Planta

Planta (I 986) 167: 54z1~553

9 Springer-Verlag 1986

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonaseugametosgametes A. Musgrave, P. de Wildt, I. van Etten, H. Pijst, C. Scholma, R. Kooyman, W. Homan and H. van den Ende Department of Plant Physiology, University of Amsterdam, Kruislaan 318, SM 1098 Amsterdam, The Netherlands

Abstract.Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or 0~, ~'-dipyridyl become non-aggtutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-/t-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-fi-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-fi-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.

Key words: Chlamydomonas (agglutination) - Flagellum - Glycoprotein - Membrane barrier. Abbreviations and symbols: CHI=cycloheximide; GTC=guanidine thiocyanate; mt+/mt-=mating type plus or minus; PAS = Periodic-acid-Schiff reagent; SDS = sodium dodecyl sulphate

Introduction During sexual reproduction, Chlamydomonas eugametos gametes agglutinate together in large numbers before eventually fusing in pairs. Agglutination is gamete-specific, vegetative cells do not take part, and it only occurs between cells of the opposite mating type, referred to as mt+ and rot-. The cells adhere together via glycoproteins exposed on their naked flagellar membranes. Both the m t agglutination factor (Musgrave et al. 1981; Lens et ai./982; Homan et al./982) and the mt + factor (Klis et al. 1985) have been identified as high-molecular-weight glycoproteins. The m t - factor has been shown to be present not only on the flagellar membrane but also in relatively large quantities on the surface of the cell body (Pijst et al. 1983). These cell-body agglutination factors cannot be involved in situ in agglutination, for the cell bodies are invariably surrounded by cell walls. However, they could represent a pool of agglutination sites that are mobilised or diffuse from the cell body to the flagella. Since the two membranes are continuous and since there is a pronounced turnover of active agglutination factors in the flagella during sexual agglutination (Snell and Moore 1980; Pijst et al./983), this concept of a readily available large pool of active factors is very appealing. Solter and Gibor (1978) were the first to suggest this idea for C. reinhardtii and more recently we also mentioned this possibility (Pijst et al. 1983). Nonetheless, there is a complication: Weiss et al. (/977) showed that a bracelet of intramembranous particles completely surrounds the base of each flagellum of C. reinhardtii. More recently, Bray et al. (1983) have shown that a similar structure exists in the transition zone of C. eugametos flagella. Based purely on the morphology of this structure, Weiss and Goodenough postulated that it could form a diffusion barrier between the plasma mem-

A. Musgrave et al. : Membrane barrier in Chlamydomonas flagella

branes of the flagella and the cell body. Indeed, some simple observations indicate that these membranes are different from each other and therefore separate. For example, the cell wall is only associated with the membrane of the cell body. Wall material is invariably found in preparations of isolated flagella but this is due to their co-purification and not due to a natural association, as we have shown (Musgrave et al. 1983). Similarly, the flagellar membranes also possess appendages not present on the cell body. For example, the mastigonemes and the glycocalyx in C. reinhardtii have been visualised under the electron microscope as a mat of prominent projections that is unique to the flagella (Monk et al. 1983). In this manuscript we present further evidence that the two membranes are functionally distinct and also show that agglutination factors on the cell body seem to be unable to either diffuse or to be transported to the flagellar surface. It is therefore most unlikely that the cellbody surface can form a pool for the convenience of the flagella. In accordance, evidence is presented that the pool lies within the cell. Material and methods Cell Culture. Chlamydornonas eugametos strains Utex 9 (mt +) and Utex 10 (mr-) from the Algal Collection at the University of Texas at Austin, Tex., USA, were cultivated in Petri dishes on an agar-containing medium in a 12 h light/12 h dark regimen as described by Mesland (1976). Gamete suspensions were obtained by flooding two-week-old cultures with sterile distiIled water just before the start of the dark period. The cells were harvested the following morning. To produce partially fused cells, or vis-~t-vis pairs as they are called, for immunofluorescence staining, i ml mt+ and 1 ml rnt- gamete suspensions were mixed and left in the light for 1 h to form pairs. Cells were then fixed in 1.25% glutaraldehyde for 30 min before washing and mounting for treatment with antisera. In order to mass produce vis-fi-vis pairs for the isolation of their flagella and for the extraction of agglutination factors, several mt+ and rot- culture suspensions were mixed in large plastic Petri dishes and left for up to 4 h in the light to pair. Newly formed vis-fi-vis pairs tended to swim to and aggregate at the surface of the culture medium, after which they swam to the bottom of the dish where they aggregated and adhered to the plastic. At this stage, all cells which were not adhered to the dish bottom were washed away in a gentle stream of water. Vis-fi-vis pairs were then scraped from the Petri dish with a glass rod. Isolation o f flagella and cell bodies. Gametes and vis-fi-vis pairs were deflagellated by the pH-shock technique described by Witman et. al. (1972). This same reference also describes how the flagella can be separated from the cell bodies over a cushion of 25% sucrose. Isolation and assay of agglutination factors. The mt agglutination factor was extracted from cell bodies in 3 M guanidine thiocyanate (GTC) as described earlier (Musgrave et al. 1981).

545 This method of extraction inactivated the mt + agglutination factor. However, it was effectively extracted in 1% Triton X-100 by shaking a suspension of cell bodies or flagella for 30 rain on a wrist-action shaker. The suspension was then centrifuged at 50000 g for 30 min when the supernatant was removed, freed of Triton X-100 by chromatography over a column of Phenyl-Sephadex packed in a Pasteur pipette, and tested for biological activity. Vis-A-vis pairs were extracted in Triton X-100 for mt + as well as mt ~ activity. In general, GTC is a more effective solvent than Triton for rot- activity but the choice was dictated by the need to extract the mt+ factor in an active form. Both mt+ and mt activity in the extracts were determined by adsorbing the active components onto activated charcoal and by then testing the charcoal for isoagglutination activity using gametes of the opposite mating type. The test has been described before (Musgrave et al. 1981). A quantitative measure of the activity in an extract was made by serially diluting the extract 1:1 (v/v) in water and then determining the highest dilution that was still active. Activity is expressed as a titer in the form 2 ~ 21, 2 z etc. Antisera. Mating-type-specific antisera were used in this study. Anti-PAS-1.2, described before (Lens et al. 1982), was used as a rot--specific serum. The mt § -specific antiserum was raised in rabbits against fractions from a Triton extract of approx. 10 l~ rnt + gametes. The extract was chromatographed in several batches over Sepharose 2B-CL in 5 m M NaC1. The biologically active fractions, which eluted just after the void volume, were pooled, freeze-dried and taken up in 3 ml 10 m M phosphate buffer, pH 7.6. Two rabbits were subcutaneously and intramuscularly injected with 1 ml of the extract mixed with Freund's complete adjuvant. The rabbits were re-immunised after two and four weeks. Immune sera were collected a week later. They are referred to as anti-rnt + serum. Monoclonal antibodies specific for the mt+ strain (code number 44.42) or m t - strain (44.2) were also used in this study. The production and characterization of these monoclonaIs wiIl be described elsewhere. lmmunofluorescence test. The indirect-immunofluorescence test was performed as described by Lens et al. (1980) except that the cells were not washed in acetone before applying the antiserum. Antibodies do not penetrate the cell wall of C. eugametos. Therefore, to test the antigenicity of the cell-body surface, the wall had to be removed. This was accomplished by suspending cells fixed in 1.25% glutaraldehyde in 0.6 M N a O H for 30 rain at 40 ~ C. Both polyclonal antisera described here reacted non-specifically with cell-wall fragments that remained after the N a O H treatment. In order to adsorb these antibodies and ensure that the sera reacted specifically with only one of the mating-type strains, the sera were incubated with NaOH-treated gametes of the opposite mating type. Although they are reffered to as mating-type specific, even adsorbed sera produced a very weak non-specific reaction with the opposite mating type. Etectrophoresis. Sodium dodecyl sulphate (SDS)-gel electrophoresis was performed as described by Laemmli (1970) using a slab gel (14.18 cm z) containing a 3 20% gradient of acrylamide. The slab was formed so that approx, the first I cm of the gel was 3 % acrylamide. Glycoprotein bands were stained using the periodic acid-Schiff (PAS) technique (Zacharius et al. 1969). Electron microscopy. Fused gametes were fixed in 1.25% glutaraldehyde for 1 h at room temperature, dehydrated through an

546

A. Musgrave et al.: Membrane barrier in Chlamydomonas flagella

ethanol series at - 2 0 ~ and --35 ~ C, and infiltrated with Lowicryl K4M as recommended by the manufacturers (Chemische Werke Lowi, Waldkraiburg, FRG). This procedure and the polymerisation of the resin was carried out as described by Robertson et al. (1984). Thin sections were cut on glass knives, mounted on 200-mesh gold grids bearing a carbon-Parlodion film which had been glow-discharged and inverted onto drops of antibody solution in 0.5 M 2-amino-2-(hydroxymethyl)-l,3propanediol (Tris) buffer (pH 7.4), 0.1% gelatine, 1% Tween 20, 1% ovalbumin and 0.02% azide. After 1 h, the sections were washed for 15 s in a gentle stream of distilled water and treated for I h with goat anti-mouse IgG-Au (20) (Janssen Pharmaceutica, Beerse, Belgium) diluted 1:20 (v/v) in Tris buffer-gelatine etc. The sections were again washed in a stream of water, blotted dry and post-stained with 20 rag. ml - 1 uranyl acetate in distilled water for 10 min. The grids were washed on four consecutive drops of water and then stained in lead citrate (Reynolds 1963), again washed on drops of water and then dried and viewed in a Jeol 1200 EX electron microscope at 80 kV.

Results

Glycoprotein composition. If the plasma membranes of the flagella and the cell bodies form a continuous lipid bilayer in which components can laterally diffuse or be transported, then one would expect the composition of both membranes to be similar. Alternatively, if they are separated by a barrier at the base of each flagellum, then the compositions of the two membranes could be very different. Figure i shows that the glycoprotein compositions of mt- flagella and cell bodies differ greatly. Many major cell-body glycoproteins do not occur at all in the flagella and similarly, most of the flagellar glycoproteins are absent from the cell body or only present as relatively minor components. The glycoproteins represented in Fig. 1 are all deemed to be extracellular membrane proteins based on the fact that they are all present in isolated flagellar membranes or extracts of the cell body made with 2 M guanidine thiocyanate (GTC) or 0.1% Triton. These extractions leave the cell virtually intact (see also Goodenough 1983). (Note that 3 M GTC and 1% Triton were usually used for extraction in the study presented here). Although the results presented are all of mt- material, similar differences in glycoprotein composition also exist between the flagella and cell bodies of mt+ gametes. Independent agglutinability of flagella and cell bodies. One may expect that all membranes with agglutination factor exposed on their surfaces are capable of agglutinating gametes of the opposite mating type. Thus both attached and isolated flagella can induce agglutination. Cell bodies of the mt- gametes are also covered with agglutination factor (Pijst et al. 1983) and when flagella-less cell bodies

Fig. 1. Glycoproteins in rot- cell bodies and flagella separated in a SDS-polyacrylamide (3-20% gradient) slab gel using the technique described by Laemmli (1970). Flagella were separated from the cell bodies via the pH-shock and sucrose-cushion methods of Witman et al. (1972). They were further purified of most of the cell-wall material by centrifuging over a caesiumchloride cushion (Musgrave et al. 1981). The cell bodies were extracted in 1% Triton for 1 h and the extract freed of Triton using Phenyl-Sepharose. The extract was freeze-dried and, like the isolated flagella, taken up in sample buffer and the components subjected to electrophoresis. Glycoprotein bands were stained via the PAS-technique whereafter the gel was dried and photographed. The typical flagella glycoprotein bands arc numbered in accordance with previous reports. Molecular-weight markers are provided with the suffix KDa (103 Da). Despite the attempts to purify the flagella of cell-wall material, the major cell-wall protein (PAS-5) is visible as a contaminant of the flagellar preparation

were homogenised by vortexing them with glass beads, the membranes disintegrated into vesicles which could be used to isoagglutinate mt+ gametes. However, the most convenient way of demonstrating and quantitating these cell-body agglutination factors is to extract them into 3 M GTC and use the charcoal bioassay. The activity in mt§ cells cannot be extracted in this way, it is inactivated but can be extracted into 1% Triton. Since mt activity can also be extracted into Triton (although the quantity is usually only a quarter of that extracted in GTC) it is convenient to use Triton when mixtures of gametes have to be extracted for both activities. Using these extraction techniques it was possible to demonstrate that treatments which destroyed the agglutinability of the flagella, did not affect the amount of agglutination activity that could be extracted from the cell bodies. This was to be expected if the two membranes

A. Musgrave et al. : Membrane barrier in Chlamyclomonas flagella Table 1. The effect of glycoprotein-synthesis inhibitors on flagellar agglutinability and the agglutination factors present on the cell-body surface of C. eugarnetos gametes. Gametes from several cultures were either treated with tunicamycin (5gg.ml-1), cycloheximide (10gg-ml 1) or ~, ~'-dipyridyl (0.3 mM) or left untreated for 48 h. After 24 and 48 h, ceils were tested for agglutinability with untreated gametes of the opposite mating type. The reaction was graded from ( - ) to ( + + + ) dependent upon whether none or all of the cells were involved in agglutination. After 18 h, cells treated with dipyridyl were all motile but sluggish. Some were still weakly agglutinable. To enhance the turnover of agglutination sites on the flagella, isolated rnt + flagella were added to isoagglutinate the gametes. By 24 h they were unable to agglutinate. At the indicated times, batches of cells (approx. 2-108) were deflagellated, the cell bodies separated from the flagella and the bodies in each batch extracted in 1 ml 1% Triton (rot +) or i ml 3 M GTC (mt-). The concentration of agglutination factors in the extract was determined in the charcoal assay after first absorbing the Triton onto Phenyl-Sepharose or dialysing out the GTC. The concentration is expressed as the agglutination titer Treatment

Flagellar agglutinability

Cell-body extract (agglutination titer)

Control Tunicamycin Control Tunicamycin

+ + + + + + + -

27 2s 27 26

Control Cycloheximide

+ + + -

27 26

Control Dipyridyl

+ + + -

26 26

mt +

24 h 48 h mr24 h mt24 h

were separated by a barrier. For example, the results in Table 1 indicate that mt+ gametes treated for 24-48 h in tunicamycin, a glycosylation inhibitor, completely lost their ability to agglutinate via their flagellar surfaces. The rot- gametes treated in the same manner remained fully agglutinable (see also Wiese and M a y e r / 9 8 2 ) but when treated for 24 h with cycloheximide (CHI), a protein-synthesis inhibitor, they became non-agglutinable. Similarly, rnt- gametes treated with 0r o(-dipyridyl. a ferrous-iron chelator which, among other effects, inhibits the hydroxylation and consequently the glycosylation of proline, also became non-agglutinable. Dipyridyl had no effect on mt+ agglutinbility. In all these treatments, when the rnt + and rntflagella were no longer active, the amount of agglutination activity that could be extracted from cell bodies was not appreciably reduced (Table 1). Thus, one must conclude that although there were

547

active agglutination factors present on the cellbody surfaces, they did not diffuse or were not transported to the flagellar surfaces. In all these experiments, biosynthesis inhibitors were used that have the disadvantage that they could inhibit the synthesis of a component necessary for transport of the agglutination factors to the flagella. This seems unlikely though, because tunicamycin and dipyridyl produced mating-typespecific effects while such a transport mechanism would be the same in both mating types. Since the results from using inhibitors are subject to the negative type of interpretation just described, examples of natural inactivation of flagellar agglutinability were studied for their effects on the cell-body agglutination factors. For example, gamete flagella become essentially non-agglutinable soon after cell fusion, which allows the vis-itvis pairs to escape from the agglutinating clumps of gametes. When isolated flagella from vis-fi-vis pairs were extracted for agglutination activity, only low levels of activity were found compared with the activity extracted from the equivalent amount of gamete flagella, as indicated in Table 2. In contrast, the cell bodies of vis-fi-vis pairs contained just as much activity as those of free-swimming gametes. A second example of the natural inactivation of flagellar agglutinability is seen when light-sensitive strains of C. eugametos or C. moewusii are placed in the dark. After a period of time, the flagella become completely non-agglutinable although they are reactivated after 30 min when they are returned to the light. When agar cultures of a light-sensitive rot- strain were flooded and kept for 16 h in the dark and the non-agglutinable flagella were then isolated and extracted for agglutination activity, none could be found, but the gamete cell bodies yielded just as much activity as usual (Table 2). Yet another example of this type has recently been described by Tomson et al. (1985). Vegetative cells of C. eugametos in liquid culture naturally undergo gametogenesis as they reach the stationary growth phase. However, the gametes are short lived and after a few days revert to a sexually incompetent form. Nonetheless, the amount of agglutination factor that can be isolated from these incompetent cells is equivalent to that from competent cells. Indeed, even using agar cultures, the amount of active agglutination factor that can be isolated from a gamete culture is more or less constant, while the agglutinability of the flagella can vary considerably, sometimes being non-existent. Thus even under natural conditions, the flagella

548

A. Musgrave et al. : Membrane barrier in Chlamydomonas flagella

Fig. 2. Immunofluorescence images of mt+ gametes fixed during isoagglutination with m t isolated flagella. The cells were labeled with the monoclonal 44.42, which is specific for mt+ antigens. Although antibodies do not penetrate the cell wall to label the cell-body surface, when part of the cell body (eg. the papillae, see arrows) penetrates the wall, that part of the cell-body surface is readily labeled. Similar images were obtained when live cells were directly labeled with a fluorescein isothiocyanate-antibody complex. Bar=10 pro; x 1 500

Table 2. Agglutination factors present on the flagellar and cellbody surfaces of gametes, light-sensitive gametes and vis-/t-vis pairs of C. eugametos, Gametes and vis-~t-vis pairs were deflagellated via the pH-sbock technique. Cell bodies were separated from the flagella over a sucrose cushion and then sedimented at 40000 g for 30 min in a sealed plastic micropipette tip (Finnpipette) to determine the packed volume. Cell bodies and flagella were then extracted separately in 1% Triton and the concentration of agglutination factors in the extract determined via the charcoal assay, after absorbing the Triton onto PhenylSepharose. The concentration is expressed as the agglutination titer of a 1 ml extract of t00 gl packed cell bodies, or their flagella. Since half the volume of vis-~i-vis pairs is mt+ and the other half r o t - , twice as much material (200 pl packed volume) was extracted to make the results comparable with those from free gametes. The preparation of vis-/t-vis pairs also contained 5% non-fused gametes Material extracted

Agglutination titer

Gametes mt +

Flagella Bodies

22 25

mt-

Flagella Bodies

23 26

mt +

Flagella Bodies

inactive 25

mt

Flagella Bodies

20 2~

Vis-a-vis pairs

Light-sensitive gametes mt-

Dark flagella Dark bodies

inactive 27

and cell-body membranes seem to be isolated from each other. Imrnunogenicity of flagella and cell bodies. Both the polyclonal and monoclonal antibodies used were

relatively specific for the mt + or mt strains. When these antibodies were used in the indirect-immunofluorescence test on whole fixed gametes, only the flagella were labeled. The mating-type-specific antigens on the cell-body surface were obscured by the cell wall. If, during sexual agglutination, papillae were formed that penetrated the cell wall, then they were also labeled by these antibodies (Fig. 2). Since the antibodies were excluded by the cell wall, we were able to test whether flagellar antigens, labeled on living gametes, were able to diffuse via the membrane onto the cell body. These experiments were performed using monoclonal antibodies that were directly labeled with fluorescein isothiocyanate. Even when incubated at different concentrations with m t - gametes for 60 min at 20 ~ C, the cell-body surface did not become labeled, only the flagella fluoresced, usually in a manner similar to that presented in Fig. 2. These monoclonal antibodies bind to strainspecific antigens present on several high-molecular-weight flagellar glycoproteins. While they undoubtedly form large molecular complexes on the flagellar surface, this does not inhibit their motility, for under certain circumstances the complexes were redistributed, becoming concentrated at the flagellar tips. Therefore, if the antigens had been able to diffuse or be transported onto the cell-body surface, that would have been detected using this technique. In complementary experiments, we were also able to show that mating-type-specific antigens (or, strictly speaking, strain-specific antigens) on the cell bodies were unable to migrate onto the flagellar surfaces, as will now be illustrated. Since the cell wall excluded antibodies from the cell body,

A. Musgrave et al. : Membrane barrier in Chlamydomonas flagelIa

549

Fig. 3A, B, C. Immunofluorescence images of fused gamete bodies and their flagella treated with anti-PAS-1.2 (polyclonal antiserum) to reveal mr--specific antigens. A Preparation fixed in 1.25% glutaraldehyde 30 min after mixing the gametes and treated with 0.6 M N a O H to remove the cell wall and expose the cell-body surface. The fluorescence reaction is restricted to one half of each vis-/~-vis pair. B Preparation fixed 180 min after mixing the gametes and treated with NaOH. Both halves of each vis-a-vis pair react with the antiserum. C Preparation deflagellated 180 min after mixing gametes and left another 90 min to regenerate new flagella. The cells were fixed but not treated with NaOH. Only two flagella per vis-a-vis pair reacted strongly with the antiserum. The weak fluorescence over the cell bodies is due to the natural fluorescence of the chloroplasts. The photograph was focused on the flagella in the top left-hand corner where the two mt+ flagella can be seen to give a very weak non-specific reaction. This illustrates that pairs regenerated four flagella even though they are not usually visible. Bars = 10 gm; x 750

the wall of fixed gametes was first removed in 0.6 M NaOH, after which the antigens on the cellbody membrane could be readily detected by the indirect-immunofluorescence test. Alternatively, material was embedded in Lowicryl K4M, sectioned and the antigenicity of the cell-body surface tested via the immuno-gold labeling technique. In this way, one could monitor changes in the antigenicity of the cell-body surface after gametes had fused to form vis-fi-vis pairs. It was found that cell bodies maintained their individual mating-type specificity for up to 3 h after fusing (Figs. 3A, 4A) but thereafter, both bodies exhibited an increasing mixed antigenicity (Figs. 3B,4C). The flagella, in contrast, did not acquire this mixed reactivity but maintained a strict mating-type specificity. Even when vis-fi-vis pairs were kept in the dark for 24 h to prevent the pairs fusing completely to form zygotes (Lewin 1954), the two pairs of flagella still reacted in a sex-specific fashion. The mating-type specificity of flagella regenerated from vis-fi-vis pairs was also tested. Pairs were first left for 3-4 h until both bodies exhibited a mixed antigenic character, as was checked by fixing a sample and later testing in the indirect-immunofluorescence test. The flagella were then amputated from the bodies using the pH-shock technique and the latter, after resuspending in fresh culture medium, were left 1.5 h to regenerate new flagella. Even though these flagella were regenerated from bodies

exhibiting a mixed antigenic character, the new flagel!a only exhibited either mt + or m t - antigenicity. Thus, although the vis-fi-vis pairs regenerated four new flagella, only two of them reacted in the immunofluorescence test with any one of the matingtype-specific antibodies (Fig. 3C). The photographs shown in Fig. 3 and 4 were obtained using absorbed anti-PAS-1.2 serum and monoclonal 44.2 to demonstrate the presence of rot--specific antigens, but equivalent, complementary results were also obtained using anti-mt + antibodies.

Intracellular pool of agglutination factors. It has been argued that agglutination factors do not move from the cell-body surface to the flagellar surface and there cannot be a pool on the cell-body surface. If this is really the case, then it must be possible to demonstrate that the pool lies somewhere else, somewhere within the cell. The following results indicate that this is the case. We have made use of dry agar cultures where the cells do not possess flagella. The cells were suspended in water or CHI solution to prevent protein synthesis. Flagellar growth and the development of flagellar agglutinability were then followed with time. The data represented in Fig. 5 show that CHI partially inhibited (30%) flagellar regeneration as previously reported (Pijst et al. 1983). When the flagella of these CHI-treated cells were amputated and regenerated, again in the presence of CHI,

550

A. Musgrave et al. : Membrane barrier in Chlamydomonas flagella

Fig. 4 A - C . Immuno-gold-labeled mt antigens on the surface of fused gametes. The monoclonal antibody 44.2 was used as a label for rot--specific antigens. It was then labeled with 20-nm gold particles bound to goat antimouse IgG. The specimens in A and B were fixed 2 h after mixing the gametes when the rnt--specific antigens were more or less restricted to the m t - cell body (left). In C, fixed 4 h after mixing the gametes, rot--specific antigens can be seen labeled on both cell bodies. In A, the section did not pass through the fused papillae but the juxtaposition of the anterior ends indicates that the cells had fused. It illustrates that both the flagellar and cell-body surfaces were labeled in a sexspecific manner. White bars in A - C represent 1 gin, 500 n m and 1 gin, respectively; A x 18000, B x28000, C x 14000

then the flagella only grew out as stumps of 3.5 gm. Such results are usually interpreted as meaning that CHI inhibits protein synthesis but since the cells maintain a pool of flagellar components, they are able to generate flagella to approx. 60-70% of the length of the controls. The pool is then practically empty and after deflagellation in the presence of CHI, the cells are only able to regenerate short stumps. Treating agar cultures with CHI did not affect the development of fiagellar agglutinability. This implies that the cells also contain a pool of agglutination factors, part of which is transported to and incorporated into the flagellar membrane during flagellar growth. The important question now is whether the pool of agglutination

factors in dry cells is present on the outside of the cell body, or not. Using the criterion that 2 M GTC efficiently extracts the rot- agglutination factor from the cell-body surface, cell bodies were extracted over this same time period. As is clear from Fig. 6, no activity was extracted into GTC from dry cells or from cells during the first hour after suspending them in a liquid medium. Thereafter, increasing quantities of agglutination activity were extracted. The increase in activity was due to the appearance of the agglutination factor (PAS-I.2) on the cell surface and not due to the activation of a factor already present, for extracts of dry cells and cells suspended up to 2 h did not produce a distinguishable PAS-1.2 band when sub-

A. Musgrave et al. : Membrane barrier in Chlamydomonas flagella 15 agglutinability

28 E = 10

flageIlar length

26

ck-_ /

r

/

LL

2L L~ o Cs C

2

22 < 2O

0

0

i

i

I

2

4

6

inactive

Time ( h )

Fig. 5. Growth of flagella and development of aggtutinability after suspending agar cultures in water or cycloheximide solution (5 ~tg-ml 1). Cells were scraped from the surface of individual Petri cultures with a glass spatula, suspended in 15 ml and returned to an empty Petri dish. The lengths of the flagella (o o) were determined after fixing the cells in 1.25% glutaraldehyde by viewing them under a microscope fitted with phasecontrast optics and an ocular micrometer. Agglutinability (zx-zx) was tested by mixing cell samples with isolated mt§ flagella diluted in a standard binary series. The greatest dilution that still gave agglutination was recorded as the agglutination titer of the cells. Cells treated with cycloheximide are represented by closed symbols and a broken line ( o - - - o )

r

26

control 2~

22

7//--~-

o 20 inactive

0

i

i

;

24

Time ( h ]

Fig. 6. Extraction of mt- agglutination factor from cell bodies after suspending the cells in water or cycloheximide solution (CHI) (5 gg.ml i). Cells were suspended in solution as described in Fig. 5. At regular intervals, the cell bodies from a single Petri culture (2-108 cells) were isolated and extracted in i ml 2 M GTC which only extracts components from the outside of the cell. After dialysing out the GTC, the quantity of the agglutination factor was determined using the charcoal assay

jected to SDS-gel electrophoresis (data not shown). Extracts of older cells produced an increasingly dense PAS-1.2 band. (See Fig. 1 for extract of cells flooded for 24 h). Agglutination factors were also extracted from cells flooded with CHI solution but

551

only one-sixteenth to one-eighth of the activity extracted from controls. In the absence of protein synthesis, this fraction must represent that originally present in the pool. Thus the major fraction of activity that eventually appears on the outside of normal cells is synthesised after flooding. However, the most important conclusion from these results is that the pool does not exist on the outside of the cell, but inside the cells. On flooding, the pool is mobilised so that agglutination factors become incorporated into the flagellar and cell-body plasma membranes at approximately the same time, an hour after flooding the cells. Although the pool of agglutination factors in dry cells could not be extracted into GTC, it could be extracted into 1% SDS, when the cell contents were more or less completely dissolved. On average, a single agar culture (2.108 cells) extracted into I ml SDS, produced a titer of 2 2 in the charcoal assay. This value is more or less equivalent to the activity that could eventually be extracted into GTC from the cell bodies in a CHI-flooded culture, and supports the contention that the activity that appears on the outside of CHI-treated cells originates from a pool and not from protein synthesis. Discussion

The idea that the plasma membrane of a singlecelled green alga can be differentiated into two separate, independent areas is not new. That some membrane appendages such as mastigonemes and membrane scales (for a review, see Moestrup 1982) are restricted to the flagellar membranes while cell walls and other types of scales are restricted to the cell bodies, has long been recognised as implying membrane differentiation. However, this phenomenon has not often been studied and therefore we do not know to what extent the two membrane regions are truly separated from each other. An exception is the work of Bouck and his colleagues (see for example Bouck and Chen 1984) on Euglena, where they have shown that the flagellar membrane, together with that of the neighbouring reservoir, are metabolically and immunogenically distinct from the membrane surrounding the rest of the cell body. The results presented here on C. eugametos indicate a similar separation of the plasma membrane into two distinct regions, with the component parts being unable to migrate from one region to the other. The idea of the plasma membrane of the cell body functioning as a pool for the convenience of the flagella seems therefore untenable. Of course, it is possible that part of the cell-body membrane is internalised and trans-

552 ported via the cytoplasm to the flagella. Coated vesicles have recently been reported to exist in C. reinhardtii (Weiss 1983), which indicates that such a mechanism is available, but the results presented here indicate that there is no, or only very limited mixing of components between these two regions. Even the sexual agglutinins, which occur in both membranes and in particularly high concentrations on the cell-body surface, are not transported to the flagella under a variety of natural and unnatural conditions. Therefore, the isolation of the flagellar membrane seems to be absolute. If one accepts the differentiation of the plasma membrane into two regions, then some interesting questions immediately arise. How does the cell differentiate between components destined for the two membranes and how is their separate incorporation organised? More fundamentally, how does the flagellar membrane grow? In Euglena, new membrane material can be fed into the reservoir region from which the flagellar membrane protrudes but in algae such as Chlamydomonas, no equivalent region adjacent to the flagella has been identified. What is more, the flagellar membrane seems to be isolated from that of the cell body in the region of the transition zone at the base of each flagellum. Here the bracelet and necklaces of intramembrane particles appear to form a diffusion barrier (Weiss et al. 1977) and it is here that the flagellar collars are so tightly bound to the flagellar membrane that they prevent large molecules or particles passing from the external medium into the periplasmatic space. Thus it seems that membrane material coming from the Golgi apparatus can only fuse with the flagellar membrane by passing into the cytoplasmic space of the flagellum and fusing with the naked flagellar membrane on the distal side of the flagellar collars. However, it is difficult to envisage even small Golgi vesicles passing into the flagellum between the closely juxtapositioned flagellar axoneme and membrane. This emphasises the need for more research into this aspect of membrane genesis. If the agglutination factors on the cell-body surface do not form a pool for the flagella, then where in the body is the pool and what is the function of these cell-surface agglutinins ? Since we are dealing with membrane glycoproteins that are presumably synthesised in the endoplasmic reticulum and Golgi bodies, it is safe to assume that they are stored somewhere within the system, for example in Golgi vesicles. The second question is more difficult to answer. It is probable that the two papillae of pairing gametes must first recognise each other before fusing and that the cell-body aggluti-

A. Musgraveet al. : Membranebarrier in Chlamydomonas flagella nation factors provide such a recognition system. However, since large quantities are present (as much as 25 times that on the flagella, Pijst et al. 1983) while each papilla is extremely small (Mesland 1976), this would seem extraordinarily inefficient. Possibly the agglutination factors fulfil another, as yet unknown function. Fused mt+ and rot- cells of C. eugametos maintain a high level of mutual independence. The cells are confluent but partly maintain their own identity. The best known example is the motility of the rnt + flagella and the immotility of the m rflagella (Lewin 1952). We have now shown that the mating-type-specific antigenicity of the flagella was maintained even when the flagella were excised and new ones regenerated. In contrast, a clear mixing of antigens on the cell-body surfaces was demonstrated, probably due to lateral diffusion within the plasma membrane. This means that flagellar membrane material cannot freely pass to and fro between the cell bodies, otherwise all four regenerated flagella would have exhibited mixed antigenicity. This is the first report on the effect of c~, c(dipyridyl on C. eugametos agglutinability. Whereas in C. reinhardtii it prevents the synthesis of mt+ activity (Cooper et al. 1983), in C. eugametos the effect is specific for m t - activity. Dipyridyl is a Fe + + chelator and consequently affects several aspects of cell metabolism. However, the specific effect on rot- activity, without seriously affecting the vitality of the cells, indicates that prolyl hydroxylase, an iron-containing enzyme, was inhibited and that the hydroxylation and consequently glycosylation of proline is essential for rot- activity. The lack of effect o n mt+ activity is consistant with the idea that only N-glycosidically linked oligosaccharides are important for mt+ activity, hence the sensitivity to tunicamycin, as first reported by Wiese and Mayer (1982). In contrast, rot- activity seems dependent on sugars O-glycosidically bound to hydroxyproline, and in addition, those bound to serine-threonine as well, for rot- activity is immediately destroyed by dilute alkali ( > pH 10). Agglutination factors isolated in Triton from a mixture of mt+ and m r- gametes could be tested independently for activity. They did not block each others agglutination sites by binding to each other, as one might predict. Apparently, an isolated factor can only bind to its partner when it is present on a living cell. This result is similar to the betterknown example of isolated rot- flagella that are capable of isoagglutinating mI + gametes but incapable of agglutinating isolated flagella from the s a m e mt+ cells.

A. Musgrave et al. : Membrane barrier in Chlamydomonas flagella Part of the work presented here was made possible by a short term European Molecular Biology Organization fellowship awarded to A.M. to work at the Cell Biology Department, John Innes Institute, Norwich. We gratefully acknowledge the help given by several members of that department.

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