Abstract. 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, crossreactivity 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"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 flageUar 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.
Abstract. Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydratebinding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanavalin 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. THERE is an emerging view that cell surfaces serve as sensory receptors for motile cells; in particular, cells can detect physical and chemical cues in their environment and respond by activating cytoskeletal machinery that modulates cell adhesion, cell spreading and whole cell locomotion (Lackie, 1986;Singer and Kupfer, 1986;Bray and Hollenbeck, 1988;Stossel, 1989). Contact-induced information transfer across the plasma membrane is thought to be mediated by specific transmembrane glycoproteins (such as integrins) and second messenger pathways often involving protein phosphorylation and dephosphorylation (Damsky and
Strains of Neisseria gonorrhoeae grown on agar (designated 1/2 GPH) containing peptone, hemoglobin extract, Isovitalex (BBL Microbiology Systems, Cockeysville, Maryland), and 0.05% glucose were stained with Alcian blue and examined by electron microscopy. Three of five strains had capsular material surrounding the majority of organisms. Growth of organisms to log phase and fixation and staining with Alcian blue-glutaraldehyde on the agar surface before manipulation of the organisms were important variables for the morphologic demonstration of intact capsules. Two of the three strains that were encapsulated when grown on 1/2 GPH agar were poorly encapsulated when grown on a standard gonococcal agar. Gentle washing of encapsulated organisms before fixation removed most of the capsular material. The gonococcal capsule stained with ruthenium red, which suggested that it was composed of acidic polysaccharide. Visualization of capsules on both N. gonorrhoeae and Neisseria meningitidis with ruthenium red was not as consistent as with Alcian blue, presumably because Alcian blue-glutaraldehyde not only stains but also fixes the polysaccharide.
Summary. The biflagellate green alga Chlamydomonas can exhibit substrate-associated gliding motility in addition to its ability to swim through a liquid medium. The flagella are the organelles responsible for both forms of whole-cell locomotion although the mechanism in each case is very different. In this study, we demonstrate that the binding of polystyrene microspheres to the flagellar surface of Chlamydomonas initiates clustering of the major flagellar-membrane glycoprotein, which is known to be involved in motility-associated substrate adhesion. In addition, we demonstrate that microsphere binding to the flagellar surface initiates the same transmembrane signaling pathway that is initiated by antibody-or lectin-induced crosslinking of the major flagellar-membrane glycoprotein. In each case, the signaling pathway involves the activation of a calciumdependent protein phosphatase that dephosphorylates a flagellar phosphoprotein known to be associated with the major flagellarmembrane glycoprotein. Bound microspheres are translocated along the flagellar surface at approximately the same velocity as whole-cell gliding motility. Previous observations suggest that microsphere binding and translocation along the flagellar surface may be a reflection of the same force-transducing system responsible for whole-cell gliding motility. In which case, these observations suggest that the transmembrane signaling pathway initiated by crosslinking the major flagellar-membrane glycoprotein is the same one that is activated when the cell contacts a physiological substrate by its flagellar surface.
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