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Experimental
INHIBITION
Cell Research 110 (1977) 337-346
OF CONJUGATION PYRZFORMZS Localization
IN TETRAHYMENA
BY ConA
of ConA-binding
sites
A. FRISCH and A. LOYTER Department
of Biological
Chemistry,
The Hebrew
University
of Jerusalem,
Jerusalem,
Israel
SUMMARY Two patterns of ConA binding to starved mating types of Tetrahymena pyriformis were observed depending on its time of addition. When ConA was added upon mixing of the mating types, at zero time of conjugation, it was first bound to the oral region and subsequently was taken into intracellular vacuoles. When it was added to conjugants, it was specifically bound as a ring around the conjugation area. The ability of the cells to form vacuoles, assayed by addition of carmine particles, declined prior to pair formation. The relationship between the above phenomena and the ability of ConA to inhibit conjugation is discussed.
Several membrane fusion processes such as fertilization [ 11,secretion of enzymes [2], hormones [3] or neurotransmitters [4] appear to be limited to specific membrane regions and do not necessarily involve the entire surface of the cell. In fertilization of mammalian cells, during which spermatozoa fuse with ova, the membrane event is restricted to the acrosomal region of the sperm cells [l], while in all secretion processes, intracellular granules fuse only with the plasma membrane facing the lumen [2, 51. This may imply that, in spite of the fluid nature of biological membranes, the plasma membrane is heterogeneous with regard to the composition and to the concentration of certain receptors. A suitable system to study the question of specific membrane changes induced at restricted regions is the conjugation process of Tetrahymena pyriformis. During this process, two starved complementary mating
types are induced to interact at specific membrane regions at which ultimately fusion will occur [6]. Recently we have reported that the lectin ConA inhibits the induction of conjugation when added to a mixture of starved complementary mating types of T. pyriformis and even partially dissociates pairs formed between them [7]. Experiments using rH]ConA showed that about 90-95% of the ConA precipitates with soluble glycoprotein secreted into the starvation medium by the two mating types [8]. By using specific anti-glycoprotein antibodies, it was shown that the secreted glycoproteins are not involved in the conjugation process [S]. It was, therefore, inferred that ConA may inhibit the conjugation process by direct binding to the surface of T. pyriformis. Addition of ConA to the cells of Stylonichia mytilus caused immobilization of the cells, probably as a result of specific bindExp
Cell
Res 110 (1977)
338
Frisch and Loyter
ing of ConA to the cilia [9, lo]. ConA also jugation, a suspension of the two starved mating types 106cells/ml) were mixed by introducing 0.5 ml interacts specifically with one of the mating (about of each suspension into a 20 ml glass flask and then types of Ulva [ll], inhibits conjugation of incubated at 30°C without shaking. The extent of conChlamydomonas [ 121, and fertilization of jugation was determined after 4 h of incubation [7]. sea urchin [13]. In all these cases the presBinding offluorescein-ConA to ence of surface glycoproteins representing single cells and to conjugants ConA receptors was assumed. Surface A suspension of either each of the mating types or of glycoproteins were also suggested to parti- conjugants (1 ml of 1Ogcells) were incubated, if not cipate in the conjugation process of T. pyri- otherwise stated, with 26 pg of fluorescein-ConA (Miles-Yeda, Rehovot, Israel) for 15 min at 30°C. At formis [ 141.Recently we have reported that the end of the incubation period the excess of fluotunicamycin, a specific inhibitor of glyco- rescein-ConA was removed by centrifugation at 500 g with 20 mM tricine NaOH, pH 7.4. The pellet obprotein biosynthesis, inhibits the conjuga- tained was suspended in 1 ml of 1% glutamldehyde dissolved in 20 mM tricine-NaOH, pH 7.4. After tion process in T. pyriformis [ 141. fixation, the cells were observed with a Zeiss WLIn the present work, we have used fluo- Research microscope with fluorescent attachment, and rescein-ConA and ferritin-ConA in order to photographs were taken at 1 min exposures on Kodak Tri-X film. study the precise localization, and the fate of ConA molecules when added to the mat- Uptake of carmine particles into ing types of T. pyriformis. It was found food vacuo[es that the ConA molecules interact with T. Conjugation was initiated by mixing 10 ml of each pyriformis at specific regions of the cell of the starved mating types (1Og cells/ml) in a 2 1 flask. At various intervals after mixing, surface. The correlation between the bind- Erlenmeyer conjugation was determined and samples of 1 ml ing of ConA and its ability to inhibit con- were withdrawn and mixed with 0.1 ml of carmine (final cont. 0.1%). After 5 min of incubajugation is discussed. In addition, the inter- particles tion at 3o”C, the cells were fixed by 2% formalderelationship between the conjugation pro- hyde, observed in the light microscope and the perof cells containing carmine particles was escess and another membranous event, name- centage timated. The experiments were performed in duplicate and about 200 cells were scanned in each sample. ly vacuole formation, is analyzed.
MATERIALS
AND METHODS
Cells mating type I (strain WH-6) and mating type III (WH-52) of syngen 1 (according to the new nomenclature suggested by Nanney & McCoy [15] the organism dealt with in this investigation should be called T. rhermophila) were grown as described before [7].
T. pyriformis
Medium The medium used for washing of cells, starvation and conjugation was 20 mM tricine-NaOH, pH 7.4.
Induction and determination of conjugation Cells were starved by incubating each of the mating types (108 cells/ml) in 20 mM tricine-NaOH, pH 7.4, at 30°C for 24 h. Conjugation was induced and determined as described in a previous work [7]. For conExp Cell Res II0 (1977)
Binding of ferritin-ConA ceils and to conjugants
to single
A suspension of either of the mating types or of conjugants (5 ml of 106cells/ml) was incubated with 25 &ml of ferritin-ConA (Miles-Yeda, Rehovot) for 15 min at 30°C. At the end of the incubation period, the cells were centrifuged at 500 g and washed twice with 15 ml of 20 mM tricine-NaOH, pH 7.4. When amethyl-mannoside (arMM) was used, it was allowed to interact with the ferritin-ConA for 10 min before addition to the cell suspension. After fixation with 1% glutaraldehyde, the cells were embedded in Epon and prepared for electron microscopy as previously described [16]. Thin sections were stained with uranyl acetate for 20 min before observation.
Binding ofjluorescein isothiocyanate (FITC) to antibody against ConAbinding material (Anti-CBM) ConA-binding material (CBM) was isolated from the starvation medium of T. pyriformis, purified and injetted into rabbits, as previously described [8]. The
Inhibition
of conjugation
Fig. 1. Binding of fluorescein-ConA to starved cells
of T. pyriformis (mating type I). Incubation with fluorescein-ConA for (a) 1 min; (b) 15 min at 30°C. Note the appearance of bright fluorescent spots in a comma-like form, at the oral region.
-y-globulin fraction was isolated from the anti-serum bv ammonium sulfate precipitation and DEAE cellulose column chromatography. FITC (Sigma) was conjugated to the y-globulins accord& to McKinney [17]. Essentially, FITC was allowed to interact with the y-globulin fraction for 2 h in phosphate buffer pH 9.5. The unbound FITC was removed from the protein-bound FITC by chromatography on Sephadex G-50.
RESULTS Stimulation of vacuole formation by ConA and its relationship to the conjugation process
Recently, we have reported that addition of low ConA concentrations to the mating types of T. pyriformis strongly inhibits conjugation [7]. In order to study the localization of the bound ConA, fluorescein-ConA was used in the present study. Preliminary experiments showed that fluorescein-ConA
in Tetrahymena
pyriformis
339
Each cell contains numerous fluorescent spots. For other experimental details see Materials and Methods. X400.
inhibits conjugation at the same concentration rate as native ConA. Inhibition by fluorescein Con& like inhibition by native ConA, could be reversed by addition of (Ymethyl-mannoside @MM). When starved unmixed mating types of T. pyriformis were incubated with fluorescein-ConA for 1 min at 30°C fixed and examined by fluorescence microscope, the fluorescent stain was found only in a confined region associated with the oral area (fig. 1a). As can be seen in fig. 1a, the rest of the cell is devoid of fluorescein-ConA. The region binding ConA has a characteristic shape resembling a wedge or a comma (fig. 1a). Three minutes after incubation with fluorescein-ConA the first intracellular bright spots appeared. Their number increased rapidly with time and, by 15 min, most of ExpCellRes 110 (1977)
340
Frisch and Loyter
but did not inhibit conjugation (table 1). Addition of soluble antibody to a mixture of starved cells, causing the formation in situ of an insoluble complex with the secreted glycoproteins gave similar results. Initiation of pair formation was, however, slightly delayed. Table 1 is a summary of the effect of several agents on vacuole Fig. 2. Abscissa: time after mixing (hours); ordinate: formation and the conjugation process. As coqjugation %. The effect of carmine narticles on the kinetics of the can be seen in table 1, after a short inconjugation process. Cells of opposite mating types cubation time, the aggregates were found in were mixed in the presence (O-O) and absence (0-O) of 0.1% carmine particles A-A, 25 @g/ml intracellular vacuoles. ConA was the only ConA was added upon mixing. Conjugation was fol- agent which inhibited conjugation permalowed and determined as described in Materials and nently, while all the other insoluble comMethods. the cell population contained numerous (up to 15) shining dots (fig. 1b). Examination of sections made of cells having fluorescent spots showed that fluorescein-ConA is localized in intracellular vacuoles (not shown). These experiments were performed with mating type I but identical results were obtained when fluorescein-ConA was added either to the other mating type or directly after mixing of the two mating types. Both the binding and the uptake of fluorescein-ConA were specifically inhibited by addition of 50 mM of crMM. Inhibition of conjugation by ConA might be due to the uptake of an insoluble complex formed between the added ConA and soluble glycoproteins secreted from the cells [8], thus serving as a nutrient and ending the starvation. In order to study this question, a specific antibody against the ConA-binding glycoproteins was prepared (see Materials and Methods), and was shown to form an insoluble complex with its antigen [8]. Incubation of starved mating types with this insoluble complex resulted in the uptake of the complex (measured by fluorescent-Ab) into intracellular vacuoles Exp Cell Res 110 (1977)
Table 1. The effect of various agents on vacuole formation and on the conjugation process
Addition None Antibody against CBM Anti-CBM-antibodv+CBM (insoluble complex) Carmine particles ConA
Vacuole formation”
Appearance of the first conjugant (hours)
NP +
0.5-l 1.5-2
+ + +
1.5-2 1.5-2 >24
O1“Vacuole formation” stands for the ability of the cells to take up the different agents into intracellular vacuoles when added upon mixing of the mating types. The presence of vacuoles was checked by phase microscope (carmine) and fluorescence microscope (the fluorescent agents). It should be emphasized that during development of conjugation, vacuole formation ceases. b “NY, not investigated. Cells were starved and induced for conjugation as described under Materials and Methods. The various agents were added upon mixing, and the time up to the appearance of the first pairs was checked. Concentrations: fluorescent anti-CBM-antibody (300 pg/ ml); carmine particles 0.1%; fluorescein-ConA 25 clgl ml. An insoluble complex between CBM and the antibody was prepared as follows: the fluorescent antibody (300 pg/ml was incubated with isolated CBM (100 pg/ml) in a final volume of 5 ml at 30°C for 1 h. At the end of the incubation period the insoluble complex formed was collected by centrifugation and the pellet obtained was suspended in 0.1 ml, 0.02 ml of the insoluble complex suspension was added to each ml of the conjugation medium. For preparation of the fluorescent antibody see Materials and Methods.
Inhibition of conjugation in Tetrahymena pyrijiormis
341
Fig. 3 shows that when carmine particles _*co were added at zero time of conjugation, 100 90% of the cells contained these particles in intracellular vacuoles. When conjugation reaches its maximal value (80% conjuga50 SO tion) only 15% of the cells were capable of taking carmine up into vacuoles. Conjugants were unable to take up carmine when o-o-o the particles were added after establishment of permanent bonds between pairs. The 2 4 0 1 3 decline in vacuole formation preceded by Fig. 3. Abscissa: time after mixing (hours); ordinate: (lefr) no. of cells containing vacuoles (%), O-O; about 1 h the formation of pairs between (right) conjugation (%) (O-O). the two mating types (fig. 3). As can be Decline in vacuole formation in T. pyriformis after induction of conjugation. Conjugation and the ability seen in fig. 3 pair formation did not start to form vacuole were determined as described in Mabefore most of the cells (7080%) lost their terials and Methods. capability to take up carmine particles. Addition of ConA to a mixture of the matplexes only slightly delayed pair formation. ing types abolished conjugation and, in its A detailed study of the effect of carmine presence, the cells maintained a constant particles on the kinetics of conjugation is rate of vacuole formation (fig. 4). seen in fig. 2. As can be seen, conjugaand tion reached the same maximal degree in Specific binding offuorescein the presence or absence of carmine par- ferritin-ConA to the conjugation area ticles; although the initial lag time observed We showed above that when ConA is added before the first pairs were formed was pro- to each of the mating types or to their longed from 30 min, in the absence of car- mixture at zero time of conjugation it is internalized in food vacuoles. Binding of mine, to about 90 min, in its presence. ConA to conjugants that were shown to be Decline in vacuole formation after unable to form vacuoles was then studied. induction of conjugation a Since it was found that addition of aggre- 100 s gated particles to a mixture of T. pyriformis delayed the onset of conjugation, it was of interest to study the relationship between So vacuole formation and the conjugation process.
The capability of the cells to form vacuoles during conjugation was sstudied -. - _ by addition of carmine particles to a mixture ml of the mating types at various times after they had been mixed. It was observed that after induction of conjugation by mixing two mating types, the number of cells capable of taking up carmine gradually declined.
0,
9
& 1
Fig. ’ 4. Abscissa:
a
-
j 2
3
4
85% 0%
time after mixing (hours); ordinate: no. of cells containing vacuoles (%). Cells of opposite mating types were mixed and their ability to form vacuoles was followed up to 4 hours. O-O, Control; A-A, 25 &ml of ConA was added upon mixing of the mating types. Columns at the right show the final percent of conjugation obtained after 4 hours. Exp Cell Res 110 (1977)
342
Frisch and Loyter
Fig. 5. Binding of fluorescein-ConA to conjugants.
Fluorescein-ConA (25 pg/ml) was added to conjugants 3 h after mixing of the starved mating types. (a, al.) Firmly held conjugant pairs. The contact area shows
Incubation of fluorescein-ConA with conjugants of T. pyriformis resulted in binding of ConA molecules to a very confined region associated with the conjugation area. As can be seen in fig. 5a, a,, a bright fluorescent ring encircled the area connecting the two fused mating types. Closer study of this region revealed that the ring consists, in fact, of two rings each associated with one of the mating partners. This finding could not be recorded on film due to the low intensity of the fluorescence and the poor resolution which resulted from it. It is clear that the rest of the cell is devoid of any fluorescent dye. Under these conditions, ConA was found neither in the oral Exp CeNResl10 (1977)
fluorescence; (b) single cell, the anterior tip is fluorescing; (c) as in (a) but in the presence of 50 mM oMM. x1000.
area nor in intracellular vacuoles and all conjugants had fluorescent rings around the conjugation region. Fluorescein-ConA was Fig. 6. Specific binding of ferritin-ConA to the conjugation area. Ferritin-ConA was incubated with conjugants and thin sections were prepared as described under Materials and Methods. (a) An overall view of the conjugation area. Ferritin molecules or aggregates ktrrows~ can be seen at the two oueninas of the channel formed between the membranes 07 the two cells (1 and 2). x 19000; (b) hi& nnurnitication of the membrane area which binds sp&tic~y fenitin-ConA (details of (a)). x78000. Fe&in-ConA molecules at the entrance of the channel can be easily identified (arrow); (c) as (b) but from another cell. Arrows point to the fenitin oarticles. Note that membranes of both cells bind fen-&n molecules. x78 Ooo; (4 as (a) but in the presence of 50 mM aMM. Note that ferritin arztzregates are present in the medium but not attachid to the membranes (arrow). x 19000.
343
344
Frisch and Loyter
also attached in the form of a bright ring ence of soluble ConA-binding glycoproteins to the anterior part of some single cells in the medium [8]. present in a conjugating system (fig. 5b). Two types of binding patterns of ConA This part is the specialized area destined molecules to the mating types of T. pyrifor the conjugation process. The single cells formis were observed. When ConA was containing the fluorescent ring represent added to the unmixed mating types it was either cells before mating or ex-conjugants first attached in a wedge or comma-like dissociated by the addition of ConA. Bind- form in the oral region of the cells and was ing of fluorescein-ConA was sensitive to subsequently taken up into intracellular aMM whose addition completely abolished vacuoles. On the other hand, addition of the appearance of the fluorescent ring (fig. ConA to conjugants resulted in its binding 5C). in a ring form around the conjugation reIn order to obtain higher resolution of the gion. Conjugants were unable to form vacuConA-binding sites, ferritin-ConA was oles and were devoid of intracellular ConA. used. Fig. 6 shows a thin section through It is clear that the two types of ConAthe conjugation area. Two cells connected binding sites could not coexist at the same via cytoplasmic bridges can be seen in fig. time on the same cells. This observation 6. Close examination of the cells shows that may reflect the changes in the membrane, numerous ferritin molecules or aggregates occurring during development of conjugaare observed either in close proximity to the tion. The inhibition of conjugation by ConA plasma membranes of each cell or associ- may be related to either of the above patated with amorphous material just next to terns. The first observable event, occurring 20 the contact area. Since this picture is a thin section these regions represent a cross sec- min after mixing of the mating types, is tion of a ring. A higher magnification (fig. cessation of vacuole formation. It appears 6b, c) shows that ferritin-ConA is close to that there is a time lapse of about 1 h bethe plasma membranes of both mating tween suppression of vacuole formation types. and the appearance of the first pairs. ThereCareful examination of the cells by elec- fore, it may be assumed that vacuolization tron microscopy revealed that no ferritin must be inhibited before conjugation can particles could be found in any part of the occur. Since both vacuolization and concells beside the above-mentioned conjuga- jugation are intensive membrane events, it tion region. Binding of ConA to specific is likely that the cells must stop one beregions, other than the oral area, could not fore starting the other. Therefore, any stimbe detected in either of the starved mating ulation of vacuolization would result in intypes before mixing, and was first observed hibition of the conjugation process. Indeed, 30-45 min after mixing. it was observed that aggregated material, such as carmine particles or insoluble antigen-antibody complex, which are quickly DISCUSSION taken up into food vacuoles delayed the onThe use of ferritin-ConA and fluorescein- set of pair formation. Eventually, vacuole ConA made it possible to study the localiza- formation stopped (not shown) and contion of ConA receptors during the conjuga- jugation occurred. ConA, which was unique tion of T. pyriformis, in spite of the exist- in continuously either stimulating vacuole Exp Cell Res 110 (1977)
Inhibition of conjugation in Tetrahymena pyriformis formation or inhibiting its suppression before mating, also caused a perpetual inhibition of conjugation. The fact that ConA was taken up into food vacuoles at relatively low concentrations and the fact that its effect was sensitive to aMM may indicate that vacuolization by ConA is due to its direct binding to the membrane of the oral apparatus. It is possible to explain the inhibition of conjugation by ConA by two mechanisms: ConA may inhibit pair formation by constantly inducing vacuole formation thus preventing the rearrangement of membranes necessary for mating. Alternatively, ConA may exert its inhibitory effect by other mechanisms and as a result vacuolization continues. In a previous paper we have reported that ConA is able to inhibit conjugation when added at different times during the conjugation process, and is even able to dissociate loosely bound pairs formed between the mating types [7]. From the data of the present work, it can be inferred that at these stages, vacuolization has already been suppressed. Under these conditions, ConA did not induce vacuole formation although it did inhibit conjugation. It has been shown here that addition of ConA to conjugants resulted in its binding to specific membrane sites around the conjugation region. We suggest that when ConA is added after rearrangement has occurred and vacuoles can no longer be formed, the inhibition of conjugation is due to direct binding of ConA to receptors in the conjugation area. Single cells in an actively conjugating population also showed specific binding of fluorescein-ConA to the conjugation area. These single cells may represent cells just before pairing or just after dissociation of pairs by ConA.
345
ConA receptors were not found in the anterior part of growing cells or in starved cells before mixing. Therefore, it can be assumed that either their number is small and beyond the resolution power of our methods or that the ConA receptors are induced only during the “co-stimulation” period. The “co-stimulation” period was previously designated as the time from mixing of the mating types until pairing [ 181.It is possible that the meeting between the opposite mating types during the co-stimulation period is mediated by their ConA receptors. We suggest that the number of ConA receptors is increased by their mutual interaction. As soon as the number of ConA receptors reaches a minimal number which allows permanent binding between the opposite mating types, cell pairs will be formed. Based on the use of inhibitors of protein synthesis, it was suggested that during the co-stimulation period specific proteins, required for pairing, are induced and synthesized [l&20]. In a previous paper we have suggested that these proteins could be glycoproteins [14] and may coincide with the ConA receptors observed in the present work. Addition of ConA during the costimulation period will cause masking of the receptors and prevent the specific interaction between the opposite mating types, thereby inhibiting further development of the surface receptors. In this way, both pairing and development of the conjugation region are inhibited by the ConA molecules. Similarly, dissociation of the pairs formed between the mating types by addition of ConA might be due to its ability to bind to the receptors and compete with the complementary receptors of the opposite mating types. Single cells containing fluorescein-ConA may be the result of such process (fig. 5). A preliminary work in our laboratory has Exp Cell Res 110 (1977)
346
Frisch and Loyter
shown that the contact area between the conjugants is completely devoid of intramembrane particles (IMP) [21]. Lately we have found that this area is encircled by a ring composed of several rows of IMP [A. Frisch & A. Loyter, unpublished results]. Similar structures were first reported by Satir [22] in fusion of mucocysts with plasma membranes of T. pyriformis and by Weiss et al. [23] during conjugation of ChlaIt is tempting to mydomonas reinhardi. speculate that ConA receptors which are visualized as a ring by using fluorescentConA are expressed in freeze-etching as a ring of IMP. The two conjugants are held together by a double ring of receptors which have a function in recognition and attachment and the fusion itself occurs in the area encircled by the ring, consisting mainly of phospholipids. Attempts to study the induction of the ConA receptors, using SDS-polyacrylamide slab gels are under way in our laboratory. This method would allow us the investigation of the kinetics of their appearance and may contribute to further understanding of the nature of these receptors.
REFERENCES Austin, C R, J reprod fert 44 (1975) 155. 2. Amsterdam, A, Ohad, I & Schramm, M, J cell biol 41 (1%9) 753. 3. Abrahams, S J & Holtzman, E, J cell bio156 (1973) 540. 4. De Robertis, E, Histophysiology of synapses and neurosecretion. Pergamon Press, Oxford (1964). 5. Chi, E Y, Lagunoff, D & Koehler, J K, Proc natl acad sci US 73 (1976) 2823. 6. Elliott. A M. Bioloav of Terrahymena (ed A M Elliottj, pp ‘259-277 Dowden,. Hutchison and Ross, Stroudsburg, PA (1973). 7. Ofer, L, Levkovitz, H & Loyter, A, J cell biol 70 (1976) 287. 8. Frisch, A, Levkovitz, H & Loyter, A, Exp cell res 106(1977) 293. 9. Frisch, A, Bessler, W, Lipps, H J & Ammermann, D, J protozoo123 (1976) 427. 10. Bessler, W & Lipps, H J, J gen microb 92 (1976) 221. 11. Lsvlie, A 62Bryhni, E, Nature 263 (1976) 779. 12. Wiese, L & Shoemaker, D W, Biol bull 138 (1970) 13. ?keta K, Exp cell res 90 (1975) 56. 14. Frisch, A, Levkovitz, H & Loyter, A, Biochem bioohvs res commun 72 (1976) 138. 15. Nanndy, D L & McCoy, J W, Trans Am micros sot 95 (1976) 664. 16. Yanovski, A & Loyter, A, J biol them 247 (1972) 4021. 17. McKinney, R M, Spillane, J T & Pearce, G W, J immuno193 (1964) 232. 18. Bruns, P J & Palestine, R F, Dev bio142 (1975) 75. 19. Allewell, N M, Oles, J &Wolfe, J, Exp cell res 97 (1976) 394. 20. Ofer, L, Mercazi, M & Loyter, A, Biol cell 29 (1977). In press. 21. Wolfe, J & Loyter, A, J cell biol67 (1975) 459~. 22. Satir, B, Schooley, C & Satir, P, J cell biol 56 (1973) 153. 23. Weiss. R L. Goodenough, D A & Goodenough, U W, J cell bio172 (1973 144. Received April 29, 1977 Revised version received June 15, 1977 Accepted July 6, 1977
Exp Cell Res 110 (1977)
Experimental
INHIBITION
Cell Research 110 (1977) 337-346
OF CONJUGATION PYRZFORMZS Localization
IN TETRAHYMENA
BY ConA
of ConA-binding
sites
A. FRISCH and A. LOYTER Department
of Biological
Chemistry,
The Hebrew
University
of Jerusalem,
Jerusalem,
Israel
SUMMARY Two patterns of ConA binding to starved mating types of Tetrahymena pyriformis were observed depending on its time of addition. When ConA was added upon mixing of the mating types, at zero time of conjugation, it was first bound to the oral region and subsequently was taken into intracellular vacuoles. When it was added to conjugants, it was specifically bound as a ring around the conjugation area. The ability of the cells to form vacuoles, assayed by addition of carmine particles, declined prior to pair formation. The relationship between the above phenomena and the ability of ConA to inhibit conjugation is discussed.
Several membrane fusion processes such as fertilization [ 11,secretion of enzymes [2], hormones [3] or neurotransmitters [4] appear to be limited to specific membrane regions and do not necessarily involve the entire surface of the cell. In fertilization of mammalian cells, during which spermatozoa fuse with ova, the membrane event is restricted to the acrosomal region of the sperm cells [l], while in all secretion processes, intracellular granules fuse only with the plasma membrane facing the lumen [2, 51. This may imply that, in spite of the fluid nature of biological membranes, the plasma membrane is heterogeneous with regard to the composition and to the concentration of certain receptors. A suitable system to study the question of specific membrane changes induced at restricted regions is the conjugation process of Tetrahymena pyriformis. During this process, two starved complementary mating
types are induced to interact at specific membrane regions at which ultimately fusion will occur [6]. Recently we have reported that the lectin ConA inhibits the induction of conjugation when added to a mixture of starved complementary mating types of T. pyriformis and even partially dissociates pairs formed between them [7]. Experiments using rH]ConA showed that about 90-95% of the ConA precipitates with soluble glycoprotein secreted into the starvation medium by the two mating types [8]. By using specific anti-glycoprotein antibodies, it was shown that the secreted glycoproteins are not involved in the conjugation process [S]. It was, therefore, inferred that ConA may inhibit the conjugation process by direct binding to the surface of T. pyriformis. Addition of ConA to the cells of Stylonichia mytilus caused immobilization of the cells, probably as a result of specific bindExp
Cell
Res 110 (1977)
338
Frisch and Loyter
ing of ConA to the cilia [9, lo]. ConA also jugation, a suspension of the two starved mating types 106cells/ml) were mixed by introducing 0.5 ml interacts specifically with one of the mating (about of each suspension into a 20 ml glass flask and then types of Ulva [ll], inhibits conjugation of incubated at 30°C without shaking. The extent of conChlamydomonas [ 121, and fertilization of jugation was determined after 4 h of incubation [7]. sea urchin [13]. In all these cases the presBinding offluorescein-ConA to ence of surface glycoproteins representing single cells and to conjugants ConA receptors was assumed. Surface A suspension of either each of the mating types or of glycoproteins were also suggested to parti- conjugants (1 ml of 1Ogcells) were incubated, if not cipate in the conjugation process of T. pyri- otherwise stated, with 26 pg of fluorescein-ConA (Miles-Yeda, Rehovot, Israel) for 15 min at 30°C. At formis [ 141.Recently we have reported that the end of the incubation period the excess of fluotunicamycin, a specific inhibitor of glyco- rescein-ConA was removed by centrifugation at 500 g with 20 mM tricine NaOH, pH 7.4. The pellet obprotein biosynthesis, inhibits the conjuga- tained was suspended in 1 ml of 1% glutamldehyde dissolved in 20 mM tricine-NaOH, pH 7.4. After tion process in T. pyriformis [ 141. fixation, the cells were observed with a Zeiss WLIn the present work, we have used fluo- Research microscope with fluorescent attachment, and rescein-ConA and ferritin-ConA in order to photographs were taken at 1 min exposures on Kodak Tri-X film. study the precise localization, and the fate of ConA molecules when added to the mat- Uptake of carmine particles into ing types of T. pyriformis. It was found food vacuo[es that the ConA molecules interact with T. Conjugation was initiated by mixing 10 ml of each pyriformis at specific regions of the cell of the starved mating types (1Og cells/ml) in a 2 1 flask. At various intervals after mixing, surface. The correlation between the bind- Erlenmeyer conjugation was determined and samples of 1 ml ing of ConA and its ability to inhibit con- were withdrawn and mixed with 0.1 ml of carmine (final cont. 0.1%). After 5 min of incubajugation is discussed. In addition, the inter- particles tion at 3o”C, the cells were fixed by 2% formalderelationship between the conjugation pro- hyde, observed in the light microscope and the perof cells containing carmine particles was escess and another membranous event, name- centage timated. The experiments were performed in duplicate and about 200 cells were scanned in each sample. ly vacuole formation, is analyzed.
MATERIALS
AND METHODS
Cells mating type I (strain WH-6) and mating type III (WH-52) of syngen 1 (according to the new nomenclature suggested by Nanney & McCoy [15] the organism dealt with in this investigation should be called T. rhermophila) were grown as described before [7].
T. pyriformis
Medium The medium used for washing of cells, starvation and conjugation was 20 mM tricine-NaOH, pH 7.4.
Induction and determination of conjugation Cells were starved by incubating each of the mating types (108 cells/ml) in 20 mM tricine-NaOH, pH 7.4, at 30°C for 24 h. Conjugation was induced and determined as described in a previous work [7]. For conExp Cell Res II0 (1977)
Binding of ferritin-ConA ceils and to conjugants
to single
A suspension of either of the mating types or of conjugants (5 ml of 106cells/ml) was incubated with 25 &ml of ferritin-ConA (Miles-Yeda, Rehovot) for 15 min at 30°C. At the end of the incubation period, the cells were centrifuged at 500 g and washed twice with 15 ml of 20 mM tricine-NaOH, pH 7.4. When amethyl-mannoside (arMM) was used, it was allowed to interact with the ferritin-ConA for 10 min before addition to the cell suspension. After fixation with 1% glutaraldehyde, the cells were embedded in Epon and prepared for electron microscopy as previously described [16]. Thin sections were stained with uranyl acetate for 20 min before observation.
Binding ofjluorescein isothiocyanate (FITC) to antibody against ConAbinding material (Anti-CBM) ConA-binding material (CBM) was isolated from the starvation medium of T. pyriformis, purified and injetted into rabbits, as previously described [8]. The
Inhibition
of conjugation
Fig. 1. Binding of fluorescein-ConA to starved cells
of T. pyriformis (mating type I). Incubation with fluorescein-ConA for (a) 1 min; (b) 15 min at 30°C. Note the appearance of bright fluorescent spots in a comma-like form, at the oral region.
-y-globulin fraction was isolated from the anti-serum bv ammonium sulfate precipitation and DEAE cellulose column chromatography. FITC (Sigma) was conjugated to the y-globulins accord& to McKinney [17]. Essentially, FITC was allowed to interact with the y-globulin fraction for 2 h in phosphate buffer pH 9.5. The unbound FITC was removed from the protein-bound FITC by chromatography on Sephadex G-50.
RESULTS Stimulation of vacuole formation by ConA and its relationship to the conjugation process
Recently, we have reported that addition of low ConA concentrations to the mating types of T. pyriformis strongly inhibits conjugation [7]. In order to study the localization of the bound ConA, fluorescein-ConA was used in the present study. Preliminary experiments showed that fluorescein-ConA
in Tetrahymena
pyriformis
339
Each cell contains numerous fluorescent spots. For other experimental details see Materials and Methods. X400.
inhibits conjugation at the same concentration rate as native ConA. Inhibition by fluorescein Con& like inhibition by native ConA, could be reversed by addition of (Ymethyl-mannoside @MM). When starved unmixed mating types of T. pyriformis were incubated with fluorescein-ConA for 1 min at 30°C fixed and examined by fluorescence microscope, the fluorescent stain was found only in a confined region associated with the oral area (fig. 1a). As can be seen in fig. 1a, the rest of the cell is devoid of fluorescein-ConA. The region binding ConA has a characteristic shape resembling a wedge or a comma (fig. 1a). Three minutes after incubation with fluorescein-ConA the first intracellular bright spots appeared. Their number increased rapidly with time and, by 15 min, most of ExpCellRes 110 (1977)
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Frisch and Loyter
but did not inhibit conjugation (table 1). Addition of soluble antibody to a mixture of starved cells, causing the formation in situ of an insoluble complex with the secreted glycoproteins gave similar results. Initiation of pair formation was, however, slightly delayed. Table 1 is a summary of the effect of several agents on vacuole Fig. 2. Abscissa: time after mixing (hours); ordinate: formation and the conjugation process. As coqjugation %. The effect of carmine narticles on the kinetics of the can be seen in table 1, after a short inconjugation process. Cells of opposite mating types cubation time, the aggregates were found in were mixed in the presence (O-O) and absence (0-O) of 0.1% carmine particles A-A, 25 @g/ml intracellular vacuoles. ConA was the only ConA was added upon mixing. Conjugation was fol- agent which inhibited conjugation permalowed and determined as described in Materials and nently, while all the other insoluble comMethods. the cell population contained numerous (up to 15) shining dots (fig. 1b). Examination of sections made of cells having fluorescent spots showed that fluorescein-ConA is localized in intracellular vacuoles (not shown). These experiments were performed with mating type I but identical results were obtained when fluorescein-ConA was added either to the other mating type or directly after mixing of the two mating types. Both the binding and the uptake of fluorescein-ConA were specifically inhibited by addition of 50 mM of crMM. Inhibition of conjugation by ConA might be due to the uptake of an insoluble complex formed between the added ConA and soluble glycoproteins secreted from the cells [8], thus serving as a nutrient and ending the starvation. In order to study this question, a specific antibody against the ConA-binding glycoproteins was prepared (see Materials and Methods), and was shown to form an insoluble complex with its antigen [8]. Incubation of starved mating types with this insoluble complex resulted in the uptake of the complex (measured by fluorescent-Ab) into intracellular vacuoles Exp Cell Res 110 (1977)
Table 1. The effect of various agents on vacuole formation and on the conjugation process
Addition None Antibody against CBM Anti-CBM-antibodv+CBM (insoluble complex) Carmine particles ConA
Vacuole formation”
Appearance of the first conjugant (hours)
NP +
0.5-l 1.5-2
+ + +
1.5-2 1.5-2 >24
O1“Vacuole formation” stands for the ability of the cells to take up the different agents into intracellular vacuoles when added upon mixing of the mating types. The presence of vacuoles was checked by phase microscope (carmine) and fluorescence microscope (the fluorescent agents). It should be emphasized that during development of conjugation, vacuole formation ceases. b “NY, not investigated. Cells were starved and induced for conjugation as described under Materials and Methods. The various agents were added upon mixing, and the time up to the appearance of the first pairs was checked. Concentrations: fluorescent anti-CBM-antibody (300 pg/ ml); carmine particles 0.1%; fluorescein-ConA 25 clgl ml. An insoluble complex between CBM and the antibody was prepared as follows: the fluorescent antibody (300 pg/ml was incubated with isolated CBM (100 pg/ml) in a final volume of 5 ml at 30°C for 1 h. At the end of the incubation period the insoluble complex formed was collected by centrifugation and the pellet obtained was suspended in 0.1 ml, 0.02 ml of the insoluble complex suspension was added to each ml of the conjugation medium. For preparation of the fluorescent antibody see Materials and Methods.
Inhibition of conjugation in Tetrahymena pyrijiormis
341
Fig. 3 shows that when carmine particles _*co were added at zero time of conjugation, 100 90% of the cells contained these particles in intracellular vacuoles. When conjugation reaches its maximal value (80% conjuga50 SO tion) only 15% of the cells were capable of taking carmine up into vacuoles. Conjugants were unable to take up carmine when o-o-o the particles were added after establishment of permanent bonds between pairs. The 2 4 0 1 3 decline in vacuole formation preceded by Fig. 3. Abscissa: time after mixing (hours); ordinate: (lefr) no. of cells containing vacuoles (%), O-O; about 1 h the formation of pairs between (right) conjugation (%) (O-O). the two mating types (fig. 3). As can be Decline in vacuole formation in T. pyriformis after induction of conjugation. Conjugation and the ability seen in fig. 3 pair formation did not start to form vacuole were determined as described in Mabefore most of the cells (7080%) lost their terials and Methods. capability to take up carmine particles. Addition of ConA to a mixture of the matplexes only slightly delayed pair formation. ing types abolished conjugation and, in its A detailed study of the effect of carmine presence, the cells maintained a constant particles on the kinetics of conjugation is rate of vacuole formation (fig. 4). seen in fig. 2. As can be seen, conjugaand tion reached the same maximal degree in Specific binding offuorescein the presence or absence of carmine par- ferritin-ConA to the conjugation area ticles; although the initial lag time observed We showed above that when ConA is added before the first pairs were formed was pro- to each of the mating types or to their longed from 30 min, in the absence of car- mixture at zero time of conjugation it is internalized in food vacuoles. Binding of mine, to about 90 min, in its presence. ConA to conjugants that were shown to be Decline in vacuole formation after unable to form vacuoles was then studied. induction of conjugation a Since it was found that addition of aggre- 100 s gated particles to a mixture of T. pyriformis delayed the onset of conjugation, it was of interest to study the relationship between So vacuole formation and the conjugation process.
The capability of the cells to form vacuoles during conjugation was sstudied -. - _ by addition of carmine particles to a mixture ml of the mating types at various times after they had been mixed. It was observed that after induction of conjugation by mixing two mating types, the number of cells capable of taking up carmine gradually declined.
0,
9
& 1
Fig. ’ 4. Abscissa:
a
-
j 2
3
4
85% 0%
time after mixing (hours); ordinate: no. of cells containing vacuoles (%). Cells of opposite mating types were mixed and their ability to form vacuoles was followed up to 4 hours. O-O, Control; A-A, 25 &ml of ConA was added upon mixing of the mating types. Columns at the right show the final percent of conjugation obtained after 4 hours. Exp Cell Res 110 (1977)
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Frisch and Loyter
Fig. 5. Binding of fluorescein-ConA to conjugants.
Fluorescein-ConA (25 pg/ml) was added to conjugants 3 h after mixing of the starved mating types. (a, al.) Firmly held conjugant pairs. The contact area shows
Incubation of fluorescein-ConA with conjugants of T. pyriformis resulted in binding of ConA molecules to a very confined region associated with the conjugation area. As can be seen in fig. 5a, a,, a bright fluorescent ring encircled the area connecting the two fused mating types. Closer study of this region revealed that the ring consists, in fact, of two rings each associated with one of the mating partners. This finding could not be recorded on film due to the low intensity of the fluorescence and the poor resolution which resulted from it. It is clear that the rest of the cell is devoid of any fluorescent dye. Under these conditions, ConA was found neither in the oral Exp CeNResl10 (1977)
fluorescence; (b) single cell, the anterior tip is fluorescing; (c) as in (a) but in the presence of 50 mM oMM. x1000.
area nor in intracellular vacuoles and all conjugants had fluorescent rings around the conjugation region. Fluorescein-ConA was Fig. 6. Specific binding of ferritin-ConA to the conjugation area. Ferritin-ConA was incubated with conjugants and thin sections were prepared as described under Materials and Methods. (a) An overall view of the conjugation area. Ferritin molecules or aggregates ktrrows~ can be seen at the two oueninas of the channel formed between the membranes 07 the two cells (1 and 2). x 19000; (b) hi& nnurnitication of the membrane area which binds sp&tic~y fenitin-ConA (details of (a)). x78000. Fe&in-ConA molecules at the entrance of the channel can be easily identified (arrow); (c) as (b) but from another cell. Arrows point to the fenitin oarticles. Note that membranes of both cells bind fen-&n molecules. x78 Ooo; (4 as (a) but in the presence of 50 mM aMM. Note that ferritin arztzregates are present in the medium but not attachid to the membranes (arrow). x 19000.
343
344
Frisch and Loyter
also attached in the form of a bright ring ence of soluble ConA-binding glycoproteins to the anterior part of some single cells in the medium [8]. present in a conjugating system (fig. 5b). Two types of binding patterns of ConA This part is the specialized area destined molecules to the mating types of T. pyrifor the conjugation process. The single cells formis were observed. When ConA was containing the fluorescent ring represent added to the unmixed mating types it was either cells before mating or ex-conjugants first attached in a wedge or comma-like dissociated by the addition of ConA. Bind- form in the oral region of the cells and was ing of fluorescein-ConA was sensitive to subsequently taken up into intracellular aMM whose addition completely abolished vacuoles. On the other hand, addition of the appearance of the fluorescent ring (fig. ConA to conjugants resulted in its binding 5C). in a ring form around the conjugation reIn order to obtain higher resolution of the gion. Conjugants were unable to form vacuConA-binding sites, ferritin-ConA was oles and were devoid of intracellular ConA. used. Fig. 6 shows a thin section through It is clear that the two types of ConAthe conjugation area. Two cells connected binding sites could not coexist at the same via cytoplasmic bridges can be seen in fig. time on the same cells. This observation 6. Close examination of the cells shows that may reflect the changes in the membrane, numerous ferritin molecules or aggregates occurring during development of conjugaare observed either in close proximity to the tion. The inhibition of conjugation by ConA plasma membranes of each cell or associ- may be related to either of the above patated with amorphous material just next to terns. The first observable event, occurring 20 the contact area. Since this picture is a thin section these regions represent a cross sec- min after mixing of the mating types, is tion of a ring. A higher magnification (fig. cessation of vacuole formation. It appears 6b, c) shows that ferritin-ConA is close to that there is a time lapse of about 1 h bethe plasma membranes of both mating tween suppression of vacuole formation types. and the appearance of the first pairs. ThereCareful examination of the cells by elec- fore, it may be assumed that vacuolization tron microscopy revealed that no ferritin must be inhibited before conjugation can particles could be found in any part of the occur. Since both vacuolization and concells beside the above-mentioned conjuga- jugation are intensive membrane events, it tion region. Binding of ConA to specific is likely that the cells must stop one beregions, other than the oral area, could not fore starting the other. Therefore, any stimbe detected in either of the starved mating ulation of vacuolization would result in intypes before mixing, and was first observed hibition of the conjugation process. Indeed, 30-45 min after mixing. it was observed that aggregated material, such as carmine particles or insoluble antigen-antibody complex, which are quickly DISCUSSION taken up into food vacuoles delayed the onThe use of ferritin-ConA and fluorescein- set of pair formation. Eventually, vacuole ConA made it possible to study the localiza- formation stopped (not shown) and contion of ConA receptors during the conjuga- jugation occurred. ConA, which was unique tion of T. pyriformis, in spite of the exist- in continuously either stimulating vacuole Exp Cell Res 110 (1977)
Inhibition of conjugation in Tetrahymena pyriformis formation or inhibiting its suppression before mating, also caused a perpetual inhibition of conjugation. The fact that ConA was taken up into food vacuoles at relatively low concentrations and the fact that its effect was sensitive to aMM may indicate that vacuolization by ConA is due to its direct binding to the membrane of the oral apparatus. It is possible to explain the inhibition of conjugation by ConA by two mechanisms: ConA may inhibit pair formation by constantly inducing vacuole formation thus preventing the rearrangement of membranes necessary for mating. Alternatively, ConA may exert its inhibitory effect by other mechanisms and as a result vacuolization continues. In a previous paper we have reported that ConA is able to inhibit conjugation when added at different times during the conjugation process, and is even able to dissociate loosely bound pairs formed between the mating types [7]. From the data of the present work, it can be inferred that at these stages, vacuolization has already been suppressed. Under these conditions, ConA did not induce vacuole formation although it did inhibit conjugation. It has been shown here that addition of ConA to conjugants resulted in its binding to specific membrane sites around the conjugation region. We suggest that when ConA is added after rearrangement has occurred and vacuoles can no longer be formed, the inhibition of conjugation is due to direct binding of ConA to receptors in the conjugation area. Single cells in an actively conjugating population also showed specific binding of fluorescein-ConA to the conjugation area. These single cells may represent cells just before pairing or just after dissociation of pairs by ConA.
345
ConA receptors were not found in the anterior part of growing cells or in starved cells before mixing. Therefore, it can be assumed that either their number is small and beyond the resolution power of our methods or that the ConA receptors are induced only during the “co-stimulation” period. The “co-stimulation” period was previously designated as the time from mixing of the mating types until pairing [ 181.It is possible that the meeting between the opposite mating types during the co-stimulation period is mediated by their ConA receptors. We suggest that the number of ConA receptors is increased by their mutual interaction. As soon as the number of ConA receptors reaches a minimal number which allows permanent binding between the opposite mating types, cell pairs will be formed. Based on the use of inhibitors of protein synthesis, it was suggested that during the co-stimulation period specific proteins, required for pairing, are induced and synthesized [l&20]. In a previous paper we have suggested that these proteins could be glycoproteins [14] and may coincide with the ConA receptors observed in the present work. Addition of ConA during the costimulation period will cause masking of the receptors and prevent the specific interaction between the opposite mating types, thereby inhibiting further development of the surface receptors. In this way, both pairing and development of the conjugation region are inhibited by the ConA molecules. Similarly, dissociation of the pairs formed between the mating types by addition of ConA might be due to its ability to bind to the receptors and compete with the complementary receptors of the opposite mating types. Single cells containing fluorescein-ConA may be the result of such process (fig. 5). A preliminary work in our laboratory has Exp Cell Res 110 (1977)
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shown that the contact area between the conjugants is completely devoid of intramembrane particles (IMP) [21]. Lately we have found that this area is encircled by a ring composed of several rows of IMP [A. Frisch & A. Loyter, unpublished results]. Similar structures were first reported by Satir [22] in fusion of mucocysts with plasma membranes of T. pyriformis and by Weiss et al. [23] during conjugation of ChlaIt is tempting to mydomonas reinhardi. speculate that ConA receptors which are visualized as a ring by using fluorescentConA are expressed in freeze-etching as a ring of IMP. The two conjugants are held together by a double ring of receptors which have a function in recognition and attachment and the fusion itself occurs in the area encircled by the ring, consisting mainly of phospholipids. Attempts to study the induction of the ConA receptors, using SDS-polyacrylamide slab gels are under way in our laboratory. This method would allow us the investigation of the kinetics of their appearance and may contribute to further understanding of the nature of these receptors.
REFERENCES Austin, C R, J reprod fert 44 (1975) 155. 2. Amsterdam, A, Ohad, I & Schramm, M, J cell biol 41 (1%9) 753. 3. Abrahams, S J & Holtzman, E, J cell bio156 (1973) 540. 4. De Robertis, E, Histophysiology of synapses and neurosecretion. Pergamon Press, Oxford (1964). 5. Chi, E Y, Lagunoff, D & Koehler, J K, Proc natl acad sci US 73 (1976) 2823. 6. Elliott. A M. Bioloav of Terrahymena (ed A M Elliottj, pp ‘259-277 Dowden,. Hutchison and Ross, Stroudsburg, PA (1973). 7. Ofer, L, Levkovitz, H & Loyter, A, J cell biol 70 (1976) 287. 8. Frisch, A, Levkovitz, H & Loyter, A, Exp cell res 106(1977) 293. 9. Frisch, A, Bessler, W, Lipps, H J & Ammermann, D, J protozoo123 (1976) 427. 10. Bessler, W & Lipps, H J, J gen microb 92 (1976) 221. 11. Lsvlie, A 62Bryhni, E, Nature 263 (1976) 779. 12. Wiese, L & Shoemaker, D W, Biol bull 138 (1970) 13. ?keta K, Exp cell res 90 (1975) 56. 14. Frisch, A, Levkovitz, H & Loyter, A, Biochem bioohvs res commun 72 (1976) 138. 15. Nanndy, D L & McCoy, J W, Trans Am micros sot 95 (1976) 664. 16. Yanovski, A & Loyter, A, J biol them 247 (1972) 4021. 17. McKinney, R M, Spillane, J T & Pearce, G W, J immuno193 (1964) 232. 18. Bruns, P J & Palestine, R F, Dev bio142 (1975) 75. 19. Allewell, N M, Oles, J &Wolfe, J, Exp cell res 97 (1976) 394. 20. Ofer, L, Mercazi, M & Loyter, A, Biol cell 29 (1977). In press. 21. Wolfe, J & Loyter, A, J cell biol67 (1975) 459~. 22. Satir, B, Schooley, C & Satir, P, J cell biol 56 (1973) 153. 23. Weiss. R L. Goodenough, D A & Goodenough, U W, J cell bio172 (1973 144. Received April 29, 1977 Revised version received June 15, 1977 Accepted July 6, 1977
Exp Cell Res 110 (1977)
