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.

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.

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.

Hydrophobicity, adhesion, and surface-exposed proteins of gliding bacteria.

The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure.

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.

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 and its mutants defective in adhesion and motility.

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 that make contact with glass substrata were radioiodinated, using a substratum-immobilized catalyst (Iodo-Gen). At least 15 polypeptides were iodinated, fewer than the number labeled by surface biotinylation of whole cells; these polypeptides define the set of possible candidates for the surface protein(s) that mediates gliding-associated substratum adhesion. The labeling of three adhesion-defective mutants exhibited two characteristic patterns of surface iodination which involved addition, loss, or alteration of several polypeptides of high molecular weight. An adhesion-competent revertant of mutant Adh3 and one of Adh2 exhibited the wild-type labeling pattern. Two other Adh2 revertants resembled their adhesion-defective parent. The labeling pattern of surface polypeptides of a nongliding but adhesive cell strain was similar to that of the wild type.

Calcium influx regulates antibody-induced glycoprotein movements within the Chlamydomonas flagellar membrane.

The Chlamydomonas flagellar surface exhibits a number of dynamic membrane phenomena associated with whole-cell gliding locomotion and the early events in fertilization. Crosslinking of a specific population of flagellar surface-exposed glycoproteins with the lectin concanavalin A or an anti-carbohydrate mouse monoclonal antibody, designated FMG-1, results in a characteristic pattern of glycoprotein redistribution within the plane of the flagellar membrane. Recent evidence suggests that flagellar membrane glycoprotein movements are associated with both whole-cell gliding motility and the early events in mating. It is of interest to determine the transmembrane signaling pathway whereby crosslinking of the external domains of flagellar glycoproteins activates the intraflagellar machinery responsible for translocation of flagellar membrane glycoproteins. The redistribution of flagellar membrane glycoproteins requires micromolar levels of free calcium in the medium; lowering the free calcium concentration to 10(-7) M results in complete but reversible inhibition of redistribution. Redistribution is maximal in the presence of 20 microM free calcium in the medium. Redistribution is inhibited in the presence of 20 microM free calcium by the calmodulin antagonists trifluoperazine, W-7 and calmidazolium, the calcium channel blockers diltiazem, methoxyverapamil (D-600) and barium chloride, and the local anesthetics, lidocaine and procaine. The actions of all of these agents can be interpreted in terms of a requirement for calcium in the signaling mechanism associated with flagellar glycoprotein redistribution. In particular, the requirement for micromolar calcium in the external medium and the effects of specific calcium channel blockers suggest that flagellar membrane glycoprotein crosslinking may induce an increase in calcium influx, which may be the initial trigger for activating the flagellar machinery responsible for active movement of flagellar membrane glycoproteins.

Beta-tubulin epitope expression in normal and malignant epithelial cells.

The expression of a unique beta-tubulin isoform (class III) was monitored in squamous cell carcinoma (SCC) and normal epithelial cells using a monoclonal tubulin antibody called TuJ1. Whole tissue homogenates of SCC, normal tissue, SCC grown in nude mice, and SCC cultured cells were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. TuJ1 antibody localization was performed using peroxidase immunostaining on paraffin sections of SCC, normal tissue, nude mouse SCC, and immunofluorescent microscopy of SCC cultured cells. The malignant tissues examined stained positive with TuJ1 and a general beta-tubulin antibody, whereas the normal tissues stained positively only for the general beta-tubulin antibody. TuJ1 epitope expression may be a useful marker for SCCs and may assist in understanding differences between normal and malignant squamous cells.

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)

Membrane-cytoskeleton interactions in the flagellum: a 240,000 Mr surface-exposed glycoprotein is tightly associated with the axoneme in Chlamydomonas moewusii.

The flagellar surface of Chlamydomonas moewusii is a dynamic structure involved in several adhesive and motile events. In this report, we describe for the first time the flagellar membrane components of vegetative C. moewusii. A glycoprotein (or pair of glycoproteins) with an apparent molecular weight of 240 x 10(3) is the dominant flagellar protein (other than the tubulins) in this species of Chlamydomonas. Both a rabbit polyclonal antibody (designated P-19) and the lectin concanavalin A recognize this 240K (K = 10(3) Mr) glycoprotein on nitrocellulose transblots of flagellar proteins. Fluorescence microscopic studies using these same two probes suggest that the 240K glycoprotein is exposed at the flagellar surface. Direct evidence that the 240K glycoprotein is exposed at the flagellar surface is provided by vectorial labelling with a N-hydroxysuccinamide derivitized biotin reagent (NHS-LC-biotin). Nonionic detergent extraction of isolated flagella fails to solubilize most of the 240K glycoprotein, although it completely removes the flagellar membranes as demonstrated by transmission electron microscopy. Furthermore, immunofluorescence microscopy of isolated axonemes demonstrates that both P-19-defined epitopes and surface-biotinylated proteins continue to be associated with the axoneme structure after detergent treatment. These observations demonstrate that the 240K flagellar protein is a glycoprotein that is both exposed at the flagellar surface and tightly coupled to the underlying cytoskeleton (axoneme). Because of its cell surface orientation and axonemal linkage, it is likely that the 240K glycoprotein plays an important role in the adhesive and/or motile phenomena exhibited by the C. moewusii flagellar surface.

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.

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.

Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation.

A method is described for determining whether particular monoclonal antibodies are specific for carbohydrate or non-carbohydrate antigenic determinants. In a model system consisting of the Lewis a human blood group determinant attached to either protein or lipid, mild periodate oxidation destroyed the carbohydrate determinant without altering protein or lipid epitopes. The technique was readily applied to antigens bound to plastic wells for ELISA, to nitrocellulose sheets for Western blots, and to thin layer chromatography (TLC) plates for TLC immunostaining. Mild periodate oxidation can prove useful during the early stages of hybridoma screening in order to select for or against anti-carbohydrate antibodies.

Preferential turnover of membrane proteins in the intact Chlamydomonas flagellum.

Radioactive labeling studies demonstrate a continuous incorporation of newly synthesized proteins and glycoproteins into the intact flagella of Chlamydomonas. This apparent turnover is preferentially occurring for membrane components. In particular, two classes of flagellar membrane components, one a high molecular weight (HMW) group of closely migrating glycoproteins and the other a protein with a MW around 65 kD, are continuously turning over in the vegetative cell. This selective protein turnover may explain the ability of Chlamydomonas to rapidly recover from proteolytic modification of the flagellar surface and to change its flagellar surface properties during the early events in mating.

Evidence against surf-riding as a general mechanism for surface motility.

The mechanism responsible for the energy-dependent movement of membrane components (ie, surface motility) is unknown. Recently a potentially unifying model, termed "surf-riding" [Hewitt, 1979] or "surf-boarding" [Berlin and Oliver, 1982], has been proposed to explain surface motility. Using phase-contrast light microscopy and membrane surface markers (polystyrene microspheres), we have tested the surf-riding/surf-boarding hypothesis on two protozoan systems: the axopodia of the heliozoan Echinosphaerium nucleofilum and the reticulopodial networks of the allogromiid foraminiferans Allogromia laticollaris and Allogromia sp, strain NF. Our evidence indicates that surface motility, as displayed by these organisms, does not occur by a surf-riding/surf-boarding mechanism. Previous observations on surface motility associated with the Chlamydomonas flagellum indicate that this system is also incompatible with the surf-boarding/surf-riding hypothesis.

A flagellar surface glycoprotein mediating cell-substrate interaction in Chlamydomonas.

The Chlamydomonas flagellar surface exhibits interesting adhesive properties that are associated with flagellar surface motility. This dynamic surface property can be exhibited as the binding and movement of small polystyrene microspheres or as the interaction of the flagellar surface with a solid substrate followed by whole cell locomotion, termed "gliding". In order to identify flagellar surface proteins that mediate substrate interaction during flagellar surface motility, two immobilized iodination systems were employed that mimic the conditions for flagellar surface motility: small polystyrene microspheres derivatized with lactoperoxidase, and large glass beads derivatized with Iodogen. Use of these iodination conditions resulted in preferential iodination of a high-molecular-weight glycoprotein with apparent molecular weight of 300,000-350,000. These results suggest this glycoprotein as a major candidate for the surface-exposed adhesive component that directly interacts with the substrate and couples the substrate to a system of force transduction presumed to be located within the flagellum.