Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas

In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.     

Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.     

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.     

Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes

A quantitative assay for cell wall release in Chlamydomonas has been used to study the timing of release of cell wall degrading enzyme (lysin) during adhesion. Lysin activity, which shows a broad pH range and requires divalent cations, is released as a pulse within 1–2 min after mixing of mt^? and mt⁺ gametes. Thereafter, there is no further lysin release. Gametes of both mating types release the activity during aggregation with isolated gametic flagella of the opposite mating type, although mt⁺ gametes appear to release more lysin activity than mt^? gametes. Electrophoretic analysis of cell wall proteins before and after lysin degradation indicate that the major wall proteins are unchanged after wall breakdown.     

Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling

Interactions between adhesion molecules, agglutinins, on the surfaces of the flagella of mt+ and mt- gametes in Chlamydomonas rapidly generate a sexual signal, mediated by cAMP, that prepares the cells for fusion to form a zygote. The mechanism that couples agglutinin interactions to increased cellular levels of cAMP is unknown. In previous studies on the adenylyl cyclase in flagella of a single mating type (i.e., non-adhering flagella) we presented evidence that the gametic form of the enzyme, but not the vegetative form, was regulated by phosphorylation and dephosphorylation (Zhang, Y., E. M. Ross, and W. J. Snell. 1991. J. Biol. Chem. 266:22954-22959; Zhang, Y., and W. J. Snell. 1993. J. Biol. Chem. 268:1786-1791). In the present report we describe studies on regulation of flagellar adenylyl cyclase during adhesion in a cell-free system. The results show that the activity of gametic flagellar adenylyl cyclase is regulated by adhesion in vitro between flagella isolated from mt+ and mt- gametes. After mixing mt+ and mt- flagella together for 15 s in vitro, adenylyl cyclase activity was increased two- to threefold compared to that of the non-mixed (non- adhering), control flagella. This indicates that the regulation of gametic flagellar adenylyl cyclase during the early steps of fertilization is not mediated by signals from the cell body, but is a direct and primary response to interactions between mt+ and mt- agglutinins. By use of this in vitro assay, we discovered that 50 nM staurosporine (a protein kinase inhibitor) blocked adhesion-induced activation of adenylyl cyclase in vitro, while it had no effect on adenylyl cyclase activity of non-adhering gametic flagella. This same low concentration of staurosporine also inhibited adhesion-induced increases in vivo in cellular cAMP and blocked subsequent cellular responses to adhesion. Taken together, our results indicate that flagellar adenylyl cyclase in Chlamydomonas gametes is coupled to interactions between mt+ and mt- agglutinins by a staurosporine- sensitive activity, probably a protein kinase.     

Kinesin-II is required for flagellar sensory transduction during fertilization in Chlamydomonas

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt-) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt- flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32 degrees C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32 degrees C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32 degrees C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32 degrees C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.     

Flagellar adhesion between mating type plus and mating type minus gametes activates a flagellar protein-tyrosine kinase during fertilization in Chlamydomonas

When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas.     

Mating aggregates in Escherichia coli conjugation.

Mating mixtures of Escherichia coli cells were shown to contain mating aggregates of two to 20 cells each rather than only mating pairs of two cells each. The mating aggregate size distribution shows two broad peaks, at two to four cells and at eight to 13 cells. The quantitative mating aggregate size distribution and the proportion of male cells in mating aggregates are dependent on the input ratio of male to female cells. At an input ratio of one to one, the average mating aggregate contains equal proportions of male and female cells and most of the cells involved in mating are in large aggregates of seven or more cells each. The deoxyribonucleic acid (DNA) transfer efficiency per mating aggregate cell was constant regardless of average aggregate size or of the ratio of male to female cells in the aggregate. Under optimal conditions essentially every male cell or every female cell in a mating aggregate can be involved in DNA transfer. A comparison of light microscopy, sucrose gradient centrifugation, and analysis with a modified Coulter counter indicated that the number of cells in mating aggregates is best equantitated using a modified Coulter counter.     

Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Agglutination and glycosyltransferase activity of isolated gametic flagella from Chlamydomonas reinhardii

Isolated flagella from gametes of both mating types (mt⁺ and mt^-) of Chlamydomonas reinhardii were suspended in buffer containing 7% sucrose. After mixing instantaneous agglutination occurred, giving rise to clumps which seem to be stable for at least 24 h. Control experiments show that no aggregates are formed when gametic flagella of one mating type are mixed with flagella prepared from vegetative cells of the other mating type.This in vitro agglutination is inhibited by a number of salt solutions in the same concentration range in which the agglutination of live gametes is affected. Moreover the clumps of flagella tend to disaggregate completely when the salt solutions are added after agglutination has occurred, or by treatment with trypsin. These observations suggest that the in vitro agglutination of isolated gametic flagella indeed reflects their physiological role in the recognition step of the mating process, which appears to be possible without participation of live gametes.We have also investigated the activity of glycosyl transferases on isolated gametic flagella before and during the in vitro agglutination reaction. As there was no detectable increase in the activity of glycosyl transferases, our results do not favour the hypothesis that these enzymes are involved in the primary step of recognition between gametic flagella.Dedicated to Prof. Dr. Otto Kandler on the occasion of his 60th birthday     

The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ~3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 10⁶ fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.     

Mating in Chlamydomonas: a system for the study of specific cell adhesion. II. A radioactive flagella-binding assay for quantitation of adhesion

To measure the flagellar adhesiveness of Chlamydomonas gametes in a more quantitative manner than agglutination assays permit, a binding assay was developed which measured the binding of radioactive flagella of one mating type to unlabeled gametes of the opposite mating type. With the appropriate assay conditions, the number of [3H] flagella specifically bound was shown to be proportional to the number of cells in the incubation mixture and, therefore, to the number of binding sites that were present. The assay was used to study the effects of trypsin treatment on the loss and development of flagellar binding sites. It was shown that after trypsin treatment at least 9 h were required for the return of a full complement of binding sites to the flagellar surface; moreover, the results indicated that these sites reappeared on existing, extended flagella.     

Elongation of the fertilization tubule in Chlamydomonas: new observations on the core microfilaments and the effect of transient intracellular signals on their structural integrity

Experimental manipulations of gametes of Chlamydomonas reinhardi and ultrastructural observation were used to examine the composition of the microfilaments in the fertilization tubule, their probable mode of formation, and their interaction with intracellular signals. Decoration with myosin subfragment-1 was used to demonstrate that the microfilaments in the fertilization tubule were actin filaments having uniform polarity: Myosin subfragment-1 arrowheads pointed away from the membrane at the tip of the process. Filaments were attached to the cone- shaped "doublet zone" at the base of the process by their pointed ends. Discrete attachment sites for filaments on the surface of the doublet zone were seen in stereo view. To test whether actin polymerization might accompany elongation of the fertilization tubule, mating gametes were exposed to cytochalasin D in an attempt to block actin polymerization. Treatment of mating type "plus" gametes with cytochalasin D prior to and during mating inhibited the appearance of actin filaments in fertilization tubules, suppressed fertilization tubule outgrowth, and lowered mating efficiency from 90 to 15%. The role of signals generated by flagellar adhesion in maintaining the structural integrity of the microfilament-doublet zone complex was examined by correlating flagellar disadhesion with the kinetics of breakdown of the complex. In zygotes, where flagellar disadhesion occurred after cell fusion, the complex disassembled within 3 h after mating. In gametes that had been agglutinated by isolated mating type "minus" flagella, microfilaments and fertilization tubules progressively disassembled over a 3-h time course following flagellar disadhesion. Disassembly of microfilaments was inhibited by maintaining flagellar agglutination, suggesting that signals generated by flagellar adhesion were necessary to maintain microfilaments intact.     

Transient increase in calcium efflux accompanies fertilization in Chlamydomonas

Mating in Chlamydomonas is a complex process initiated by contact of gametic flagellar surfaces, resulting in transmission of a signal from the flagella to the cell bodies. This signal triggers later events of cell wall loss, mating structure activation, and cell-cell fusion. Little is known about the nature of the signal or the role of Ca in these events. It was found that extracellular Ca is not necessary for successful mating in Chlamydomonas. However, cells will take up Ca from the medium in a linear manner for many hours and will accumulate micromolar concentrations, presumably by sequestering Ca within intracellular storage sites. If gametic cells of one mating type (preloaded with 45Ca) are mated with gametes of the opposite mating type (preloaded with unlabeled calcium), there is a rapid, transient increase in calcium efflux rate (20 times that of the control) that lasts approximately 6 min. This effect is not associated with cell-cell fusion, since the same observation is made if (+) gametes preloaded with 45-Ca are agglutinated by isolated flagella from (-) gametes preloaded with unlabeled Ca. Other experiments have shown that the increased efflux rate is not a simple consequence of cell wall release. Ca efflux in unmated gametes is greatly reduced in deflagellated cells, suggesting that much of the Ca movement is associated with the flagellar membrane. Although signaling itself may involve Ca fluxes across the flagellar membrane, it is also possible that a consequence of signaling is release of Ca from intracellular storage sites (perhaps functional equivalents of the sarcoplasmic reticulum). The observed transient increase in Ca efflux rate may reflect a transient increase in the cytoplasmic free-Ca concentration. This increase in cytoplasmic Ca may regulate the later events in mating (such as cell wall release and mating structure activation).     

Cell-cell coordination in conjugatingChlamydomonas gametes

Summary During conjugation, complementaryChlamydomonas gametes [mating type plus (mt+) and mating type minus (mt–)] are mutually attached via specific adhesion molecules, called agglutinins, which are located at the surface of the flagella. By these contacts the gametes are stimulated to fuse. It is demonstrated that fusion is preceded by a compulsary sequence of events: first, the flagellar swimming beat is arrested, next the flagellar contact is reinforced and finally, the position of the cell bodies is adjusted to permit fusion. Evidence is presented that each consecutive step of the mating process requires a higher level in cell-cell signalling, which is obtained by the formation of additional agglutinin contacts. It is shown that the mt+ and mt– traverse their conjugation process in synchrony, probably because the two sexes acquire new agglutinin contacts at equal rates. It is proposed that this symmetrical behavior is due to the complementarity of the mt+ and mt– agglutinin molecules. A scenario of the conjugation process inC. eugametos, incorporating the recent findings, is provided.Abbreviations EM electron microscopy - FTA flagellar tip activation - FITC fluorescein isothiocyanate - GA glutaraldehyde - IA vesicle iso-agglutinin vesicle - mAb monoclonal antibody - mt mating type - TRITC tetramethylrhodamine Bisothiocyanate - UrAc uranyl acetate     

Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier

Fertilization in Chlamydomonas reinhardtii is initiated when gametes of opposite mating types adhere to each other via adhesion molecules (agglutinins) on their flagella. Adhesion leads to loss of active agglutinins from the flagella and recruitment of new agglutinins from a pool associated with the cell body. We have been interested in determining the precise cellular location of the pool and learning more about the relationship between agglutinins in the two domains. In the studies reported here we describe methods for purification of mt+ cell body agglutinins by use of ammonium sulfate precipitation, chromatography (molecular sieve, ion exchange, and hydrophobic interaction), and sucrose gradient centrifugation. About 90% of the total agglutinins were associated with the cell body and the remainder were on the flagella. Cell body agglutinins were indistinguishable from mt+ flagellar agglutinins by SDS-PAGE, elution properties on a hydrophobic interaction column, and in sedimentation properties on sucrose gradients. The nonadhesiveness of cell bodies suggested that the cell body agglutinins would be intracellular, but our results are not consistent with this interpretation. We have demonstrated that brief trypsin treatment of deflagellated gametes destroyed all of the cell body agglutinins and, in addition, we showed that the cell body agglutinins were accessible to surface iodination. These results indicated that C. reinhardtii agglutinins have a novel cellular disposition: active agglutinins, representing approximately 10% of the total cellular agglutinins, are found only on the flagella, whereas the remaining 90% of these molecules are on the external surface of the cell body plasma membrane in a nonfunctional form. This segregation of cell adhesion molecules into distinct membrane domains before gametic interactions has been demonstrated in sperm of multicellular organisms and may be a common mechanism for sequestering these critical molecules until gametes are activated for fusion. In experiments in which surface-iodinated cell bodies were permitted to regenerate new flagella, we found that the agglutinins (as well as the 350,000 Mr, major flagellar membrane protein) on the newly regenerated flagella were iodinated. These results indicate that proteins destined for the flagella can reside on the external surface of the cell body plasma membrane and are recruited onto newly forming flagella as well as onto preexisting flagella during fertilization.     

Fusion of influenza virus with cardiolipin liposomes at low pH: mass action analysis of kinetics and extent

The kinetics and extent of low pH induced fusion between influenza virus and large unilamellar cardiolipin liposomes were investigated with an assay for lipid mixing based on fluorescence resonance energy transfer. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of virus-liposome adhesion or aggregation followed by the first-order fusion reaction itself. The fluorescence development during the course of the fusion process was calculated by numerical integration, employing separate rate constants for the initial aggregation step and for the subsequent fusion reaction. Analytical solutions were found for several limiting cases. Deaggregation of virus--liposome aggregates was explicitly taken into account but was found to be a minor effect under the conditions studied. The calculations gave good simulations and predictions for the kinetics and extent of fusion at different virus/liposome concentrations and ratios. At pH 5.0 and 37 degrees C, very high rate constants for aggregation and fusion were obtained, and essentially all of the virus particles were involved in the fusion process. Experiments at different virus/liposome ratios showed that fusion products may consist of a single virus particle and several liposomes but not of a single liposome and several virus particles. At pH 6.0, the rate constant for aggregation was the same as at pH 5.0, but the rate constant of fusion was about 5-fold lower, and only 25-40% of the virus particles were capable of fusing with the liposomes. The analytical procedure presented enables elucidation of the crucial role of the composition of target membrane vesicles in the initial adhesion and subsequent fusion of the virus at various pH values.