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PubMed2001

Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan.

Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania. This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface-expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3'nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid-phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.

Animals↗

PubMed1983

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.

Actins↗

PubMed1985

Aberrant distribution of the peri-axonemal structures in the human spermatozoon: possible role of the axoneme in the spatial organization of the flagellar components.

A quantitative ultrastructural study combined with stereology was performed on semen samples from four men selected for apparently isolated anomalies of the peri-axonemal structures. Comparison of the results with those of a control group revealed a decrease: in the mean length of the principal piece; in the mean length of the 9 dense fibres and in the difference in length between the 9 dense fibres, all the fibres being approximately as long as the medium length fibres of the normal spermatozoon. In addition, longitudinal columns were single and/or in abnormal position. However, the extent of the dense fibres (along 60% of the principal piece) and their proportion within the flagellum (35.1% of the principal piece per fibre on average) were normal. Analysis of the results suggests that: A-tubules of the axonemal doublets are involved in the spatial arrangement of the peri-axonemal structures; axonemal microtubule-associated proteins (MAPs) may be responsible for this structural function; and three different types of longitudinal doublet differentiations may exist along the flagellar axoneme.

Humans↗

PubMed1977

Comparison of the flagellins from different flagellar morphotypes of Escherichia coli.

The molecular weights of the flagellins of 13 strains of Escherichia coli, each with a different H antigen, were estimated using polyacrylamide gel electrophoresis. In each case only one major polypeptide was demonstrated, although some strains possessed apparently sheathed flagella. Considerable differences in the molecular weight of flagellin accompanied the previously described structural differences between flagella from strains with different H antigens. The relationship between flagellar diameter and the molecular weight of the corresponding flagellins was similar for both unsheathed and apparently sheathed flagella. Crosss-polymerization occurred between seed consisting of fragment of unsheathed flagella and flagellin solution from apparently sheathed flagella and vice versa. Co-polymerization of flagellin from unsheathed flagella and flagellin from apparently sheathed flagella was also demonstrated. These polymerization experiments indicate that the assembly pattern of flagellin molecules is probably the same in all E. coli flagella. The above and other evidence suggests that there is no true sheath, but that the differences in flagellar surface structure between different E. coli flagella are the result of differences in the superficial parts of the flagellin molecules.

Antigens, Bacterial↗

PubMed2004

Dual flagellar systems enable motility under different circumstances.

Flagella are extremely effective organelles of locomotion used by a variety of bacteria and archaea. Some bacteria, including Aeromonas, Azospirillum, Rhodospirillum, and Vibrio species, possess dual flagellar systems that are suited for movement under different circumstances. Swimming in liquid is promoted by a single polar flagellum. Swarming over surfaces or in viscous environments is enabled by the production of numerous peritrichous, or lateral, flagella. The polar flagellum is produced continuously, while the lateral flagella are produced under conditions that disable polar flagellar function. Thus at times, two types of flagellar organelles are assembled simultaneously. This review focuses on the polar and lateral flagellar systems of Vibrio parahaemolyticus. Approximately 50 polar and 40 lateral flagellar genes have been identified encoding distinct structural, motor, export/assembly, and regulatory elements. The sodium motive force drives polar flagellar rotation, and the proton motive force powers lateral translocation. Polar genes are found exclusively on the large chromosome, and lateral genes reside entirely on the small chromosome of the organism. The timing of gene expression corresponds to the temporal demand for components during assembly of the organelle: RpoN and lateral- and polar-specific sigma(54)-dependent transcription factors control early/intermediate gene transcription; lateral- and polar-specific sigma(28) factors direct late flagellar gene expression. Although a different gene set encodes each flagellar system, the constituents of a central navigation system (i.e., chemotaxis signal transduction) are shared.

Bacteria↗

PubMed1989

Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279-285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium-binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers of the transition zone. These observations have led us to question whether microtubule severing may be a more general phenomenon than previously suspected and to suggest that microtubule severing may contribute to the dynamic behavior of cytoplasmic microtubules in other cells.

Animals↗

PubMed1989

Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids.

The flagellar membrane of trypanosomatids has certain ultrastructural and antigenic characteristics that make its biochemical analysis very interesting. We have obtained a highly purified flagellar membrane fraction from epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas samuelpessoai. The fractions consisted of regular spherical vesicles. Membrane fractions fixed in a glutaraldehyde solution containing filipin and freeze-fractured showed few intramembranous particles, and many protuberances indicative of filipin-sterol complexes. The protein composition of all the subflagellar fractions was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The flagellar membrane fraction showed more than twenty bands. We wish particularly to point out the presence of six bands corresponding to proteins of 160, 135, 41, 31, 26 and 18 kDa which were not present in the axoneme-paraxial structure fraction. The blot of flagellar membrane proteins successively incubated with concanavalin A and horseradish peroxidase showed intense binding of the lectin to proteins of 117 and 87 kDa, and less strong binding to several other minor bands. Our results showed that although the membrane of the flagellum of trypanosomatids had few proteins, it seemed rich in glycosylated elements.

Animals↗

PubMed2000

The spirochete FlaA periplasmic flagellar sheath protein impacts flagellar helicity.

Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.

Alleles↗

PubMed1980

The paraxial structure of the flagellum of trypanosomatidae.

The flagella of several Trypanosomatidae (Trypanosoma cruzi, Herpetomonas samuelpessoai, Leptomonas samueli, Herpetomonas megaseliae, and Crithidia harmosa) were studied. Besides the axoneme, they have a filamentous, latticelike structure, the paraxial or paraflagellar rod. This structure was not observed in the flagellum of the intracellular spheromastigote form of Trypanosoma cruzi, but it appeared when the transformation of epimastigote and trypomastigote stages occured. Cross sections of the flagella show that the paraxial structure maintains a fixed position relative to the axonemal microtubules, being localized in a region comprised between doublets 4 and 7. Flagella of T. cruzi and H. samuelpessoai were isolated using the detergent lubrox PX. Most of them did not have a flagellar membrane. However, the paraxial structure remained associated with the axoneme. By negative staining, short projections were seen connecting the paraxial structure to the axonemal microtubules. The paraxial structure did not stain with phosphotungstic acid as occurs with the peripheral doublet microtubules, but it is formed by microfilaments longitudinally oriented in relation to the axoneme crossed in two directions by oblique filaments which make an angle of 45 degrees with the longitudinal ones.

Animals↗

PubMed2007

Flagellar apparatus of south-seeking many-celled magnetotactic prokaryotes.

Magnetotactic bacteria orient and migrate along geomagnetic field lines. Each cell contains membrane-enclosed, nano-scale, iron-mineral particles called magnetosomes that cause alignment of the cell in the geomagnetic field as the bacteria swim propelled by flagella. In this work we studied the ultrastructure of the flagellar apparatus in many-celled magnetotactic prokaryotes (MMP) that consist of several Gram-negative cells arranged radially around an acellular compartment. Flagella covered the organism surface, and were observed exclusively at the portion of each cell that faced the environment. The flagella were helical tubes never as long as a complete turn of the helix. Flagellar filaments varied in length from 0.9 to 3.8 micro m (average 2.4 +/- 0.5 micro m, n = 150) and in width from 12.0 to 19.5 nm (average 15.9 +/- 1.4 nm, n = 52), which is different from previous reports for similar microorganisms. At the base of the flagella, a curved hook structure slightly thicker than the flagellar filaments was observed. In freeze-fractured samples, macromolecular complexes about 50 nm in diameter, which possibly corresponded to part of the flagella basal body, were observed in both the P-face of the cytoplasmic membrane and the E-face of the outer membrane. Transmission electron microscopy showed that magnetosomes occurred in planar groups in the cytoplasm close and parallel to the organism surface. A striated structure, which could be involved in maintaining magnetosomes fixed in the cell, was usually observed running along magnetosome chains. The coordinated movement of the MMP depends on the interaction between the flagella of each cell with the flagella of adjacent cells of the microorganism.

Chemotaxis↗

PubMed2002

Structural properties of FliH, an ATPase regulatory component of the Salmonella type III flagellar export apparatus.

FliH is a regulatory component for FliI, the ATPase that is responsible for driving flagellar protein export in Salmonella. FliH consists of 235 amino acid residues, has a quite elongated shape, exists as a homodimer and together with FliI forms a heterotrimer. Here, we have investigated the structural properties of the FliH homodimer in further detail. Like intact His-tagged FliH homodimer, fragment His-FliH(N2) (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliH(C1) (consisting of amino acid residues 119-235), but to a much lesser degree. Thus the elongated shape of FliH appears to derive primarily from its N-terminal region. A deletion version of N-His-FliH, lacking amino acid residues 101-140, does not dimerize and so we were able to establish the gel filtration properties of an almost full-size monomeric form; it also exhibited anomalous elution behavior. We performed trypsin proteolysis of the FliH homodimer and subjected the cleavage products to gel filtration chromatography. FliH was degraded by trypsin and a contaminating protease into two stable fragments: FliH(Prt1) (missing both the first ten and the last 12 amino acid residues), and FliH(Prt2) (missing both the first ten and the last 63 amino acid residues); however, substantial amounts remained undigested even after 24 hours. Under native conditions, both FliH(Prt1) and FliH(Prt2) co-eluted with undigested His-FliH from the gel filtration column, indicating that the fragments exist as a hybrid dimer with intact FliH. These results suggest that the two subunits within the dimer differ in their proteolytic susceptibility. No heterotrimer was observed by gel filtration chromatography when His-FliI was mixed with either His-FliH/FliH(Prt1) or His-FliH/FliH(Prt2) hybrid dimers. A hybrid dimer of FliH and His-FliHDelta1 (lacking the first ten amino acid residues) retained the ability to form a complex with His-FliI. In contrast, hybrid dimers consisting of FliH and either His-FliH(W223ochre) or His-FliH(V172ochre) failed to complex to His-FliI, demonstrating that the C-terminal region of both FliH monomers within the FliH dimer are required for heterotrimer formation.

Adenosine Triphosphatases↗

PubMed1975

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.

Agglutination↗

PubMed1998

The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-sigma28 domain.

The flagellar-specific anti-sigma factor, FIgM, inhibits the expression of late flagellar genes until the hook-basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins). FIgM monitors this assembly checkpoint by being a substrate for export via the hook-basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FIgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FIgM. Deletion of this region (S3-P11) resulted in lower intracellular FIgM levels, but did not prevent recognition and export by the flagellar-specific secretion system. Mutations were isolated in a second region of FIgM spanning residues K27 to A65 that exhibited increased anti-sigma28 activity. These FIgM 'hyperinhibitor' mutants were secreted less than wild-type FIgM. Mutations that interfere with the secretion of FIgM without abolishing anti-sigma28 activity have a negative effect upon the secretion of a His-tagged FIgM mutant that lacks anti-sigma28 activity. Models are proposed to explain the dominant negative phenotype of the FIgM secretion mutants reported in this study.

Alanine↗

PubMed2004

Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus.

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.

Antigens, Bacterial↗

PubMed1997

Centrin and the cytoskeleton of the protist Holomastigotoides.

The cytoskeleton of the parabasalid protozoan Holomastigotoides was investigated by epifluorescence, scanning confocal, and transmission electron microscopy using antibodies to centrin, tubulin, and MPM-2 epitopes. Previous microscopic analysis of Holomastigotoides spp. has shown that up to 10,000 flagella are arranged in 2-8 spiral bands encircling the cell. Spindle poles are associated with two flagellar bands. Sheets of cytoplasmic microtubules (MTs) called axostyles originate in the cell apex and extend to the cell base. Antibodies to centrin, a member of the EF-hand family of calcium-binding proteins, labeled a number of structures in Holomastigotoides, namely axostyles, the mitotic spindle, and portions of flagellar bands. The identity of these structures was confirmed by transmission electron microscopy and by immunofluorescence microscopy using antibodies to tubulin and MPM-2 epitopes. Antibodies to tubulin labeled MTs in basal bodies, flagella, axostyles, and the mitotic spindle. MPM-2 antibodies labeled spindle poles and short segments of flagellar bands to which the spindle poles are attached. Centrin is known to show calcium-sensitive contractile behavior. The pattern of flagellar band staining by antibodies to centrin was affected by [Ca2+]. In detergent-extracted cell fragments, the centrin staining pattern could be changed by changing [Ca2+]. This Ca2+ effect was modulated by a monoclonal antibody to centrin (20H5), indicating that centrin plays a role in altering flagellar band structure. These results show that centrin is located in key positions for maintaining cell polarity and directing cell movement through interactions with other cytoskeletal elements. Calcium may regulate the morphology of centrin-containing structures.

Animals↗

PubMed2005

Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription.

The hetero-oligomeric complex of the FlhD and FlhC proteins (FlhDC) regulates transcription from several flagellar and non-flagellar operons in bacteria. The crystallographic structure of the Escherichia coli FlhDC complex has been solved to 3.0 A resolution, revealing a hexameric FlhD4FlhC2 assembly. In the complex, each FlhC protomer binds an FlhD2 dimer; the conformation of the dimer in the complex differs significantly from its conformation in the absence of FlhC. FlhC has a novel tertiary fold that includes a heretofore unrecognized zinc-binding site in which the ion is ligated by four cysteine residues. Gel shift experiments show that binding of the FlhDC complex to a cognate promoter bends the DNA by approximately 111 degrees . The structure of the FlhDC complex is compatible with models in which a fragment of operator DNA, at least 48 base-pairs in length, wraps around the complex and bends significantly when binding.

Base Sequence↗

PubMed1988

Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays.

The testis-specific beta 2 tubulin of Drosophila is required for assembly and function of at least three architecturally different microtubule arrays (Kemphues et al., 1982). Two recessive male-sterile mutations in the B2t locus that encode partially functional, stable, variant forms of beta 2 tubulin cause defects in only certain microtubule-based processes during spermatogenesis. These mutations could thus identify aspects of beta tubulin primary structure critical for function only in specific microtubule arrays. In males carrying the B2t6 mutation, meiotic chromosome segregation and nuclear shaping are normal and flagellar axonemes are formed, but there is a subtle defect in axoneme structure; the outer doublet microtubules fill in with a central core normally seen only in the central pair and accessory microtubules. In homozygous B2t7 males, chromosome movement is usually normal during meiosis but cytokinesis often fails, cytoplasmic microtubules are assembled and nuclear shaping appears to be normal, but the flagellar axoneme lacks structural integrity. In contrast, the B2t8 allele affects a general property of tubulin, the ability to form normal side-to-side association of protofilaments (Fuller et al., 1987), and causes defects in meiosis, axoneme assembly and nuclear shaping. Certain combinations of these beta 2 tubulin mutations show interallelic complementation; in B2t6/B2t8 males functional sperm are produced and both variant subunits are incorporated into mature sperm, in the absence of wild-type beta 2 tubulin. Comparison of the phenotypes of the three partially functional beta 2 tubulin alleles reveals some aspects of tubulin primary structure more important for function in specific subsets of microtubule arrays, and other aspects required for the construction of microtubules in general.

Alleles↗

PubMed1988

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.

Actin Cytoskeleton↗