determination of secretory vesicle production rate … · 2005-08-25 · packed secretory vesicles...

J. Cell Set. 49, 261-272 (1981) 261Printed in Great Britain © Company of Biologists Limited 1 081

DETERMINATION OF SECRETORY VESICLE

PRODUCTION RATES BY DICTYOSOMES IN

POLLEN TUBES OF TRADESCANTIA

USING CYTOCHALASIN D

JILL M. PICTON AND MARTIN W. STEER

Department of Botany, The Queen's University of Belfast, Belfast, Northern Ireland

SUMMARY

Pollen tubes of Tradescantia were grown in vitro and exposed to 0-3 /xg/ml cytochalasin Dfor 5 or 10 min. Fine-structural observations revealed no visible effect of the drug on theorganelles. Stereological analysis, using a method recently developed by Rose (1980) to obtainsphere size-distributions corrected for section thickness, revealed a substantial increase in thenumber of secretory vesicles present in the cytoplasm around the dictyosomes. Equating therate of vesicle accumulation with the rate of vesicle production, a total of 5388 vesicles perminute are formed by a growing tube. This corresponds to 2-4 vesicles per minute perdictyosome, and a turnover rate of 3-7 min for a single dictyosome cisterna, or about 15-18-5min for a complete dictyosome. The calculated vesicle production rate agrees well with thatrequired to sustain the observed growth rate of such tubes, based on the addition of membraneor wall material to the tube tip.

INTRODUCTION

Apical growth, by means of the secretion of the contents from dictyosome-producedvesicles, occurs in several types of plant cell; for example, fungal hyphae tips (Grove,Bracker & Morre, 1970), algal rhizoids (Herth, Franke & Van Der Woude, 1972),root hairs (Bonnet & Newcomb, 1966; Herth et al. 1972; Sawhney & Srivastava,1974) and pollen tubes (Sassen, 1964; Larson, 1965; Dashek & Rosen, 1966; Rosen,Gawlick, Dashek & Siegesmund, 1964).

This apical growth, and other examples of dictyosome vesicle secretion togetherwith cytoplasmic streaming, is one of the several cellular processes to be inhibitedby the cytochalasins (Herth et al. 1972; Franke, Herth, Van Der Woude & Morre, 1972;Mascarenhas & Lafountain, 1972; Sawhney & Srivastava, 1974; Mollenhauer &Morre, 1976; Pope, Thorpe, Al-Azzawi & Hall, 1979). These secondary metabolitesof fungi are believed to interfere with the functioning of microfilaments (e.g. seeTannenbaum, 1978), possibly by either their disruption (e.g. see Wessells et al. 1971)or their detachment from the plasma membrane (Spooner, 1973), or by producinga state of sustained contraction in the actomyosin meshwork (Miranda, Godman &Tanenbaum, 1974).

Although the exact mode of action of the cytochalasins has still to be elucidated,it is assumed that growth is prevented by the inability of the cell to transport vesiclesto the plasma membrane, and as a result they accumulate at their site of production(Pope et al. 1979; Mollenhauer & Morre, 1976). Provided that vesicle production is

262 J. M. Picton and M. W. Steer

not affected by cytochalasin, the rate of vesicle accumulation around the dictyosomesshould reflect their rate of synthesis.

Pollen tubes grown in vitro provide an ideal closed system with which to investigatethis phenomenon. We have attempted to quantify the rate of vesicle production inTradescantia virginiana pollen tubes by inhibiting tube growth with cytochalasin D.Vesicle production rates, as determined by this method can then be compared tothose necessary for the observed tube growth rates.

MATERIALS AND METHODS

Pollen tube culture and electron microscopy

Pollen from open flowers of T. virginiana was sown on a medium consisting of io-2 M CaCl2,5 x io-5 M KHjPO,, 0 0 1 % boric acid, io% sucrose and o-6% agar. After about 30 mingrowth, the cultures were treated with 0-3 jug/ml cytochalasin D in o-oi % dimethyl sulphoxidefor o, s or 10 min. The tubes were fixed for 2 h in 2-5 % glutaraldehyde in PIPES buffer,followed by 2 h post fixation in 2 % osmium tetroxide. They were then dehydrated in anacetone series and embedded in Epon-Araldite resin. Sections were cut on a Riechert OMU4Ultramicrotome, stained with 2 % aqueous uranyl acetate and lead citrate, and viewed witha Hitachi H 300 electron microscope.

Stereology

Electron micrographs from the zone of vesicle production (Fig. 1) were analysed. Fromeach micrograph the following measurements were recorded:

(i) Pollen tube area (within the plasma membrane), (ii) Number of dictyosomes. (iii) Volumefraction of dictyosomes. (iv) Height and diameter of those dictyosome profiles not sectioned

— To nuclei,callose plugs

and grain

Morevacuolateregion

'Active' dictyosomesand

other organelles

Densemitochondrial

zone

50-100 ̂ mSample micrographs takenfrom this region

Fig. i. Zonation of a pollen tube showing approximate extent of each zone and regionfrom which the sample micrographs were taken.

Fig. 2. Dictyosomes (d) from the 'active' zone showing the production of typicalwall-forming secretory vesicles (v). The 2-layered structure of the pollen tube wall(top) can be seen. Mitochondria (m), a plastid (p), lipid droplets (/), and roughendoplasmic reticulum (er) are also present, x 58 500.Fig. 3. Structure of the apical vesicle zone in a control pollen tube. Note the denselypacked secretory vesicles, x 36000.Fig. 4. Structure of the apical vesicle zone of a pollen tube treated with 0-3 /xg/mlcytochalasin D for 5 min. The density of secretory vesicles has been markedlyreduced compared to control tubes, x 36000.

Pollen vesicle formation 263


ssr


obliquely, (v) Volume fraction of vacuoles. (vi) Number and diameter of all vesicle profiles.Volume fractions were obtained using the 'point-count' method using a dot-pattern overlaywith spacing 075 cm. (For theoretical principles see Weibel & Bolender, 1973; Steer, 1981.)

Vesicle size distributions were determined using the method of Rose (19S0). This involvesmeasuring a large number of vesicle profile diameters in a known area {A) of cytoplasm andassigning them to size classes. The number of profiles (n) in each class (1, 2, 3 . . . ft) is deter-mined and used to find the number of vesicles (N) with mean diameter at the midpoint ofeach class (1, 2, 3 . . . m) from:

Number per unit volume (Nm) = — [Pmk] [nk],

where A is the area and A the class-interval size, both in the same units. The advantage ofthis method is that it takes into account section thickness (T), and Rose (1980) suppliesa conversion matrix for the calculation for a series of different 7*/A values. For our calculations,however, we used a class interval of 40 nm and a section thickness of 60 nm giving a value for!T/A of 1 "5. The necessary conversion matrix for this value is not included in those suppliedby Rose (1980) and was calculated from first principles. For a more complete explanationof the problem of section thickness see Steer (1981).

It is important to note that all calculations are based on unit volume of 'cytoplasm' withvacuoles, nuclei and cell walls excluded.

RESULTS

Pollen tube structure and the effects of cytochalasin D

Tradescantia pollen tubes exhibit the same characteristic zonation as that found inother pollen tubes (Fig. 1). The extreme tip region contains closely packed secretoryvesicles. Behind these is found a dense zone of mitochondria interspersed with smoothendoplasmic reticulum. Following these are found the dictyosomes and all the othernormal cell organelles, i.e. plastids, microbodies, rough endoplasmic reticulum andnumerous lipid droplets. The dictyosomes produce the secretory vesicles (Fig. 2),which migrate to the tube tip, where they fuse with the plasma membrane and resultin tube extension. In the older regions of the tube the vegetative nucleus followed bythe generative nuclei move towards the tip. Beyond these the cytoplasm becomesmore vacuolate and eventually callose plugs are formed. The pollen tube wall consistsof 2 distinct layers (Fig. 2). The outer cellulose fibrillar layer is found all alongthe tube, while the inner callose wall is not formed until a short distance behindthe tip.

Cytochalasin D inhibits cytoplasmic streaming and pollen tube growth. A con-centration of 0-3 /<g/ml is the lowest capable of immediate inhibition and thisconcentration was therefore chosen for use throughout this work. At the electronmicroscope level, the closely packed vesicles at the tip in control tubes (Fig. 3) are

Fig. 5. The active dictyosome region of a control pollen tube, x 22000.

Fig. 6. The active dictyosome region of a pollen tube treated with 0 3 /xg/mlcytochalasin D for 5 min. Note the similarity of the organelles to those in thecontrol, x 22000.

Fig. 7. As Fig. 6, but treated with 0-3 /j.g/ml cytochalasin D for 10 min. Again, this issimilar to the control tube, x 22000.


lost from this region in tubes treated with cytochalasin D within 5 min (Fig. 4). Theremainder of the tube appears indistinguishable from control tubes (Figs. 5, 6), asdo tubes for 10 min (Fig. 7).

Vesicle production

Vesicle size distribution analysis (Fig. 9) of control tubes reveals the presence ofvesicles with a range of diameters. This range corresponds to a maturation sequence,starting with small (50 nm) vesicles, formed at the dictyosomes, which enlarge to

100 200 300 400

Mean vesicle diameter, nm

500

Fig. 8. Graph showing secretory vesicle size distribution in Tradescantia pollen tubes.Control ( # # ) and tubes treated with 0-3 /ig/ml cytochalasin D for 5 min(O O) and 10 min (A A). The number of vesicle profiles counted in eachcase were 733, 845 and 843, giving total numbers of vesicles per /Am3 of i4'27, 24-93and 34'14, respectively. The rate of vesicle accumulation per fj.m3 is 10/5 min or 2/min.

about 200 nm in the cytoplasm. Vesicle numbers increase with increasing length ofexposure to cytochalasin D at a rate of approximately 10 vesicles per fim3 per 5 min.By assuming that the rate of vesicle accumulation is equivalent to the rate of theirformation, a production rate of 2 vesicles per /tm3 per minute can be obtained.

Dictyosome density

The number of dictyosomes per /4m3 can be calculated from the following:

No. per unit volume =Nj 1—^y x -


where iVA is the number of profiles per unit area, Vv is the volume fraction of thecomponent and /? is a shape coefficient determined from the standard curves ofWeibel (1969), assuming the dictyosomes to be flattened cylinders. The volumefractions and numerical densities of dictyosomes for control and treated tubes aregiven in Table 1. The mean density of dictyosomes in all pollen tubes is 0-849 per/<m3

of 'active' cytoplasm.

Table 1. Dictyosomes in Tradescantia pollen tubes

Cytochalasin D

Control S min

Volume fraction of dictyosomes 00308 0-0322 0-0331Number of dictyosomes//xm3 0-780 o-68o 1-088

Mean = 0849 dictyosomes//um3.

Volume fractions and density of dictyosomes in the 'active' dictyosome region of controland tubes treated with 03 /xg/ml cytochalasin D for 5 min and 10 min.

Dictyosome activity

Combining the results of the two preceding sections, it can be seen that 2 vesiclesare produced per /im3 per min, and that there are 0-85 dictyosomes per/<m3; thereforeeach dictyosome is forming 2-35 vesicles per minute.

To obtain a figure for the overall rate of vesicle production of a growing pollentube, it is necessary to establish the volume of cytoplasm that contains the dictyosomeswhich contribute vesicles for the growth of the tube. Although dictyosomes are foundthroughout the length of the pollen tube (except for the extreme tip) they are mostconcentrated in a region behind the mitochondria-rich zone up to the point of theappearance of small vacuoles. The length of this region varies in different tubes, butis typically about 70 //m (Fig. 1). A tube of diameter 7-0//m (not including wall) willcontain a volume of 'active' cytoplasm (minus vacuoles) of 2694/(m3. Assumingthat 2 vesicles are produced per /im3 per minute, as determined above, the vesicleproduction rate of such a pollen tube would be 5388 vesicles per minute.

DISCUSSION

Although the build-up of vesicles around dictyosomes caused by cytochalasininhibition has been noted on several occasions, a quantitative study of the rate ofaccumulation has not previously been attempted. Also, our observation that thehigh density of vesicles at the tube tip is lost with cytochalasin D treatment has notpreviously been reported. This observation clearly indicates that, while vesicletransport has been halted by cytochalasin treatment, the ability of vesicles to fusewith the plasma membrane has not been affected.

In using cytochalasin to provide estimates of vesicle production rates, it was as-sumed that it does not affect the rate of vesicle production. We believe this assumption


is justified on the basis both of our own observations and those of other workers.We could detect no significant change in dictyosome structure, dimensions or densityafter cytochalasin D treatment. Similar conclusions were reached by Mollenhauer &Morre (1976) for maize root tips treated with cytochalasin B and also by Pope et al.(1979) after treatment of wheat coleoptiles and maize roots with cytochalasin B.

An alternative method of determining vesicle production rates is to calculatevesicle numbers necessary to sustain the observed growth rates, based on eithermembrane surface area production or wall volume production. This was done byVan Der Woude & Morre (1968) for Lilium pollen tubes. They arrived at a figureof 2000 vesicles per minute per pollen tube. They found identical vesicle requirementsfor both wall and membrane production, but this would occur for only the precisevesicle diameter used in their calculation (0-3 jam diameter).

Similar calculations can be made for Tradescantia, using the parameters given inTable 2. The results of these calculations show that there is a remarkable similarity

Table 2. Secretory vesicle fusion rates

Structure to be formed Fusion rate, vesicles/min

Plasma membrane 3629Inner wall layer (o-i /urn thick) 4177Outer wall layer (0-07 (ira thick) 2960Total wall 7137

Fusion rates of secretory vesicles, diameter 0-15/tm, required to produce the observedgrowth rate of 6 /im/min in Tradescantia pollen tubes (7 /im in diameter, to the plasma mem-brane), based on surface area of plasma membrane and volume of the 2 wall layers.

between the necessary vesicle fusion rate and that determined for vesicle production(5388 vesicles per minute). Our estimate of vesicle production is clearly sufficientto account for the growth rates observed.

As might be expected, pollen-tube wall formation requires a greater rate of vesiclefusion than does plasma membrane synthesis. The balance is presumably maintainedby membrane recycling (Morre, Mollenhauer & Bracker, 1971). It is difficult tocompare these results with those determined by Van Der Woude & Morre (1968)since both the growth rate (12/tm/min) and vesicle diameter (0-3/tm) for Liliumare larger than those determined here for Tradescantia.

The reliability of the results depends upon the accuracy in determining the size ofthe various structures involved. A critical parameter is vesicle diameter. The Rose(1980) analysis has reduced the errors caused by section thickness. Selection of themean vesicle size, from the range observed, for use in the calculations in Table 2 is,however, somewhat arbitrary since insufficient information is available on the sizesof the mature vesicles fusing with the tube tip. There is also some difficulty indistinguishing true secretory vesicles from profiles of small vacuoles. The variationin the extent of vacuolation in different tubes also necessitated basing all calculationson unit volume of cytoplasm minus vacuole. Wall thickness determination alsoproved difficult, as the extent of condensation of the pollen-tube wall was found to


be very sensitive to fixation procedures. Total wall thickness varied from about0-06 to 0-17 fim. The values used in the calculations are means of several differentfixations.

In calculating vesicle requirements for growth it is assumed that the volume ofwall material in a vesicle accounts for the same volume of pollen tube wall. This seemsa natural assumption to make; however, there is no evidence that this is in fact thecase. A further difficulty with this method is the fact that 2 distinct wall layers areformed from a single type of secretory vesicle, with one, the inner callose layer, notbeing formed until several micrometres behind the tip. An interesting point is thatin higher plants generally it is believed that callose is secreted into the wall fromendoplasmic reticulum adjacent to the plasma membrane, while secretory vesiclescontribute hemicellulose and pectin to the wall, cellulose being synthesized at theplasma membrane surface. This throws further doubt on the assumption that a unitvolume of secretory vesicles can be equated to a unit volume of tube wall.

Dictyosome activity has also been measured by Schnepf (1961) in Drosophyllummucilage glands. His value of 3 vesicles per dictyosome per minute agrees well withthe value we obtained for Tradescantia, 2-35 vesicles per dictyosome per minute.

An alternative measure of dictyosome activity is that of dictyosome turnover rates.By the use of time-lapse photography and morphometric analysis, Brown (1969) hasshown that during scale production in Pleurochrysis scherffelii, a new dictyosomecisterna is formed every 2 min. A similar value, a cisterna every 1-2 min, wascalculated by Chrispeels (1976) from pulse-chase experiments of his own and fromkinetic analysis of polysaccharide secretion in pea stems by Eisinger & Ray (1972).Also Heinrich (1973) found that in the trichome hydathode of Monarda fistulosa,1 dictyosome yields 1 cisterna per minute and in about 8 min the entire dictyosomeis renewed. Bowles & Northcote (1974), however, found turnover to be more rapidin root cap slime and hemicellulose plus pectin secretion. They calculated thatturnover times for complete dictyosomes were 0-3 min and 2-5 min, respectively.

In Tradescantia an approximate dictyosome turnover time may be calculated byassuming that each cisterna is a flattened cylinder, and that its surface area of 0-07 /<m2

is completely used in producing vesicles of diameter 50 nm (surface area approx.0-008 /im2). One cisterna can therefore produce 8-75 vesicles at a rate of 2-35 permin, giving a turnover time of about 3-7 min. For a dictyosome with 4-5 cisternaethe complete turnover time will be 15-18-5 min. This is close to the range, estimatedby Morre et al. (1971), for a turnover time for a complete dyctyosome of 20-40 minand a single cisternal turnover time of 1-4 min.

A serious difficulty in working with pollen tubes is the problem of determiningthe extent of the zone of dictyosomes actually contributing to the growth of the tube.Dictyosomes are present throughout the tube except for the extreme tip region, butfrom how great a distance can vesicles travel to the tip? This is one of the manyquestions regarding pollen tube growth that still remain unanswered.

The question of what provides the motive force for the movement of secretoryvesicles has been considered previously. Franke et al. (1972) proposed 3 possibleguide elements in the form of microfilaments, microtubules and cisternae of the


endoplasmic reticulum. The inability of colchicine to disrupt secretion (Franke et al.1972; Mollenhauer & Morre, 1976) apparently discounts microtubules.

Pollen tube growth has generally been associated with cytoplasmic streaming andmore recently with the contractile protein system. Williamson (1980) suggests thatif actin (identified in pollen tubes by Condeelis, 1974) could be found associated withthe plasma membrane in plants, as it is in animal cells, it could control the locationof vesicle secretion. Our findings clearly contradict this. If microfilaments wereinvolved in vesicle fusion, it would not be expected that this could continue in thepresence of cytochalasin D. Fusion is clearly occurring in Tradescantia, asdemonstrated by the rapid reduction of vesicle numbers at the tube tip after treatmentwith cytochalasin D.

An alternative hypothesis for the control of vesicle fusion has been proposed byQuatrano (1978). He suggests that general cell polarity (clearly demonstrated inpollen tubes) is determined by the position of certain membrane 'patches'. Thesecontrol the uptake of Ca2+ into the cell, and their initial location is controlled by thephysical environment of the young cell. These patches are stabilized in a certainregion by microfilaments, and by their localized ion accumulation they cause thebuild-up of an electrical potential gradient and, as a result, cell polarity. The anchoredmicrofilaments could serve as guide elements for vesicle transport and other polarizedevents.

The general tendency to associate vesicle movement with cytoplasmic streamingmay not, of course, be valid, and there have been reports of these 2 processes occurringindependently. Sawhney & Srivastava (1974) could overcome cytochalasin B inhibitionof growth in root hairs with benzyl-adenine, but cytoplasmic streaming could not berestored. Herth (1978) and Reiss & Herth (1979), using the ionophore A 23187, couldhalt pollen tube growth in Lilium before cytoplasmic streaming was affected. Theyinterpreted the effect of the ionophore as causing the disruption of the localizationof polar vesicle fusion. This agrees well with the ideas of Quatrano (1978), and theionophore would presumably disrupt the Ca2+ ion balance and break downpolarity.

The ability of the dictyosomes to continue producing vesicles when they are notbeing removed from their vicinity seems to discount a build-up of vesicle numbersas a control mechanism on dictyosome activity. The question as to what does controltheir activity, therefore, remains unanswered.

The technical assistance of Mr George McCartney is gratefully acknowledged. This workwas supported by a research grant from the Science Research Council and a post-graduatestudentship (to J.M.P.) from the Northern Ireland Department of Education.

REFERENCESBONNET, H. T. Jr & NEWCOMB, E. H. (1966). Coated vesicles and other cytoplasmic com-

ponents of growing root hairs of radish. Protoplasma 62, 59-75.BOWLES, D. J. & NORTHCOTE, D. H. (1974). The amounts and rates of export of poly-

saccharides found within the membrane system of maize root cells. Biochem. J. 142, 139-144.


BROWN, R. M. Jr (1969). Observations on the relationship of the Golgi apparatus to wallformation in the marine Chrysophycean alga Pleurochrysis scherffelii Pringsheim. J. Cell Biol.41, 109-123.

CHRISPEELS, M. J. (1976). Biosynthesis, intracellular transport, and secretion of extracellularmocromolecules. A. Rev. PL Physiol. 27, 19-38.

CONDEELIS, J. S. (1974). The identification of F actin in the pollen tube and protoplast ofAmaryllis belladonna. Exptl Cell Res. 88, 435-439.

DASHEK, W. V. & ROSEN, W. G. (1966). Electronmicroscopial localisation of chemical com-ponents in the growth zone of Lilium pollen tubes. Protoplasma 61, 192-204.

EISINGER, W. & RAY, P. M. (1972). Function of Golgi apparatus in synthesis and transportof cell wall polysaccharides. PL Physiol. 49, 2 (suppl.).

FRANKE, W. W., HERTH, W., VAN DER WOUDE, W. J. & MORRE, D. J. (1972). Tubularand filamentous structures in pollen tubes: possible involvement as guide elements inprotoplasmic streaming and vectorial migration of secretory vesicles. Planta 105, 317-341.

GROVE, S. N., BRACKER, C. E. & MORRE, D. J. (1970). An ultrastructural basis for hyphal tipgrowth in Pythium ultimum. Am. J. Bot. 57, 245-266.

HEINRICH, G. (1973). Die Feinstruktur der Trichom-Hydathoden von Monarda fistulosa.Protoplasma 77, 271-278.

HERTH, W. (1978). Ionophore A23187 stops tip growth but not cytoplasmic streaming, inpollen-tubes of Lilium longiflorum. Protoplasma 96, 275-282.

HERTH, W., FRANKE, W. W. & VAN DER WOUDE, W. J. (1972). Cytochalasin stops tip growthin plants. Naturwissenschaften 59, 38-39.

LARSON, D. A. (1965). Fine-structural changes in the cytoplasm of germinating pollen.Am.J. Bot. 52, 139-154.

MASCARENHAS, J. P. & LAFOUNTAIN, J. (1972). Protoplasmic streaming, cytochalasin B, andgrowth of the pollen tube. Tissue and Cell 4, 11-14.

MIRANDA, A. F., GODMAN, G. C. & TANENBAUM, S. W. (1974). Action of cytochalasin D oncells of established lines. II. Cortex and microfilaments. J. Cell Biol. 62, 406-423.

MOLLENHAUER, H. H. & MORRE, D. J. (1976). Cytochalasin B, but not colchicine, inhibitsmigration of secretory vesicles in root tips of maize. Protoplasvia 87, 39-48.

MORRE, D. J., MOLLENHAUER, H. H. & BRACKER, C. E. (1971). Origin and continuity of Golgiapparatus. In Results and Problems in Cell Differentiation. 2. Origin and continuity of cellorganelles (ed. J. Reinhert & H. Ursprung), pp. 82-126. Berlin: Springer.

POPE, D. G., THORPE, J. R., AL-AZZAWI, M. J. & HALL, J. L. (1979). The effect of cytochalasinB on the rate of growth and ultrastructure of wheat coleoptiles and maize roots. Planta 144,373-383-

QUATRANO, R. S. (1978). Development of Cell Polarity. A. Rev. Physiol. 29, 487-510.REISS, H-D. & HERTH, W. (1979). Calcium ionophore A23187 affects localized wall secretion

in the tip region of pollen tubes of Lilium longiflorum. Planta 145, 225-232.ROSE, P. E. (1980). Improved tables for the evaluation of sphere size distribution including

the effect of section thickness. .7. Microscopy 118, 135-141.ROSEN, W. G., GAWLIK, S. R., DASHEK, W. V. & SIEGESMUND, K. A. (1964). Fine structure

and cytochemistry of Lilium pollen tubes. Am.J. Bot. 51, 61-71.SASSEN, M. M. A. (1964). Fine structure of petunia pollen grain and pollen tube. Ada bot. neerl.

13. 17S-181.SAWHNEY, V. K. & SRIVASTAVA, L. M. (1974). Cytochalasin B-induced inhibition of root-hair

growth in lettuce seedlings and its reversal by benzyladenine. Planta 119, 165-168.SCHNEPF, E. (1961). Quantitative Zusammenhange zwischen der Sekretion des Fangschleimes

und den Golgi-Strukturen bei Drosophyllum lusitanicum. Z. Naturf. 16 B, 605-610.SPOONER, B. S. (1973). Cytochalasin B: toward an understanding of its mode of action. Devi.

Biol. 35, f i3 - f i8 .STEER, M. W. (1981). Understanding Cell Structure. Cambridge University Press (In press).TANNENBAUM, J. (1978). Approaches to the molecular biology of cytochalasin action. In

Cytochalasins, Biochemical and Cell Biological Aspects (ed. S. W. Tanenbaum), pp.521-559. Amsterdam, New York, Oxford: Elsevier/North-Holland Biomedical.

VAN DER WOUDE, W. J. & MORRE, D. J. (1968). Endoplasmic reticulum-Dictyosome-Secretory vesicle associations in pollen tubes of Lilium longiflorum Thunb. Proc. IndianAcad. Sci. 77, 164-170.


WEIBEL, E. R. (1969). Stereological principles for morphometry in electron microscopiccytology. Int. Rev. Cytol. 26, 235-302.

WEIBEL, E. R. & BOLENDER, R. P. (1973). Stereological techniques for electron microscopicmorphometry. In Principles and Techniques of Electron Microscopy. Biological Applications,vol. 3 (ed. M. A. Hayat), pp. 237-296. New York: Van Nostrand Reinhold.

WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR,E. L., WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in Cellular and Develop-mental Processes. Science 171, 135-143.

WILLIAMSON, R. E. (1980). Actin in motile and other processes in plant cells. Can.jf. Bot. 58,766-772.

{Received 3 September 1980)

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