morphology and physiology of gamete mating and...

J. Cell Set. S3, 193-209 (1982)

Printed in Great Britain © Company of Biologists Limited 1982

MORPHOLOGY AND PHYSIOLOGY OF GAMETE

MATING AND GAMETE FUSION IN THE

FUNGUS ALLOMYCES

JEFFREY POMMERVILLE*Department of Botany, University of Georgia, Athens, Georgia 30602, U.S.A.

SUMMARYNew structural features of gametes are presented and several physiological aspects of mating

and gamete fusion in the fungus Allomyces macrogynus are described. In the posterior region ofthe male gametes, where fusion is thought to occur, thin-section electron microscopy demon-strated the presence of microfijaments that were associated with a ruffling of the otherwisesmooth plasma membrane. When the cells were treated with the microfilament-disruptive drugcytochalasin B, fusion was inhibited due to the interaction of the drug with the female gamete.Freeze-fracture replica analysis of the posterior region of the gamete plasma membrane showedan area devoid of membrane particles (termed the flagellar collar) at the basal end of theflagellum. Physiological analysis of gamete mating and fusion showed a requirement for divalentcations, and the time required for gamete mating could be lengthened or shortened by decreasingor increasing, respectively, the divalent cation concentration. Diphenhydramine and chloro-quine also increased the time required for mating or completely prevented fusion. Tertiaryamine local anaesthetics produced the same type of results, as did trypsin and Pronase. Phospho-lipases were without an inhibitory effect. When the gametes were washed free of the drugsprior to mating, it was seen that the female gametes were more sensitive to cell surface changesthan the males. In addition, 2-h-old female gametes showed a 70 % loss of fusion ability withyoung male gametes while the reverse experiment showed only a 20 % decrease. These observa-tions support the concept that gamete mating and fusion may occur at the flagellar collar areaand that mating in A. macrogynus is governed, at least in part, by cation and protein interactions.

INTRODUCTION

The mechanisms by which cell membranes 'recognize' one another and fuse toform a single bilayer have been of intensive interest to cell biologists (see Poste &Allison, 1973; Ahkong, Fisher, Tampion & Lucy, 1975; Lucy, 1975). Studies on thefusion of cytoplasmic vesicles with the plasma membrane, such as mucocyst secretionin Tetrahymena (Satir, Schooley & Satir, 1973) and other exocytotic processes (seeSatir, 1980), have demonstrated that fusion is not a random process but rather occurs atspecific sites on the plasma membrane. These sites, called' fusion rosettes', consist of in-tramembrane particle arrays surrounded by a clear area devoid of particles. The rosette isthought to act in part to align the cytoplasmic vesicle with the plasma membrane sitesand provide an ion gate mechanism to trigger fusion of the membranes (Satir, 1980).

Information on the regulation of cell to cell fusion is not as well understood. Somaticcell fusion studies on liposome-mediated fusion of membranes (Papahadjopoulos,Poste & Schaeffer, 1973) and in virally induced and chemically induced fusion systems

• Present address: Department of Biology, Texas A & M University, College Station,Texas 77843, U.S.A.

194 J- Pommerville

(Poste, 1972; Okada, Kim, Maeda & Koseki, 1974; Ahkong et al. 1973) have suggestedthat a clearing of membrane particles is necessary for the fusion of adjacent plasmamembranes. Although an increased understanding of fusion mechanisms has resultedfrom somatic cell studies, these are artifactual systems involving cells grown inunnatural culture conditions and the cell fusions obtained are the result of abnormaland possibly pathological changes.

Studies on the regulation of natural cell-cell fusion have included the myoblastsystem (Schudt& Pette, 1976; Prives& Shinitzky, 1977; Kalderon& Gilula, 1979) andgamete mating (fertilization) in the green alga Chlamydomonas reinhardtii (Weiss,Goodenough & Goodenough, 19776). Fertilization would seem to offer a good systemfor study because treatment of the cells with various chemicals and drugs can be usedto examine the molecular nature of the mating and fusion areas. The presence ofthick egg layers and the fairly elaborate structure of the sperm required to penetratethese layers makes physiological experiments on mating and fusion difficult to dowith gametes from higher organisms.

A group of organisms that has not been thoroughly examined with regard to matingand fertilization is the fungi. The water mould Allomyces has several characteristicsthat make it especially appealing for such studies. (1) The fungus possesses a motilegamete system that is morphologically simpler than sperm-egg systems of metazoa.(2) The fungus produces anisogamous, uniflagellate, motile male and female gametesthat do not possess exterior plasma membrane coverings, such as thick glycoproteincoats or a cell wall (Pommerville & Fuller, 1976). (3) The male cells do not producemacroscopic fertilization structures or contain an acrosome, and the female gametesdo not contain micropyles (Hatch, 1938; Pommerville & Fuller, 1976). (4) The femalegametes produce a sexual pheromone, called sirenin (Machlis, 1958), that attractsmale gametes to the female cells (Machlis, 1958; Carlile& Machlis, 1965; Pommerville,1978a, 1981a). Thus the mating reaction is extremely efficient and involves a seriesof rapid signals between cells.

The cytology and ultrastructure of the general fertilization stages have beenexamined (Pommerville & Fuller, 1976). This paper describes additional fine-structural and freeze-fracture replica features associated with the fusion area on thegametes of Allomyces macrogynus, and presents physiological data demonstrating thepossible involvement of cations, microfilaments and proteins for successful matingbetween the gametes. A preliminary report on some of this work has been presented(Pommerville, 19786).

MATERIALS AND METHODS

A. macrogynus (Emerson) Emerson and Wilson (strain Burma 3-35) was used throughoutthis study. Gametothallic cultures bearing the male and female gametangia were grown for 12days on full-strength Emerson YpSs agar at 23 °C. All studies were performed on male andfemale gametes collected and separated as previously described (Pommerville, 1978a).

Gamete mating and fusion in Allomyces 195

Phase microscopy

Gamete interactions during mating were photographed using phase-contrast optics at x 400on a Zeiss research microscope.

Transmission electron microscopy andfreeze-fracture

The preparation of gametes for thin-section transmission electron microscopy involved anew chemical fixation procedure for Allomyces that permits superior preservation of the gameteplasma membrane. Gametes were placed in 1 vol. of 4% (v/v) glutaraldehyde for 10 s, followedby the addition of an equal volume of 2 % (w/v) osmium tetroxide. All fixatives were made inMachlis' (1958) dilute salts (DS) solution containing 1 mM-calcium chloride and titrated topH 69 with 001 M - K O H . After fixation for 20 min at room temperature, the gametes werewashed in DS and dehydrated in an ascending acetone series. All washes and acetone up to95 % contained 1 mM-CaCl8. Infiltration and embedding were as previously described (Pommer-ville & Fuller, 1976).

Freeze-fracture replicas of male and female gametes were prepared by fixing the cells in 2 %(v/v) glutaraldehyde (in 001 M-potassium phosphate buffer, pH 69) for 15 min. The gameteswere then washed in buffer and incubated in ascending concentrations of cold glycerol (10, 20,30 %. v / v ) f°r 3° min a t each step. The gametes were pelleted by centrifugation and drops ofconcentrated gametes were placed on copper planchets. The drops were frozen in liquid Freonand stored in liquid nitrogen. The specimens were then fractured at — 115 °C and immediatelyshadowed with carbon and platinum in a Balzers 350M unit (Balzers High Vacuum Corp.,Hudson, New Hampshire). The replicas were digested overnight in concentrated HC1/Chromerge, rinsed several times in distilled water, and mounted on 300-mesh copper grids.Freeze-fracture nomenclature follows that of Branton et al. (1975).

Gamete mating assay

Mating assays were set up using a 'glass well plate' (Boerner microscope slide; ScientificProducts) so that each well contained approx. 6 x io3 male gametes. Mating reactions prior togamete fusion were observed with a dissecting microscope after adding 5 female gametes to thesuspension of male cells. As previously described (Pommerville, 1981a), the time of fusion canbe precisely determined because male gametes, which clustered over the female (see Fig. 4),will dissociate immediately once the female gamete has fused with a male cell. Further, thenew binucleate fusion cell will swim up and away from the old fertilization cluster (Pommerville,1981 b). The times reported for the length of mating are based on 3 separate experiments, eachanalysing the mating of at least 20 female gametes.

The effects of various agents on gamete fusion were determined using the mating assay con-taining the agent in question. The cells were incubated with the agent for 30 min, washed inDS, mixed with the other gamete type (with or without drug treatment), and the time of matingand the percentage of cells that fuse determined. All assays were done at 23 CC and only viable,motile gametes were used in the determinations. Viability required that the gametes be motile,the female gametes be able to generate sirenin, and the male gametes be attracted to the femalecells throughout the assay period.

Materials

Diphenhydramine hydrochloride, chloroquine diphosphate, and hydrocortisone-21-hemi-succinate were obtained from Sigma Chemical Co. (St Louis) as were phospholipase D (type I),phospholipase C (type I), phospholipase A2 (from Crotalus adamanteus), trypsin inhibitor(type I-S), procaine hydrochloride, and tetracaine hydrochloride. Trypsin (grade A) andPronase (non-specific protease from Streptomyces griseus) were obtained from Calbiochem (SanDiego) and cytochalasin B from Aldrich Chemical Co. (Milwaukee).

tip§ J. Pommerville

RESULTS

Structure of the mating area

Results obtained from previous work (Hatch, 1938; Pommerville & Fuller, 1976)suggest that the site of fusion between male and female gametes is near the pointwhere the whiplash flagellum inserts into the cell body. This can be demonstrated byaddition of female gametes to a suspension of male cells in the mating assay. Male

Figs. 1-4. Light micrographs of gamete mating in A. macrogynus. x 1500.Fig. 1. Association of a male gamete (<S) with a female ($). Note the absence of

contacts between the male flagellum (<$f) and the female flagellum (?/).Fig. 2. A male gamete positioned over the posterior portion of the female gamete.Fig. 3. A cluster of several male gametes on a single female ($).Fig. 4. A cluster of male gametes located over the posterior portion of the female

gamete ($).


gametes are attracted quickly to the sluggishly motile female cells and cluster overthe posterior portion of the female cell body (Figs. 1, 2). No male gametes are foundon the remaining portion of the female cell surface. Higher concentrations of malegamates in the assays result in more males being attracted, often completely coveringthe female cell, although the majority position themselves over the posterior half(Figs. 3, 4). No flagellar interaction can be seen that would facilitate mating. Most ofthe male gametes can be removed by either sucking the cluster into and out of amicropipet several times, or by placing the cluster in a drop of 0-5% (v/v) glutar-aldehyde. In doing either of these manipulations, or by using a dilute suspension ofmale gametes, only one or a few male gametes will remain attached to the female celland these are positioned at the posterior only. As a result of fertilization, other malegametes will no longer be attracted to or remain attached to the young fusion cell. Inaddition, few (o-i %) fusion cells are multinucleate.

Ultrastructural examination of the gamete posterior regions has shown a fewstructures that may be directly or indirectly mediating gamete mating. At the posteriorregion of the male gamete there exists a ' ruffling' of the plasma membrane (Figs. 5-7).This feature is seen only at this position and never occurs over any other portion ofthe plasma membrane (Fig. 5). Structures resembling microfilaments, measuring about6 nm in diameter, traverse the area of membrane 'ruffling' (Figs. 6, 7), the length ofruffled membrane corresponding to the length of the microfilament area. This groupof filaments is not seen in longitudinal sections that pass directly through the flagellumand kinetosome, but rather appear to be offset from these structures.

Observation of freeze-fracture replicas of the gametes provided additional detailin this posterior region. In common with almost all plasma membranes the PF-facecontains many more particles than does the EF-face (Figs. 8, 11). Most interesting isan area completely devoid of intramembrane particles that completely surrounds theinsertion point of the flagellum into the cell body (Figs. 8-10). This area, which Ihave called the 'flagellar collar', consists of 9 bumps (or depressions), which can beseen on both fracture faces (Figs. 8, 10, 11). Thin sections of A. macrogynus motilecells near the point of flagellum insertion have shown a set of struts (or props) thatextend from the kinetosome to the plasma membrane (Pommerville & Fuller, 1976).In cross-section through haploid zoospores of A. macrogynus, Olson (1973) hasdemonstrated 9 equally spaced struts in this posterior location. Therefore the flagellarcollar represents those areas where struts are associated with the plasma membrane.In all cross-fractures through the collar, the fracture always occurs on the flagellarside of the collar (Figs. 8, 10, 11). Fractures are never found where the break occurson the cell body-side of the collar. Although not always clear, linear rows of membraneparticles often surround the collar region (Figs. 9, 10).

Characteristics of the physiology of the mating area

Several different agents were added to the suspension of male or female gametesof A. macrogynus to determine some of the molecular properties required for mating.

Cation effects. The time required for mating (tm) represents the time interval fromthe first contact between a male gamete with the female cell and the formation of the

198 Jf. Pommerville


fusion cell. In the standard DS solution this time is just over 1 min (Fig. 12). Time-lapse motion picture analysis suggested that the actual fusion process takes less than50 ms, so the tm is representive of events required for gamete contact, and alignmentof plasma membranes and mating sites. This time is independent of the number ofmale gametes present in the assay.

Although the tm is relatively short, it is regulated at least in part by cations in theDS solution. Decreasing the Ca2+ concentration lengthened the tm while increasingits concentration shortened the /m 2-fold (Fig. 12). Other divalent cations will sub-stitute for Ca2+ at higher concentrations (Fig. 13) with strontium having a slightinhibitory effect at the highest concentrations.

The divalent cation experiments showed a sharp decline in Zm around io~* M (Figs.12, 13) and observations with the light microscope of male attraction and matingsupport this finding. At lower concentrations (< io"6 M) the attracted male gametesmove over or around the female cell surface for a longer time before positioning them-selves at the posterior region, while at higher concentrations this period of movementis much shorter and the cell associates with the posterior region of the female gametealmost immediately on contact.

In the following experiments, assay conditions were used that gave the best associa-tion between male and female gametes. Therefore the DS solution was modified tocontain 10-3 M-Ca2+ (Ca-DS) where the tm is 07 min (Fig. 12). All other componentsof the DS solution were left as originally described (Machlis, 1958).

Cytochalasin B. In the first part of this paper structures similar to microfilamentswere described in the posterior region of the male gamete (Figs. 6, 7). The role ofmicrofilaments in the mating process was tested by incubating the cells in the micro-filament-disruptive drug cytochalasin B. At very low concentrations the drug iseffective in blocking fusion (Table 1). Only female gametes are sensitive to the drug,since female gametes incubated in cytochalasin B, washed prior to mating, and mixedwith untreated male cells did not result in fusion even though the male gametes areattracted to the posterior region. Male gametes treated with the drug appear unaffected(Table 1).

Effect of membrane stabilisers'. There are several agents that are commonly referredto as membrane stabilizers, although their modes of action may differ or are not

Figs. 5-7. Transmission electron microscopy of the posterior region of male gametesof A. macrogynus.

Fig. 5. An oblique section through a male gamete showing mitochondria (m), lipidbodies (0, a single nucleus (mi) and nucleolus (wo), and the nuclear cap (nc), whichcontains all the cell's ribosomes. Note the ruffling (bar) of the otherwise smooth plasmamembrane, x 16000.

Figs. 6, 7. The posterior portion of male gametes showing that the ruffling isassociated with structures similar to microfilaments (arrows), x 75000.Fig. 8. Freeze-fracture replica of the PF-face of a male gamete. Freeze-fractureparticles are evenly distributed over the surface except at the position where theflagellum inserts into the cell body (flagellar collar), where few particles are observed,x 25000.

200 J. Pommerville

ra


1-5

0-5

DS

No 001addition

0-1 1 10 100

[Ca*] (MX1CT4)

Fig. 12. Reduction in the time for mating (t^ as a function of the Ca1+ concentration.The normal time for mating in dilute salts (DS) is shown. Bars show ± standard error.

completely understood. The phenothiazine tranquilizer, diphenhydramine, when addedto gametes of A. macrogynus, is effective in inhibiting fusion (Table 2) even thoughmale gametes are attracted and cluster over the female cell. As the concentration ofthis drug increases not only do fewer cells fuse, but there is an increase in the tm ofthose clusters where fusion does occur. These changes are reversible by washing thecells free of the inhibitor prior to mating (Table 2). Similar results were obtainedwith the lipophilic, lysosomotropic amine, chloroquine, except the concentrationsneeded for inhibition were much lower (Table 2). Washing the cells free of the inhibitor

Figs. 9-11. Freeze-fracture replicas of the flagellar collar.Fig. 9. A replica of the PF-face showing the collar devoid of fracture particles. The

flagellum (/) contains a random distribution of particles except at the interface withthe collar where 2 linear rows of particles can be seen (arrows), x 75 500.

Fig. 10. A replica of the collar showing several of the depressions of the collar area.Again 2 rows of particles are present (arrows), x 84000.

Fig. 11. A replica of the EF-face showing a lack of fracture particles on the mem-brane and a series of 9 depressions of the flagellar collar, x 120000.

202 J. Pommerville

before mating could restore fusion ability to some of the male gametes, but not tothe female cells. The anti-inflammatory drug, hydrocortisone, was without effect atconcentrations of 10-200 /tg/ml.

Action of local anaesthetics. Tertiary amine local anaesthetics are known to bringabout changes in membrane physiology and Ca2+ interactions (Seeman, 1972). Twosuch anaesthetics, procaine and tetracaine, were tried. As the concentration of procaine

1-5

cJE 1

E

0-5

001 0-1 1001 10or [Sr*] (M X 10"4)

Fig. 13. Effect of metal cations on mating. Both Mg1+ (O) and Sr'+ ( | ») bring aboutan abrupt change in the tm at 10"Bars show ± standard error.

M. Ca1+ was present in all solutions at 5 x io"s M.

increases the *m increases almost 7-fold while slightly higher concentrations completelyprevent gamete fusion (Table 3). Again the effects of the anaesthetic can be reversedby washing in Ca-DS solution prior to mating with non-incubated gametes. Althoughthe /m remains higher for the female gametes than controls, the majority of the gametesdo fuse (Table 3). Tetracaine shows similar effects at lower concentrations (Table 3)and reversibility is evident after washing.

Effects of proteolytic and lipolytic enzymes. The nature of contact and mating betweenthe gametes of A. macrogynus was studied further using 2 proteolytic enzymes, trypsinand Pronase. Addition of trypsin at low concentrations increases the tm while decreasingthe fusion percentage (Table 4). Washing gametes after treatment demonstrates thatthey recover some of their ability to fuse although the tm remains 2 to 3-fold higher


Table 1. The effect of cytochalasin B (CB) on gamete fusion

Treatment

(1) Control (Ca-DS)(2) Female(CB) x male(CB)

Female(CBJ x male(DS)Male(CB) x female(DS)

(3) Dimethylsulphoxide§

CB C"g/ml)

0

1

51 0

1 0

1 0

o-i %

tm(min)±S.E.»

o-7±o-iI-O±O-I

+ st+ S+ 5

I-O±O-I

0 7±o-i

Fusion (%)

1 0 0

1 0 0

0 0

0 0

0 0

1 0 0

1 0 0

• Standard error.f The ability of gametes to fuse was not observed beyond 5 min.% Gametes were first incubated in the agent for 30 min and then washed for 15 min in Ca-DS

prior to mating in Ca-DS. Gametes represented by (DS) were only incubated in Ca-DS.§ Used to solubilize CB.

Table 2. Effect of' membrane stabilizer' agents on fusion of AJlomyces gametes

Treatment /*g/ml im(min)±s.E. Fusion (%)

(1) Control (Ca-DS)(2) Diphenhydramine

Female(DPH) xmale(DPH)

Female{DPH)» xmale(DS)

Male(DPH) x female(DS)(3) Chloroquine

Female(CQ) x male(CQ)

Female(CQ x male(DS)Male(CQ) x female(DS)

0

1 0

So1 0 0

1 0 0

1 0 0

1 0

252525

• See Table 1 and MaterialsDPH, diphenhydramine; CQ,

07 ±o-i

0-9 ±021-3 ±0-426 ±0707 ±o-i

o-6±o-i

1-3 ±0-5+ 5±5

O7±o-i

and Methods.chloroquine.

1 0 0

1 0 0

1 0 0

301 0 0

1 0 0

700 0

0 0

5°

than in controls. The addition of soybean trypsin inhibitor prevents the inhibitionthat occurred with trypsin alone. The male and female gametes are very sensitive toPronase (Table 4). As with trypsin, low concentrations of Pronase are effective inpreventing gamete fusion while increasing the tm of those gametes that do fuse.Washing the gametes after treatment does not allow either type of gamete to fusewith untreated ones.

The role of phospholipid in gamete mating was analysed using phospholipases D,C and A2. Both low and fairly high concentrations (up to 200 /ig/ml) do not preventgamete fusion and have little effect on the tm and fusion percentage.

Ageing. The fusion of male and female gametes can be prevented without theaddition of any of the above agents. If male and female gametes in DS are mixed at

204 J. Pommerville

Table 3. Inhibition of gamete fusion by tertiary

Treatment

(1) Control (Ca-DS)(2) Procaine

Female(PC) x male(DS)

Female(PC) x male(DS)#

Male(PC) x female(DS)(3) Tetracaine

Female(TC) x male(TC)

Female(TC) x male(DS)Male(TC) x female(DS)

• See TablePC, procaine

AnaestheticG"g/ml) t

0

So75

1 0 0

1 0 0

1 0 0

2550SoSo

1 and Materials and; TC, tetracaine.

amine local anaesthetics

m (min)±s.E.

o-7±o-i

17 ±024-6±0'2

+ 51710-30710-1

3-1 ±0-5+ 5

O'8±o-io-8±o-i

Methods.

Table 4. Effect of proteases on fusion of Allomyces gametes

Treatment

(1) Control (Ca-DS)(2) Trypsin

Female(T) x male(T)

Female(T) x tnale(DS)*Male<T) x female(DS)(T) + SBTI +gametes

(3) PronaseFemale(P) x male(P)

Female(P) x male(DS)Male(P) x female(DS)

ProteaseCug/ml)

0

1 0

IS25151525 each

1

51 0

1 0

1 0

^(mirOfs.E.

0710-1

2-O±O-221 ±04

+ 521 ± 0 4I-3±O-2o-8 ±o-i

1-4 ± 0 22-3 ± 0 5

+ 5+ 5+ 5

• See Table 1 and Materials and Methods.T, trypsin; SBTI, soybean trypsin inhibitor; P, Pronase.

Fusion (%)

1 0 0

90

500 0

801 0 0

400 0

1 0 0

1 0 0

Fusion (%)

1 0 0

702 1

0 0

6785

1 0 0

9 0600 0

0 0

0 0

various times after release from the gametangia, the percentage of gametes that fusefalls with time (Fig. 14). Moreover, this ageing effect is a consequence of changes tothe female gamete. If young male gametes are mixed with 2-h-old female cells, thepercentage of fusion is low as compared to mixing young female gametes with 2-h-oldmale cells (Fig. 14). This effect on the female gametes does not appear to be the resultof some material secreted into the DS solution, since when young female gametesare mixed with old females for 30 min prior to the mating assay, only the young femalegametes undergo fusion with the male cells.


100 •

60 90 120Gamete age (min)

Fig. 14. Effect of ageing on gamete fusion abilities. Female gametes (O) show thelargest reduction in the percentage of cells capable of fusion ( ) while both male( x ) and female cells show an increased *„, with age ( ).

DISCUSSIONThe ultrastructural evidence has argued for the site of gamete fusion in A. macrogynus

to be at the posterior region, near the flagella. Male gametes preferentially positionthemselves over this region on the female gametes. In other flagellated organisms, asUha mutabilis, U. lactuca and C. reinhardtii, cell fusion also occurs near the flagella(Braten, 1971; Melkonian, 1980; Weiss et al. igjyb). Previous ultrastructural work(Pommerville & Fuller, 1976) and the results presented here fail to show the formationof an elaborate mating structure between gametes of A. macrogynus, suggesting thatmating competence is solely a function of the chemical nature of the plasma membranesof the 2 gametes. In U. mutabilis and C. reinhardtii, gamete nagellar agglutinationtriggers the activation of plasma membrane fusion (Levlie & Bryhni, 1976; Good-enough, 1977). Even though A. macrogynus gametes have no flagellar interaction duringmating, microscopic tubules with small radii of curvature may be formed to overcomethe net repulsion of membrane charges since small, cytoplasmic bridges are seenbetween fusing gametes (Pommerville & Fuller, 1976). Few multinucleate fusion cellsare found, suggesting the block to polyspermy must be achieved either by a loss ofsirenin production (no attraction) or through the 'erasing' of the mating area as aresult of fusion. Although A. macrogynus is polyploid, it is not a result of polyspermy.

The freeze-fracture work to date demonstrates that the only unique plasma mem-brane structure is the flagellar collar. It is generally believed that freeze-fractureparticles or intramembrane particles (IMPs) represent membrane proteins, whilesmooth areas represent the lipid bilayer (Branton, 1971). Therefore, the flagellar collaris apparently a region devoid of membrane proteins. One major problem with the

206 J. Pommerville

interpretation of freeze-fracture replicas is the generation of artifacts during specimenpreservation with glutaraldehyde. The replicas of the gametes of A. macrogynus' appear' well preserved after fixation. Membrane particles are found evenly distributedover the entire membrane surface and no 'blistering' or bulging of the membranewas seen. The flagellar collar is the only distinct membrane specialization seen on theplasma membrane replicas.

Definitive evidence that the flagellar collar is the fusion site requires further study.Ahkong et al. (1975) were the first to suggest that fusion is mediated by protein-freeregions of the plasma membrane. Studies on myoblast fusion (Kalderon & Gilula,1979), concanavalin A endocytosis (Hatae & Benedetti, 1978), and the acrosomereaction of metazoan spermatozoa (Tilney, 1980) report the clearing of IMPs fromthe area of membrane fusion. This area is then surrounded by a ring of IMPs. Tilney(1980) suggests that these particles could allow the entrance of Ca2+ or maintain theparticle-free area. As a result of freeze-fracturing, a ring of particles can be seenaround the flagellar collar of A. macrogynus and the break of the flagellum from thecell body always occurs on the flagellar side of the collar, suggesting there is structuralstabilization around the collar. On the other hand, exocytosis in Paramecium andTetrahymena (Satir, 1980), secretion in mast cells (Chandler & Heuser, 1980), andmating in C. reinhardtii (Weiss et al. 1977&), apparently do not involve the IMP-freeregion for initial fusion, although the ensuing fusion of membranes may involvethis area.

In C. reinhardtii, the flagellar base is similar in structure to the flagellar collar andis associated with strut arrays (Weiss, Goodenough & Goodenough, 1977a). The areadevoid of membrane particles is not as striking as it is in A. macrogynus and somemembrane particles are associated with the arrays in C. reinhardtii. The C. reinhardtiireplicas were the result of gametes prepared without fixatives or cryoprotectants.

The flagellar collar of A. macrogynus is protein-depleted. The role of the micro-filaments in regulating this specialized structure and, in general, the mating processremains unknown. The results with cytochalasin B certainly demonstrate an effect onthe fusion process. Ca2+ is known to cause changes in microfilament structure and,since Ca2"1" uptake in A. macrogynus is required for chemotaxis of the male gametes(Machlis, 1973; Pommerville, 1981a), the influx of Ca2 "̂ during chemotaxis may actto clear the eventual fusion area and act as the signal for activation of mating (fusion)areas.

In most fusion systems, Ca2+ alone is not sufficient to cause cell fusion. InA. macrogynus, fusion can be inhibited or accelerated by manipulation of the cations inthe DS solution, suggesting an alteration in mating alignment. Additional evidencefor a role for Ca2+ is demonstrated by the effects of procaine and tetracaine on mating.Besides having an effect on chemotaxis at higher concentrations (Pommerville, 1981 a),these tertiary amine local anaesthetics are known to displace Ca2+ from the plasmamembrane (Low, Lloyd, Stein & Rogers, 1979) and, like most local anaesthetics, theireffects can be reversed by washing the cells free of the drug.

Diphenhydramine and chloroquine are drugs that have been shown to block virus-mediated cell fusion (Greenham & Poste, 1971; Poste & Reeve, 1972), and chloroquine


will block some receptor-mediated endocytosis events (Sando, Titus-Dillon, Hall &Neufeld, 1979). From their effects on A. macrogynus, they must alter the plasmamembrane, not allowing the proper signal or alignment for mating or fusion. Theseagents have been seldom used to study natural cell fusion (Ross & Shipley, 1973)and never on the system explored here or on any other fungus.

The involvement of membrane proteins in the mating interaction was demonstratedby the studies with trypsin and Pronase. Removal of essential surface polypeptideswould not allow proper alignment of mating surfaces. In Chlamydomonas moetvusii,similar results have been reported. The minus mating type (mt~) is sensitive to trypsinand Pronase, the plus mating type (mt+) is resistant (Wiese, 1974), and gamete fusionis sensitive to trypsin treatment (Wiese & Shoemaker, 1970). Membrane phospho-lipids apparently play no essential role in mating since addition of phospholipasesdid not inhibit fusion in A. macrogynus. Conway & Metz (1976) have proposed thatendogenous phospholipases bring about a destabilization of the membrane (Poole,Howell & Lucy, 1970), allowing fusion to occur. However, some other fusion systemsthat have been investigated do not support this idea (Kent & Vagelos, 1976; Hirao &Yanagimachi, 1978).

In all the chemical treatments and ageing experiments reported in this paper, thefemale gamete is more sensitive than the male cell. It remains to be determined if thefemale gamete plays a greater role in determining mating and fusion than the malegamete.

This work was supported by a Postdoctoral Fellowship from the Department of Botany atthe University of Georgia.

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(Received 1 June 1981)

morphology and physiology of gamete mating and...

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