; bar in C,D, 15 µm.
2689Vesicle accumulation and growth rate in pollen
Inhibitors affecting tip growth redistribute the FM4-64 staining patternTwo well-characterised inhibitors known to affect tip growthin pollen tubes, in particular the number and distribution ofsecretory vesicles, were tested on FM4-64-loaded tubes toassess the effect on dye distribution.
Brefeldin A (BFA) treatment consistently disrupted theFM4-64 staining pattern and arrested growth in L. longiflorumpollen tubes (Fig. 9). The timing of growth arrest wasdependent on the concentration applied, with higherconcentrations acting faster (Fig. 9B). The decline in growthwith inhibitor concentrations ≥3.6 µM was generally rapidafter onset and coincided with the disappearance of the brightapical FM4-64 staining (Fig. 9B; 3.5-5.5 minutes). BrefeldinA was co-applied with the BFA-Bodipy FL conjugate, whichacted as a useful marker of BFA uptake after application.Staining with this dye was very rapid, appearing within thefirst minute after application (data not shown). After thedisappearance of bright apical FM4-64 staining, a weakeraccumulation of stained material emerged at the apex (Fig. 9B,
from 6.5 minutes), matching reports of the development ofan apical aggregation of membrane material (Rutten andKnuiman, 1993).
Cytochalasin D (0.5-1.5 µM) treatment of L. longiflorumpollen tubes produced distorted and reduced growth within 2minutes of application (Fig. 10). Co-incident with this was theloss of the normal cytoplasmic streaming pattern and bothrapid and severe disruption in the pattern of FM4-64 staining.Dissociation and dissipation of FM4-64 staining rapidlyprogressed throughout the apex (Fig. 10; 25-55 seconds).Within 5-10 minutes after application, FM4-64 staining hadlargely redistributed from the apex and became scattered inpatches over more subapical regions. By this time growth wasalmost completely arrested and streaming in the apicalcompartment was reduced to discontinuous and erraticmovements.
FM4-64 reveals bulk membrane flow away from theapexThe pathway of cytoplasmic streaming is well known inpollen tubes (Heslop-Harrison, 1987) and follows a ‘reversefountain’, modelled in Fig. 11J from time-lapse imaging (datanot shown) of the trajectories of large lipid storage vacuoles,amyloplasts and fluorescently labelled mitochondria. Bystudying time-lapse confocal image sequences of FM4-64-stained growing pollen tubes at rates of between one and fourframes per second, the movements of FM4-64-stained materialcould also be seen. Most obvious was the progression of‘brighter’ patches of FM4-64-stained material from close
Fig. 6. Injection of aqueous FM4-64 into a L. longiflorumpollentube. Insert shows the bright field image at 1/3 size. (A) Tip region;(B) subapical region. Bar, 15 µm.
Fig. 7.Effects of sodium azide on FM4-64 uptake by L. longiflorumpollen tubes. (A) 30 minutes after applying 2 µM dye to a tube pre-treated for 2 minutes with 500 µM sodium azide. (B) Similartreatment as in A with 1 mM sodium azide pre-treatment. Dye wasapplied in the continued presence of inhibitor. Inserts show brightfield images at 1/3 size. Bar, 15 µm.
Fig. 8.FM4-64-washout time course from a growing pollen tube ofL. longiflorumpreloaded with dye for 3 hours. Washout was achievedby continuous perfusion with dye-free medium. (A) Selected imagesshowing the pattern of decline in fluorescence; times relative to thestart of perfusion are given in minutes. (B) Plots of normalisedfluorescence intensity within four regions, defined in A by the areasa-d: (a) extreme apex, (b) subapical region, (c) plasma membrane(plotted as c minus d) and (d) periphery. Bar, 15 µm.
2690
behind the extreme apical 3-5 µm stained regionaway from the tip along the reverse cytoplasmicflow at the centre of the tube, which could befollowed over several frames (Fig. 11A-C,J-K).An apparent ‘cycling’ of patches of FM4-64-stained material from >10 µm from the extremeapex back towards the extreme apical regionwas also occasionally seen, appearing as an‘eddy’ in the general movement away from thetip (Fig. 11D-K). Movement of individualvesicles towards the apex could not be seenusing confocal fluorescence imaging; individualvesicles were subresolution but wouldcontribute to the low-level backgroundfluorescence outside the brightly stained apicalV-shaped region. However, in video sequences small patchesof FM4-64-stained material could be clearly seen movingalong the tube periphery towards the apex.
To track the movements of FM4-64-stained material, thetechnique of FRAP was employed. Irradiating the region 20-30 µm behind the tip resulted in an area of bleaching, whichwas invariably displaced away from the apex along thepathway of reverse flow, in agreement with the earlier time-course observations (Fig. 12A,B). Moderate photobleachingtreatment of regions along each flank behind the apex (lateralregion) produced a rapid transient reduction in the alreadyweak fluorescence of that region. It also subsequently causeda transient reduction in the fluorescence intensity within thebright apical 3-5 µm region and in more subapical regions,without stopping growth (Fig. 12C,D). Irradiating growingtubes within the extreme apical 5 µm region, with laserintensities sufficient to cause bleaching in other regions (or atthe apex of growth-arrested tubes), failed to produce distinctbleached ‘spots’.
FM4-64 staining fluctuations suggests a possiblecorrelation with growth fluctuations in individualpollen tubesThe typical FM4-64 staining pattern was only seen in normallygrowing pollen tubes. Staining pattern was similar in bothyoung (<700 µm long) and old (>1000 µm long) tubes and overa range of growth rates from 5 to >20 µm/minute. No obviouscorrelation between growth rate and staining pattern withinpopulations of tubes was found. In non-growing or stressedpollen tubes the apical FM4-64 stained region was variouslydisorganised or absent (data not shown).
In individual pollen tubes >3 hours after germination, thegrowth rate fluctuated in a regular oscillatory fashion (Fig.13A) with a period between 35 and 45 seconds. Quantitativeanalysis of FM4-64 staining intensity in median sectionconfocal fluorescence images (see Fig. 1; Fig. 13E) taken atregular time intervals revealed that the FM4-64 fluorescence
signal also varied in a regular oscillatory manner with a similarperiod to the oscillatory growth-rate fluctuations (Fig. 13A-D).When examined as video clips, time-lapse images showed thatthe previously described movements of material from behindthe apical 3-5 µm region accounted for the fluctuations in FM4-64 signal variation, which in turn could be related to thefluctuations in growth rate. The most obvious variation influorescence was found in the region between 5 and 15 µmbehind the extreme apex. The apical 1-3 µm sample areashowed a low-amplitude, less-clearly-defined variation influorescence. Slight differences in magnitude of the growth-rate fluctuations could be related to variations in peakfluorescence intensity. This suggested that the phase of theoscillation in FM4-64 fluorescence was shifted relative to the
JOURNAL OF CELL SCIENCE 114 (14)
Fig. 9.Time course of changes in growth rate anddye distribution of FM4-64-loaded L. longiflorumpollen tubes treated with BFA. (A) Confocalfluorescence images of a pollen tube at differenttimes (in minutes) following treatment with 3.6 µMBFA. (B) Typical growth rates for tubes treated with0.36, 3.6, 18 and 36 µM BFA. An arrow indicatesaddition of BFA. Bar, 15 µm.
Fig. 10.The effects of Cytochalsin D treatment on the FM4-64staining pattern of an L. longiflorumpollen tube; times are inseconds. Bar, 15 µm.
2691Vesicle accumulation and growth rate in pollen
Fig. 11.Movement of FM4-64-stainedmaterial in the apex of a L. longiflorumpollen tube (average growth rate 21.9µm/minute). (A-C) Median sectionconfocal images taken 2 seconds apartshowing bulk movement of FM4-64-stained material from a region ~5 µmbehind the apex away from the tip alongthe path of cytoplasmic streaming(arrows). (D-I) Images from the sametime series, taken 1 second apart,showing movement of FM4-64-stainedmaterial from a region ~25 µmsubapically back towards the apex(arrows). Diagrammatic interpretation ofthe ‘reverse fountain’ path ofcytoplasmic streaming in a growing L.longiflorum pollen tube (J) (see maintext), and of the movement of FM4-64-stained material based upon time-sequence imaging (see A-I) and FRAPexperiments (see text for details) (K).Bar, 20 µm.
Fig. 12.Fluorescence recovery afterphotobleaching (FRAP) analysisshowing the direction of movement ofFM4-64-stained material around thepollen tube. (A,B) Movement from theapex down the centre of the cell to moresubapical regions. (A) Time sequence ofconfocal fluorescence images; timerelative to bleaching is in seconds. Theoriginal area of bleaching is shown asthe boxed area. (B) Pixel intensity plotsalong a central transect through theimages in A. The original bleached zoneis indicated as the boxed area. Therelative position of the original bleachedzone to the apex is shown as the dashedboxed area; graphs have been alignedfrom the cell apex. (C,D) Movementfrom subapical peripheral regions intothe apex. (C) Time sequence of confocalfluorescence images; the first imageindicates the lateral bleaching areas (boxa). (D) Graphs of fluorescence intensityagainst time for the areas indicated a-c.The relative position of the sample areasto the apex, shown in the first image of(C), was maintained throughout. Thegrey shaded area indicates time ofbleaching. Bars, 15 µm.
2692
variation in growth rate, with thepeak FM4-64 fluorescencepreceding peak growth rate by 5-10seconds. It was also noted that‘isolation’ of the apical 3-5 µmregion of staining from the moresubapical regions of staining wasmost apparent (i.e. most clearlydefined in plots of pixel intensityalong a median transect; Fig. 1; Fig.13E) at around the time of peakgrowth rate (see grey bars over thegraphs in Fig. 13).
Growth on high sucrose medium(15%) caused a reproduciblereduction in growth and slowercytoplasmic streaming (asdescribed by Li et al., 1996), ashorter apical clear zone and also anappreciable alteration in the apical FM4-64 staining pattern(Fig. 14A). FM4-64 staining distribution was significantlyreduced, less extended subapically (lacking a significant tail)and showed far less distinct ‘reverse’ movement of materialfrom behind the apex into more subapical regions.
Growth on high calcium medium (25-10 mM) causeddelayed germination and slow growth (<1 to ~5 µm/minute)with thickening of the apical cell wall (Fig. 14B). Despite theconsiderably slowed growth, FM4-64 staining under theseconditions did not differ greatly from that observed withnormal growth.
DISCUSSION
In this study we investigated dynamic membrane movementsat the pollen tube apex in relation to tip growth, based uponFM4-64 staining. In order to establish FM4-64 staining as a
marker of membrane movement, the pathway of FM4-64internalisation and its distribution within the pollen tube wereinvestigated. Subsequent analysis of FM4-64 staining patternin the growing pollen tube revealed a quantitative relationshipwith oscillations in growth rate.
Endocytic internalisation of FM4-64 in the pollentubeIn the current study we used FM4-64 as a tracer of membranemovement in the pollen tube apex. In other cell systems thisand other related FM-dyes have been used to follow bothvesicle trafficking and endocytosis (Cochilla et al., 1999).
Our data showed a distinct intracellular localisation of FM4-64 staining, which corresponds strikingly, in both location anddistribution, to the accumulation of exocytotic vesiclescontaining cell wall material within the apical clear zone(defined by EM studies of different pollen tube species(Derksen et al., 1995; Lancelle and Hepler, 1992)).
JOURNAL OF CELL SCIENCE 114 (14)
12090
100
110
120
130
140
150
160
abso
lute
pix
el v
alu
e
70
80
90
100
110
120
130
140
abso
lute
pix
el v
alu
e
120 140 160 180 200 220 240 26050
60
70
80
90
100
110
120
abso
lute
pix
el v
alu
e
0
4
8
12
16
20
grow
th r
ate
(µm
/min
ute)
A
B
C
D Area C – 10 µm behind the apex
Area B – 5 µm behind the apex
Area A – apical 1-3 µm
Interval growth rate
Etime (seconds)
FABC
ABC
lowest growthrate
Peak growthrate
Peak growthrate
LengthPixel intensity
0
150
157
0
0
0
0
0
0
138
137
152
149
151
153
0
144
0
0
144
0
143
15 µm
Fig. 13.Quantitative analysis of therelationship between FM4-64 stainingand oscillatory growth of a L.longiflorum pollen tube. (A) Growthrate for a pollen tube derived fromconfocal images of FM4-64fluorescence. (B-D) Correspondingplots of average pixel value againsttime for the regions A-C defined indiagram (E). Heavy dashed lines showpeak growth rate; grey boxed areasshow the times when the apical 3-5 µmregion is most distinctly defined fromthe more subapical FM4-64 staining(see Results and F). (E) Sample areasA-C: apical 1-3 µm, 5 µm and 10 µmsubapically, respectively. (F) Graphs ofpixel values along a central transect ofthe pollen tube are shown (as in Fig. 1)for images covering the growth periodoutlined by the cross-hatched area inA. The maximum and minimum rategrowth phases of the period areindicated.
2693Vesicle accumulation and growth rate in pollen
Examining the time-course of dye uptake suggested that dyeis internalised from the plasma membrane by a pathwayinvolving particular endomembrane compartments: an earlyuptake compartment (possibly endosomes or the Golgiapparatus), apical vesicle membranes and ultimately thevacuolar membrane. A similar sequence of uptake has beenobserved in filamentous fungi (Fischer-Parton et al., 2000) andin yeast, where endocytic uptake of the dye has beenestablished (Vida and Emr, 1995). In animal cells localisationof membrane marker dyes internalised by endocytosis toparticular endomembranes by endogenous ‘sorting’mechanisms has been reported (Murkherjee et al., 1999).Such a mechanism acting to direct FM-dye to particularendomembranes could account for the apparently restrictedlocalisation observed in pollen tubes. Furthermore, the apexand apical vesicle cloud are sites of considerable membraneflux, whereas the vacuole membrane would be expected to beless ‘active’ explaining its slower FM4-64 accumulation.
Here we have shown that FM4-64 entry does not proceed byunfacilitated diffusion (Fig. 7). It has, however, been suggestedthat internalisation of FM-dyes may be mediated by alternativemechanisms, specifically, the activity of ‘flippases’ (Fischer-Parton et al., 2000) or mechanosensitive cation channels(Nishikawa and Sasaki, 1996). Both of these mechanismscould allow ‘free’ dye in the cytoplasm and dissociate dyedistribution from vesicle trafficking. Injection of free dye intothe cell (Fig. 6) produced a drastically different intracellularlocalisation compared to external application, arguing againstsuch entry mechanisms. To date there is no evidence thatflippase enzymes are able to translocate FM-dyes across thelipid bilayer. Dye entry by a mechanosensitive cation channel(Nishikawa and Sasaki, 1996) could also be argued against onthe grounds that: (1) contrary to earlier work (Nishikawa andSasaki, 1996), high external divalent cation concentration didnot inhibit dye uptake (Fig. 14B,C); and (2) in the pollen tubesuch channels are most likely to be active at the pollen tubeapex, yet the dye uptake sequence showed that dyeinternalisation proceeded from sites behind the apex. Washoutof dye from a loaded pollen tube (Fig. 8) shows that the V-shaped apical labelling is not the product of continual dyeinflux from the extreme apex, as this staining pattern persistedwell after external dye removal.
Exocytotic versus endocytic vesicles at the pollentube apexThe predominant FM4-64 labelling in the pollen tube coincideswith a region that consists largely of Golgi-derived membranevesicles (Lancelle and Hepler, 1992). Thus, the observedstaining pattern would be consistent with subapical endocyticuptake of the dye and redistribution through trafficking ofstained vesicles predominantly to the apex, arguably the main
region of membrane flux in the growing tube. FM-dye haspreviously been found to be associated with exocytotic vesiclesin other tip growing cells (Fischer-Parton et al., 2000; Belangerand Quatrano, 2000; Hoffmann and Mendgen, 1998; Betz etal., 1996).
Brefeldin A, which inhibits anterograde ER to Golgi traffic,and Cytochalasin D, which disrupts the actin cytoskeleton(Geitman et al., 1996 and references cited therein; Rutten andKnuiman, 1993; Picton and Steer, 1981), have effects on thenumber and distribution of secretory vesicles in pollen tubesand are well characterised by electron microscopy. Theseinhibitors produced characteristic effects on FM4-64 stainingpattern (Fig. 9; Fig. 10) consistent with labelling Golgi-derivedsecretory vesicles.
Although current observations support the contention thatFM4-64 labels secretory vesicles in the pollen tube apex,reporting their distribution and the extent of theiraccumulation, it is also possible that FM4-64 is present inendocytic vesicles. Endocytosis is now recognised as arequirement for pollen tube tip growth (Steer and Steer, 1989)and evidence to show this is already accumulating (Blackbournand Jackson, 1996; Derksen et al., 1995; O’Driscoll and Steer,1993).
Endocytosis of membrane could offer an explanation for theobserved movement of FM4-64-stained material away from thetip. However, this explanation does not fit in with currentobservations on the sequence of dye internalisation, dyewashout or, equally significantly, the effects of restrictinggrowth rate with high external sucrose or calcium conditions.
According to previous studies (Roy et al., 1998; Picton andSteer, 1983; 1982), vesicle production and delivery to the apexoccur at the same rates in normally growing tubes and underconditions of slow growth. This implies that the rate ofmembrane recycling would be increased under slow growthconditions. However, when we applied the two differentrestrictive growth conditions (Fig. 14; high calcium orsucrose), FM4-64 staining at the apex was either unchanged ordiminished. The lack of increased FM4-64 staining under theseconditions suggests that endocytosed material does notcontribute significantly to the pool of FM4-64-stained materialobserved moving away from the apex.
More recently, the concept of rapid membrane recycling bya ‘kiss and run’ mechanism has emerged from work on nerveterminals (Stevens and Williams, 2000). In this model,secretory vesicles do not completely integrate with the targetmembrane but release their contents through a short-livedfusion pore before detaching. This mechanism of rapidendocytosis is suggested to ‘avoid’ uptake of FM-seriesendocytic markers because of the short lifetime of the fusionpore, which exposes the vesicle membrane only briefly to theexternal pool of dye (Cousin and Robinson, 2000; Stevens and
Fig. 14.FM4-64 staining pattern of a L. longiflorumpollen tube grown under restrictive growthconditions. Confocal images of FM4-64 fluorescenceare shown with corresponding bright-field images.(A) 15% sucrose medium (growth 8.7 µm/minute).(B) 25 mM calcium medium (growth <1 µm/minute),note the thickened apical cell wall. (C) Growth on 15mM calcium medium (4.6 µm/minute). Bars, 15 µm.
2694
Williams, 2000). Such a mechanism could conceivably allowrapid endocytosis of membrane at the pollen tube apex withoutit being reported by FM4-64 staining of the endocytic vesicles.
Dynamics of the apical vesicle accumulation arerelated to the rate of growthExamination of many FM4-64-loaded pollen tubes showed aclear relationship between the V-shaped apical staining patternand normal growth. However, no obvious relationship could befound between the different growth speeds of ‘healthy’individuals within a population and FM4-64 staining pattern.
The apparent presence of distinct regions of FM4-64staining within the V-shaped (cone shaped in three dimensions)apical region of secretory vesicles (Fig. 1; Fig. 3; Fig. 11; Fig.12) and the movements of material within these regionsobserved here (Fig. 11; Fig. 12) suggest both an underlyingstructural basis and a relevance to the process of tip growth.The region occupying the apical 3-5 µm region of the tip showsa superficial correspondence to the region of high calcium atthe pollen tube apex defined by Pierson et al. (Pierson et al.,1996) and is believed to regulate vesicle fusion to the apex.Also reported is the existence of an actin ring near the apex ofgrowing tobacco pollen tubes (Kost et al., 1998), which woulddefine such an apical region within the overall V-shaped apicalvesicle cloud region.
The importance of our observations of apparently distinctregions in the apical FM4-64 staining and distinct patterns ofmovement of FM4-64 staining is revealed in the furtherobservation that a strict quantitative relationship exists forindividual growing cells between FM4-64 signal and theregular oscillatory growth-rate fluctuations that are a feature ofL. longiflorum pollen tube growth (Fig. 13). Interestingly, moresubapical regions of FM4-64 staining gave the most dynamicvariation in signal and showed the clearest relationship to thegrowth rate fluctuation (Fig. 13A-D).
The exact correlation between FM4-64 staining and growthfluctuations still remains to be determined by statisticalanalysis. However, it appears that peak growth rates coincidewith a decline in the FM4-64 signal immediately behindthe apical 3-5 µm region (Fig. 13F). This corresponds to aparticular phase of the movement of FM4-64-stained materialfrom behind the apical 3-5 µm apical region to more subapicalregions (see Fig. 11).
Any model that seeks to establish the relationship betweenthe dynamics of the FM4-64 apical staining pattern andoscillatory growth of individual tubes needs to take accountof the observations of a possible underlying structuralorganisation to the apical FM4-64 staining and the distinctpattern of movement of material reported here. We speculatethat the observed movement of material may be simply the‘cycling’ of an excess of secretory vesicles delivered to theapex in between the rounds of maximum tip extension. It ispossible that at the maximum rate of tip extension theconsumption of vesicles is high. Correspondingly, at the lowergrowth rate fewer vesicles are used, which results in an‘overspill’ of the excess secretory vesicles, which pass backinto the cytoplasmic stream to be eventually returned to theapex. This model fits in with the current understanding that therate at which secretory vesicles are produced and delivered tothe tip is not regulated with respect to the rate of tip extension.It also implies that the apical 3-5 µm region is a pool of limited
capacity and as such may act to regulate the rate of vesicledelivery to the apex.
The research was supported by BBSRC postdoctoral fellowships(to RMP and SF-P) and an HFSP postdoctoral fellowship (to MKW).
REFERENCES
Belanger, K. D. and Quatrano, R. S.(2000). Membrane recycling occursduring asymmetric tip growth and cell plate formation in Fucus distichuszygotes. Protoplasma212, 24-37.
Betz, W. J., Mao, F. and Smith, C. B.(1996). Imaging exocytosis andendocytosis. Curr. Opin. Neurobiol.6, 365-371.
Blackbourn, H. D. and Jackson, A. P.(1996). Plant clathrin heavy chain:Sequence analysis and restricted localisation in growing pollen tubes. J. CellSci.109, 777-786.
Cochilla, A. J., Angleson, J. K. and Betz, W. J.(1999). Monitoring secretorymembrane with FM1-43 fluorescence. Annu. Rev. Neurosci.22, 1-10.
Cousin, M. A. and Robinson, P. J. (2000). Two mechanisms of synapticvesicle recycling in rat brain nerve terminals. J. Neurochem. 75, 1645-1653.
Derksen, J., Rutten, T., Lichtscheidl, I. K., Dewin, A. H. N., Pierson, E. S.and Rongen, G. (1995). Quantitative analysis of the distribution oforganelles in tobacco pollen tubes: implications for exocytosis andendocytosis. Protoplasma188, 267-276.
Edwardson, J. M. (1998). Membrane fusion: All done with SNAREpins?Curr. Biol. 8, R390-R393.
Feijó, J. A., Sainhas, J., Hackett, G. R., Kunkel, J. G. and Hepler, P. K.(1999). Growing pollen tubes possess a constitutive alkaline band inthe clear zone and a growth-dependent acidic tip. J. Cell Biol.144, 483-496.
Feijó, J. A., Sainhas, J., Holdaway-Clarke, T, Cordeiro, M. S., Kunkel, J.G. and Hepler, P. K. (2001). Cellular oscillations and the regulation ofgrowth: the pollen tube paradigm. Bioessays23, 86-94.
Fischer-Parton, S., Parton, R. M., Hickey, P., Dijksterhuis, J., Atkinson,H. A. and Read, N. D.(2000). Confocal microscopy of FM4-64 as a toolfor analysing endocytosis and vesicle trafficking in living fungal hyphae. J.Microsc. 198, 246-259.
Geitmann, A., Wojciechowicz, K. and Cresti, M. (1996). Inhibition ofintracellular pectin transport in pollen tubes by monensin, brefeldin A andcytochalasin D. Bot. Acta109, 373-381.
Heslop-Harrison J. (1987). Pollen germination and pollen tube growth. Int.Rev. Cytol.107, 1-78.
Hoffmann, J. and Mendgen, K.(1998). Endocytosis and membrane turnoverin the germ tube of Uromyces fabae. Fungal Genet. Biol.24, 77-85.
Holdaway-Clarke, T. L., Feijó, J. A., Hackett, G. R., Kunkel, J. G. andHepler, P. K. (1997). Pollen tube growth and the intracellular cytosoliccalcium gradient oscillate in phase while extracellular calcium influx isdelayed. Plant Cell9, 1999-2010.
Kost, B., Spielhofer, P. and Chua, N. H.(1998). A GFP-mouse talin fusionprotein labels plant actin filaments in vivo and visualizes the actincytoskeleton in growing pollen tubes. Plant J. 16, 393-401.
Lancelle, S. A. and Hepler, P. H.(1992). Ultrastructure of freeze-substitutedpollen tubes of Lilium longiflorum. Protoplasma167, 215-230.
Li, H., Lin, Y. K., Heath, R. M., Zhu, M. X. and Yang, Z. B. (1999). Controlof pollen tube tip growth by a Rop GTPase-dependent pathway that leadsto tip-localized calcium influx. Plant Cell11, 1731-1742.
Li, Y.-Q., Zhang, H.-Q., Pierson, E. S., Huang, F.-Y., Linskens, H. F.,Hepler, P. K. and Cresti, M. (1996). Enforced growth-rate fluctuationscauses pectin ring formation in the cell wall of Lilium logiflorum pollentubes. Planta200, 41-49.
Malhó, R. and Trewavas, A. J.(1996). Localized apical increases of cytosolicfree calcium control pollen tube orientation. Plant Cell8, 1935-1949.
Messerli, M. A., Creton, R., Jaffe, L. F. and Robinson, K. R.(2000).Periodic increases in elongation rate precede increases in cytosolic Ca2+
during pollen tube growth. Dev. Biol.222, 84-98.Mukherjee, S., Soe, T. T. and Maxfield, F. R.(1999). Endocytic sorting of
lipid analogues differing solely in the chemistry of their hydrophobic tails.J. Cell Biol.144, 1271-1284.
Nishikawa, S. and Sasaki, F.(1996). Internalization of styryl dye FM1-43 inthe hair cells of lateral line organs in Xenopus larvae. J. Histochem.Cytochem. 44, 733-741.
JOURNAL OF CELL SCIENCE 114 (14)
2695Vesicle accumulation and growth rate in pollen
O’Driscoll, D., Hann, C., Read, S. M. and Steer, M. W.(1993). Endocytoticuptake of fluorescent dextrans by pollen tubes grown in vitro. Protoplasma175, 126-130.
Parton, R. M. and Read, N. D. (1999). Calcium and pH imaging in livingcells. In Light microscopy in biology. A practical approach.(Lacey, A. J.,ed.), pp. 221-271. Oxford University Press.
Pelham, H. R. B. (1997). SNAREs and the organization of the secretorypathway. Eur. J. Cell Biol.74, 311-314.
Picton, J. M. and Steer, M. W.(1981). Determination of secretory vesicleproduction rates by dictyosomes in pollen tubes of Tradescantiausingcytochalasin D. J. Cell Sci.49, 261-272.
Picton, J. M. and Steer, M. W.(1982). A Model for the mechanism of tipextension in pollen tubes. J. Theor. Biol.98, 15-20.
Picton, J. M. and Steer, M. W.(1983). Membrane recycling and the controlof secretory activity in pollen tubes. J. Cell Sci.63, 303-310.
Pierson, E. S., Miller, D. D., Callaham, D. A., Vanaken, J., Hackett, G. andHepler, P. K. (1996). Tip localized calcium entry fluctuates during pollentube growth. Dev. Biol.174, 160-173.
Roy, S., Jauh, G. Y., Hepler, P. K. and Lord, E. M.(1998). Effects of Yariv
phenylglycoside on cell wall assembly in the lily pollen tube. Planta 204,450-458.
Roy, S. J., Holdaway-Clarke, T. L., Hackett, G. R., Kunkel, J. G., Lord,E. M. and Hepler, P. K. (1999). Uncoupling secretion and tip growth inlily pollen tubes: evidence for the role of calcium in exocytosis. Plant J.19,379-386.
Rutten, T. and Knuiman, B. (1993). Brefeldin A effects on tobacco pollentubes. Eur. J. Cell Biol.61, 247-255.
Steer, M. W. and Steer, J. M.(1989). Tansley review no 16: Pollen tube tipgrowth. New Phytol.111, 323-358.
Stevens, C. F. and Williams, J. H.(2000). ‘Kiss and run’ exocytosis athippocampal synapses. Proc. Natl. Acad. Sci. USA97, 12828-12833.
Van der Woude, W. J., Morre, D. J. and Bracker, C. E.(1971). Isolationand characterization of secretory vesicles in germinated pollen of Liliumlongiflorum. J. Cell Sci.8, 331-351.
Vida, T. A. and Emr, S. D.(1995). A new vital stain for visualizing vacuolarmembrane dynamics and endocytosis in yeast. J. Cell Biol.128, 779-792.
Zheng, Z. L. and Yang, Z. B.(2000). The Rop GTPase switch turns on polargrowth in pollen. Trends Plant Sci. 5, 298-303.
