Partial purification of presynaptic plasma membrane by immunoadsorption

Chlamydomonas flagella exhibit force transduction in association with their surface. This flagellar surface motility is probably used both for whole cell gliding movements (flagella-substrate interaction) and for reorientation of flagella during mating (flagella-flagella interaction). The present study seeks to identify flagellar proteins that may function as exposed adhesive sites coupled to a motor responsible for their translocation in the plane of the plasma membrane. The principal components of the flagellar membrane are a pair of glycoproteins (approximately 350,000 mol wt), with similar mobility on SDS polyacrylamide gels. A rabbit IgG preparation has been obtained which is specific for these two glycoproteins; this antibody preparation binds to and agglutinates cells by their flagellar surfaces only. Treatment of cells with 0.1 mg/ml pronase results in a loss of motility-coupled flagellar membrane adhesiveness. This effect is totally reversible, but only in the presence of new protein synthesis. The major flagellar protein modified by this pronase treatment is the faster migrating of the two high molecular weight glycoproteins; the other glycoprotein does not appear to be accessible to external proteolytic digestion. Loss and recovery of flagella surface binding sites for the specific antibody parallels the loss and recovery of the motility-coupled flagellar surface adhesiveness, as measured by the binding and translocation of polystyrene microspheres. These observations suggest, but do not prove, that the faster migrating of the major high molecular weight flagellar membrane glycoproteins may be the component which provides sites for substrate interaction and couples these sites to the cytoskeletal components responsible for force transduction.     

Regulation of flagellar glycoprotein movements by protein phosphorylation

Cross-linking of surface exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. A number of observations suggest that active movements of flagellar membrane glycoproteins are associated with the processes of whole cell gliding motility and the early events of fertilization in Chlamydomonas. Protein redistribution is totally inhibited if the free calcium concentration in the medium is 10(-7) M or below or in the presence of a number of calcium channel blockers (Bloodgood, R. A., N. L. Salomonsky, J. Cell Sci. 96, 27-33 (1990]. The present report demonstrates that glycoprotein redistribution in vivo is inhibited reversibly by three different protein kinase inhibitors: H-7, H-8 and staurosporine. Taken together, these observations suggest that the flagellum uses a signaling pathway that involves calcium influx induced by glycoprotein cross-linking, calcium activation of a protein kinase and specific protein phosphorylation to initiate flagellar surface dynamics.     

Use of a novel Chlamydomonas mutant to demonstrate that flagellar glycoprotein movements are necessary for the expression of gliding motility

As an alternative to swimming through liquid medium by the coordinated bending activity of its two flagella, Chlamydomonas can exhibit whole cell gliding motility through the interaction of its flagellar surfaces with a solid substrate. The force transduction occurring at the flagellar surface can be visualized as the saltatory movements of polystyrene microspheres. Collectively, gliding motility and polystyrene microsphere movements are referred to as flagellar surface motility. The principal concanavalin A binding, surface-exposed glycoproteins of the Chlamydomonas reinhardtii flagellar surface are a pair of glycoproteins migrating with apparent molecular weight of 350 kDa. It has been hypothesized that these glycoproteins move within the plane of the flagellar membrane during the expression of flagellar surface motility. A novel mutant cell line of Chlamydomonas (designated L-23) that exhibits increased binding of concanavalin A to the flagellar surface has been utilized in order to restrict the mobility of the concanavalin A-binding flagellar glycoproteins. Under all conditions where the lateral mobility of the flagellar concanavalin A binding glycoproteins is restricted, the cells are unable to express whole cell gliding motility or polystyrene microsphere movements. Conversely, whenever cells can redistribute their concanavalin A binding glycoproteins in the plane of the flagellar membrane, they express flagellar surface motility. Since the 350 kDa glycoproteins are the major surface-exposed flagellar proteins, it is likely that most of the signal being followed using fluorescein isothiocyanate (FITC)-concanavalin A is attributable to these high molecular weight glycoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Directed movements of ciliary and flagellar membrane components: a review.

The ability to rapidly translocate polystyrene microspheres attached to the surface of a plasma membrane domain reflects a unique form of cellular force transduction occurring in association with the plasma membrane of microtubule based cell extensions. This unusual form of cell motility can be utilized by protistan organisms for whole cell locomotion, the early events in mating, and transport of food organisms along the cell surface, and possibly intracellular transport of certain organelles. Since surface motility is observed in association with cilia and flagella of algae, sea urchin embryos and cultured mammalian cells, it is likely that it serves an additional role beyond those already cited; this is likely to be the transport of precursors for the assembly and turnover of ciliary and flagellar membranes and axonemes. In the case of the Chlamydomonas flagellum, where surface motility has been most extensively studied, it appears that cross-linking of flagellar surface exposed proteins induces a transmembrane signaling pathway that activates machinery for moving flagellar membrane proteins in the plane of the flagellar membrane. This signaling pathway in vegetative Chlamydomonas reinhardtii appears to involve an influx of calcium, a rise in intraflagellar free calcium concentration and a change in the level of phosphorylation of specific membrane-matrix proteins. It is hypothesized that flagellar surface contact with a solid substrate (during gliding), a polystyrene microsphere or another flagellum (during mating) will all activate a signaling pathway similar to the one artificially activated by the use of monoclonal antibodies to flagellar membrane glycoproteins. A somewhat different signaling pathway, involving a transient rise in intracellular cAMP level, may be associated with the mating of Chlamydomonas gametes, which is initiated by flagellum-flagellum contact. The hypothesis that the widespread observation of microsphere movements on various ciliary and flagellar surfaces may reflect a mechanism normally utilized to transport axonemal and membrane subunits along the internal surface of the organelle membrane presents a paradox in that one would expect this to be a constitutive mechanism, not one necessarily activated by a signaling pathway.     

Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A

Two carbohydrate-binding probes, the lectin concanavalin A and an anti-carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.     

Reversible inhibition of Chlamydomonas flagellar surface motility

Chlamydomonas exhibits force transduction in association with its flagellar surface; this can be visualized by the saltatory movements of attached polystyrene microspheres. This flagellar surface motility has been quantitated by determining the percentage of attached microspheres in motion at the time of observation (60% in the case of control cells at 25 degrees C). A number of experimental treatments reversibly inhibit flagellar surface motility. These include an increase in sodium or potassium chloride concentration, a decrease in temperature, or a decrease in the free calcium concentration in the medium. Many of the conditions that result in inhibition of flagellar surface motility also result in an induction of flagellar resorption. Although both flagellar stability and flagellar surface motility are dependent on the availability of calcium, the two processes are separable; under appropriate conditions, flagellar surface motility can occur at normal levels on flagella that are resorbing. Inhibition of protein synthesis results in a gradual loss of both the binding of microspheres to the flagellum and the flagellar surface motility. After resumption of protein synthesis, both binding and movement return to control levels. The effect of the inhibition of protein synthesis is interpreted in terms of selective turnover of certain components within the intact flagellum, one or more of these components being necessary for the binding of the microspheres and their subsequent movement. If this turnover is inhibited by keeping the cells below 5 degrees C, the absence of protein synthesis no longer has an effect on microsphere attachment and motility, when measured immediately after warming the cells to 25 degrees C.     

The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein

Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60- kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

Calcium-regulated phosphorylation of proteins in the membrane-matrix compartment of the Chlamydomonas flagellum.

Crosslinking of surface-exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. Previous work has shown that these membrane glycoprotein movements are dependent on a critical concentration of free calcium in the medium and are inhibited reversibly by calcium channel blockers and the protein kinase inhibitors H-7, H-8, and staurosporine. These observations suggest that the flagellum may use a signaling pathway that involves calcium-activated protein phosphorylation to initiate flagellar membrane glycoprotein movements. In order to pursue this hypothesis, we examined the calcium dependence of phosphorylation of flagellar membrane-matrix proteins using an in vitro system containing [gamma-32P]ATP or [35S]ATP gamma S. Using only endogenous enzymes and endogenous substrates found in the membrane-matrix fraction obtained by extraction of flagella with 0.05% Nonidet P-40, we observed both calcium-independent protein phosphorylation and calcium-dependent protein phosphorylation in addition to an active protein dephosphorylation activity. Addition of micromolar free calcium increased the amount of protein phosphorylation severalfold. Calcium-activated protein kinase activity was inhibited by H-7, H-8, and staurosporine, the same protein kinase inhibitors that inhibit the calcium-dependent glycoprotein redistribution in vivo. A small group of polypeptides in the 26-58 kDa range exhibited a dramatic increase in phosphorylation in the presence of 20 microM free calcium. We suggest that Chlamydomonas utilizes the intraflagellar free calcium concentration to regulate the phosphorylation of specific flagellar proteins in the membrane-matrix fraction, one or more of which may be involved in regulating the machinery responsible for flagellar membrane glycoprotein redistribution.     

Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion

To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion.     

Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.     

Sex-specific glycoproteins in Chlamydomonas flagella an immunological study

To identify mating type-specific glycoproteins associated with the flagellar membrane of Chlamydomonas eugametos, which could be involved in sexual agglutination, antibodies were raised in rabbits against purified gamete flagella of either mating type. The immunoglobulin (Ig) fractions exhibited partial mating-type specificity in agglutinating gametes, in the indirect immunofluorescence test and in the crossed immunoelectrophoresis test. This specificity was strongly enhanced by absorbing the fractions with flagella of the opposite mating type. Absorbed Ig fractions produced a single precipitation line with Triton extracts of gamete flagella in the crossed immunoelectrophoresis technique. On polyacrylamide gel electrophoresis this line appeared to contain two flagellar glycoprotein fractions, PAS 1 and PAS 4. Polyacrylamide gels of flagellar extracts incubated with these Ig fractions, followed by staining with peroxidase-anti-rabbit Ig resulted in the staining of only the PAS 1 and PAS 4 bands, which confirms that these components of the flagellar membrane are mating type-specific antigens.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Flagellar surface antigens in Euglena: immunological evidence for an external glycoprotein pool and its transfer to the regenerating flagellum

Antibodies raised against the Sarkosyl-insoluble, major flagellar glycoprotein fraction, mastigonemes, were used to determine the source of flagellar surface glycoproteins and to define the general properties of flagellar surface assembly in Euglena. After suitable absorption, mastigoneme antiserum reacts with several specific mastigoneme glycoproteins but does not bind either to the other major flagellar glycoprotein, xyloglycorien, or to other Sarkosyl-soluble flagellar components. When Fab' fragments of this mastigoneme-specific antiserum were used in combination with a biotin-avidin secondary label, antigen was localized not only on the flagellum as previously described but also in the contiguous reservoir region. If deflagellated cells are reservoir pulse-labeled with Fab' antibody, this antibody appears subsequently on the newly regenerated flagellum. This chased antibody is uniformly distributed throughout the length of the flagellum and shows no preferred growth zone after visualization with either fluorescein or ferritin-conjugated secondary label. From these and tunicamycin inhibition experiments it is concluded that (a) a surface pool of at least some flagellar surface antigens is present in the reservoir membrane adjacent to the flagellum and that (b) the reservoir antigen pool is transferred to the flagellar surface during regeneration.     

Flagellar protein dynamics in Chlamydomonas

Cilia and flagella appear to be stable, terminal, microtubule-containing organelles, but they also elongate and shorten in response to a variety of signals. To understand mechanisms that regulate flagellar dynamics, Chlamydomonas cells with nongrowing flagella were labeled with (35)S, and flagella and basal body components were examined for labeled polypeptides. Maximal incorporation of label into the flagella occurred within 3 h. Twenty percent of the flagellar polypeptides were exchanged. These included tubulins, dyneins, and 80 other axonemal and membrane plus matrix polypeptides. The most stable flagellar structure is the PF-ribbon, which comprises part of the wall of each doublet microtubule and is composed of tubulin and three other polypeptides. Most (35)S was incorporated into the high molecular weight ribbon polypeptide, rib240, and little, if any, (35)S is incorporated into PF-ribbon-associated tubulin. Both wild-type (9 + 2) and 9 + 0 flagella, which lack central microtubules, exhibited nearly identical exchange patterns, so labeling is not due to turnover of relatively labile central microtubules. To determine if flagellar length is balanced by protein exchange, (35)S incorporation into disassembling flagella was examined, as was exchange in flagella in which microtubule assembly was blocked by colchicine. Incorporation of (35)S-labeled polypeptides was found to occur into flagellar axonemes during wavelength-dependent shortening in pf18 and in fla10 cells induced to shorten flagella by incubation at 33 degrees C. Colchicine blocked tubulin addition but did not affect the exchange of the other exchangeable polypeptides; nor did it induce any change in flagellar length. Basal bodies also incorporated newly synthesized proteins. These data reveal that Chlamydomonas flagella are dynamic structures that incorporate new protein both during steady state and as flagella shorten and that protein exchange does not, alone, explain length regulation.     

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.     

Reconstitution of biological activity in isoagglutinins fromChlamydomonas eugametos

Gametes ofChlamydomonas eugametos produce membrane vesicles, called isoagglutinins, which are shed into the culture fluid. It is assumed that they originate from the flagellar membrane for, like flagella, they can bind to the flagellar surface of gametes of the opposite mating type (mt). The composition ofmt ^- isoagglutinin was investigated with respect to this agglutinability. When the agglutination factor present on the surface ofmt ^- isoagglutinins (PAS-1.2) was removed, together with other membrane bound glycoproteins, the membrane vesicles were rendered inactive. They could be reactivated however by incubation with the extracted glycoproteins in a time-and concentration-dependent manner. The agglutination factor proved to be necessary yet sufficient in itself for the reactivation process to occur. Experiments with CsCl density gradients showed that the agglutination factor truly bound to the vesicles during reactivation. Inactivated vesicles derived frommt ⁺ gametes could be reactivated to gainmt ^- properties. Reactivation was inhibited by prior treatment with trypsin. The results indicate that the agglutination factor inmt ^- isoagglutinins is an extrinsic membrane protein bound to an intrinsic proteinaceous receptor.Abbreviations GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PAS periodic acid Schiff     

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.     

The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane

The Chlamydomonas FLA10 gene was shown to encode a flagellar kinesin- like protein (Walther, Z., M. Vashishtha, and J.L. Hall. 1994. J. Cell Biol. 126:175-188). By using a temperature-sensitive allele of FLA10, we have determined that the FLA10 protein is necessary for both the bidirectional movement of polystyrene beads on the flagellar membrane and intraflagellar transport (IFT), the bidirectional movement of granule-like particles beneath the flagellar membrane (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. (USA). 90:5519-5523). In addition, we have correlated the presence and position of the IFT particles visualized by light microscopy with that of the electron dense complexes (rafts) observed beneath the flagellar membrane by electron microscopy. A role for FLA10 in submembranous or flagellar surface motility is also strongly supported by the immunolocalization of FLA10 to the region between the axonemal outer doublet microtubules and the flagellar membrane.     

Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction

A structural and biochemical study is presented concerning the agglutination of gametic flagella, the initial step in the mating reaction of Chlamydomonas reinhardtii. An alteration in the distribution of the intramembranous particles revealed by freeze-fracturing of flagella membranes is shown to accompany gametic differentiation in both mating types. The isolation and electrophoretic analysis of flagellar membranes and mastigonemes are reported; no electrophoretic differences can be detected when the membrane or mastigoneme glycoproteins from vegative and gametic cells are compared, nor when glycoproteins from the two mating types are compared, and no novel polypeptides are present in gametic preparations. The membrane vesicles, after they are freed of mastigonemes by sedimentation through a discontinuous sucrose gradient, are extremely active as an isoagglutinin, indicating a direct involvement of the membrane in the mating reaction.