THE STRUCTURE, ORIGIN, ISOLATION, AND COMPOSITION OF THE TUBULAR MASTIGONEMES OF THE OCHROMONAS FLAGELLUM

The structure, assembly, and composition of the extracellular hairs (mastigonemes) of Ochromonas are detailed in this report. These mastigonemes form two lateral unbalanced rows, each row on opposite sides of the long anterior flagellum. Each mastigoneme consists of lateral filaments of two distinct sizes attached to a tubular shaft. The shaft is further differentiated into a basal region at one end and a group of from one to three terminal filaments at the free end. Mastigoneme ontogeny as revealed especially in deflagellated and regenerating cells appears to begin by assembly of the basal region and shaft within the perinuclear continuum. However, addition of lateral filaments to the shaft and extrusion of the mastigonemes to the cell surface is mediated by the Golgi complex. The ultimate distribution of mastigonemes on the flagellar surface seems to be the result of extrusion of mastigonemes near the base of the flagellum, and it is suggested that mastigonemes are then pulled up the flagellum as the axoneme elongates. Efforts to characterize mastigonemes biochemically after isolation and purification on cesium chloride (CsCl) followed by electrophoresis on acrylamide gels have demonstrated what appear to be a single major polypeptide and several differentially migrating carbohydrates. The polypeptide is not homologous with microtuble protein. The functionally anomalous role of mastigonemes in reversing flagellar thrust is discussed in relation to their distribution relative to flagellar anatomy and to the plane of flagellar undulations.     

Twist (and rotation?) of central-pair microtubules in flagella ofPoterioochromonas

Summary The chrysoflagellate algaPoterioochromonas bears two unequal flagella. There is a short naked one and a long flagellum with mastigonemes. Ultrastructural investigation reveals that the centralpair microtubules in both flagella have no fixed position with respect to the flagellar base and root system, or the mastigoneme rows in the long flagellum. The central-pair microtubules are twisted several times along the length of the flagellum. This might indicate active or passive rotation of the central-pair microtubules during flagellar beat.     

EXTRACELLULAR MICROTUBULES : The Origin, Structure, and Attachment of Flagellar Hairs in Fucus and Ascophyllum Antherozoids

Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure—a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170–190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems.     

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.     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

A TUBULAR MASTIGONEME‐RELATED PROTEIN,OCM1, ISOLATED FROM THE FLAGELLUM OF A CHROMOPHYTE ALGA,OCHROMONAS DANICA1

The phylogenetic group stramenopiles refers to the systematic groups that possess tripartite tubular hairs (stramenopiles) on their flagella. There have been a number of studies describing the fine structure of these mastigonemes and a few studies isolating the component proteins; however, these proteins and their gene sequences have not yet been identified. In the present study, we identified a mastigoneme protein (Ocm1) of the chrysophycean alga Ochromonas danica Pringsh. (UTEX LB1298). Its corresponding gene, Ocm1, was identified by using degenerate primers that correspond to the partial amino acid sequences of a protein (85 kDa) obtained from a mastigoneme‐rich fraction of isolated flagella. The polypeptide encoded by Ocm1 has four cysteine‐rich, epithelial growth factor (EGF)–like motifs, potentially involved in protein–protein interactions. It lacks obvious hydrophobic regions characteristic of transmembrane domains, suggesting that this polypeptide is not likely a protein for anchoring the mastigoneme. In addition, a polyclonal antibody against Ocm1 labeled the area where the tubular shafts of the mastigonemes are located, but not the basal portion or the terminal filaments.     

Surface organization and composition of Euglena. II. Flagellar mastigonemes

The surface of the Euglena flagellum is coated with about 30,000 fine filaments of two distinct types. The longer of these nontubular mastigonemes (about 3 micron) appear to be attached to the paraflagellar rod whereas the shorter nontubular mastigonemes (about 1.5 micron) are the centrifugally arranged portions of a larger complex, which consists of an attached unit parallel to and outside of the flagellar membrane. Units are arranged laternally in near registration and longitudinally overlap by one-half of a unit length. Rows of mastigoneme units are firmly attached to the axoneme microtubules or to the paraflagellar rod as evidenced by their persistence after removal of the flagellar membrane with neutral detergents. SDS-acrylamide gels of whole flagella revealed about 30 polypeptides, of which two gave strong positive staining with the periodic acid-Schiff (PAS) procedure. At least one of these two bands (glycoproteins) has been equated with the surface mastigonemes by parallel analysis of isolated and purified mastigonemes, particularly after phenol extraction. The faster moving glycoprotein has been selectively removed from whole flagella and from the mastigoneme fraction with low concentrations of neutral detergents at neutral or high pH. The larger glycoprotein was found to be polydisperse when electrophoresed through 1% agarose/SDS gels. Thin-layer chromatography of hydrolysates of whole flagella or of isolated mastigonemes has indicated that the major carbohydrate moiety is the pentose sugar, xylose, with possibly a small amount of glucose and an unknown minor component.     

ULTRASTRUCTURE OF THE RESERVOIR AND FLAGELLA IN PHACUS PLEURONECTES (EUGLENOPHYCEAE)1

The ultrastructure of the reservoir region of Phacus pleuronectes is described. Thin sections, ruthenium red staining, and shadow-cast preparations elucidate relationships and structural details of the flagella and flagellar hairs or mastigonemes. A heretofore undescribed structure in Phacus, the multitubular structure (MTS), with associated fibrillar projections, is reported. The MTS is located in the cytoplasm at the distal region of the reservoir near the contractile vacuole. A coordinated function of the MTS and adjacent fibrillar projections is suggested. The occurrence of mastigonemes along the entire length of the emergent flagellum is suggested, in contrast to earlier reports of their presence only on that portion of the flagellum distal to the cytostome. The present investigation postulates also that the mastigonemes are bipartite, the thicker fibrous bases becoming modified distally into the classically described, mastigonemes.     

THE OCCURRENCE OF FLAGELLATED EGGS IN LAMINARIA ANGUSTATA (PHAEOPHYTA,LAMINARIALES)1

Eggs of laminaria angustata Kjellman were shown to have two flagella. Compared with flagella of other phaeophycean swarmers, those of Laminaria eggs have several unique characters such as lack of mastigonemes, widely spaced basal bodies and no flagellar rootlets. The flagella abscise during egg liberation.     

Homologs of the sexually induced gene 1 (sig1) product constitute the stramenopile mastigonemes

The tripartite tubular mastigoneme on the anterior flagellum is a morphological feature that characterizes the stramenopiles. Mastigonemes are significant and potentially informative structures not only from the viewpoint of systematics, but also of cell biology. Nevertheless, few biochemical studies have been reported on stramenopile mastigonemes. The flagella of Scytosiphon lomentaria (Phaeophyceae) were successfully isolated and analyzed using SDS-PAGE followed by protein sequencing. The partial amino acid sequence of one flagellar protein (115kDa) showed high similarity with the sexually induced gene 1 (sig1) product of centric diatoms. A polyclonal antibody against the 115-kDa protein reacted not only to the shaft of mastigonemes in Scytosiphon lomentaria, but also another distinctly different stramenopile flagellate, Sulcochrysis biplastida (Dictyochophyceae). Therefore, we propose that the 115-kDa protein (i.e. Sig1 homologs) is a constituent of the tubular shaft of the mastigoneme.     

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.     

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.     

THE RELEASE,SETTLEMENT AND GERMINATION OF ZOOSPORES IN CHORDA TOMENTOSA (PHAEOPHYCEAE,LAMINARIALES)1,2

Details of zoospore germination in Chorda tomentosa Lyngb. are outlined. Uninucleate zoospores, when released are embedded in a mucilaginous mass of carbohydrate which dissolves and the biflagellate zoospores become motile. The long anterior flagellum is composed of a highly coiled terminal region and a rigid lower section bearing mastigonemes. The rigid, short posterior flagellum lacks mastigonemes. After initial contact by the tightly coiled region of the anterior flagellum, the zoospore draws itself to the substrate by flagellar resorbtion. After deposition of 3 wall layers the germling produces a germ tube. During this time the disc-shaped chloroplast enlarges undergoing changes in shape. As the germ tubes reach ca. 15 μm they cease forward growth and swell at their tips. The majority of cytoplasm of the original zoospore moves into the tube. Just before the nucleus enters the tube, centriole replication occurs. Mitosis is presumed to take place somewhere in the germ tube so that at 24 h, 2-celled gametophytes are produced.     

DYNAMICS OF THE HISPID FLAGELLUM OF OCHROMONAS DANICA : The Role of Mastigonemes

High speed cinephotographic techniques were used to determine the pattern of fluid flow about the hispid flagellum of Ochromonas danica and to investigate the behavior of this flagellum in media of increased viscosity. The fluid currents are consistent with the hypothesis that the mastigonemes are passive, rigid, remain normal to the flagellar surface, and lie in the plane of flagellar undulation during motility.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.     

ULTRASTRUCTURAL VARIATIONS IN CRYPTOMONAD FLAGELLA1

The arrangement of flagellar appendages in 19 cryptomonad species was examined and four new flagellar types are described. The first new type has a single row of mastigonemes on both flagella and hairs on the side opposite the mastigonemes. The second type, which is common, has unilateral rows of mastigonemes on both flagella, but no hairs. A third type has an acronematic short flagellum and a single row of mastigonemes on the long flagellum. A fourth type lacks mastigonemes but has a unilateral row of curved “spikes” on the short flagellum and hairs on both flagella. These additional flagellar variations may contribute to a more natural system of classification for cryptomonads.     

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)1

Two‐dimensional (2‐D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine‐rich, epithelial growth factor (EGF)‐like motifs, potentially involved in protein–protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up‐regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

Fibrous Flagellar Hairs of Chlamydomonas reinhardtii Do Not Enhance Swimming

The flagella of Chlamydomonas reinhardtii possess fibrous ultrastructures of a nanometer-scale thickness known as mastigonemes. These structures have been widely hypothesized to enhance flagellar thrust; however, detailed hydrodynamic analysis supporting this claim is lacking. In this study, we present a comprehensive investigation into the hydrodynamic effects of mastigonemes using a genetically modified mutant lacking the fibrous structures. Through high-speed observations of freely swimming cells, we found the average and maximum swimming speeds to be unaffected by the presence of mastigonemes. In addition to swimming speeds, no significant difference was found for flagellar gait kinematics. After our observations of swimming kinematics, we present direct measurements of the hydrodynamic forces generated by flagella with and without mastigonemes. These measurements were conducted using optical tweezers, which enabled high temporal and spatial resolution of hydrodynamic forces. Through our measurements, we found no significant difference in propulsive flows due to the presence of mastigonemes. Direct comparison between measurements and fluid mechanical modeling revealed that swimming hydrodynamics were accurately captured without including mastigonemes on the modeled swimmer’s flagella. Therefore, mastigonemes do not appear to increase the flagella’s effective area while swimming, as previously thought. Our results refute the longstanding claim that mastigonemes enhance flagellar thrust in C. reinhardtii, and so, their function still remains enigmatic.     

SPERMIOGENESIS OF THE TELEOST,ORYZIAS LATIPES,WITH SPECIAL REFERENCE TO THE FORMATION OF FLAGELLAR MEMBRANE *

In the early stage of Oryzias spermiogenesis, an axonemal bud appears at the distal end of a centriole characterized by its electron dense accessories. When the axoneme begins to grow in the cytoplasm, small vesicles come to surround it. These vesicles are similar to those produced by the Golgi apparatus which lies close to the growing axoneme. At this stage, the spermatid cell membranes disappear, causing transformation of the mononuclear spermatids into a multinucleated syncytium. As each axoneme elongates in the syncytium, it is enveloped by a cylindrical array of vesicles which are most likely derived from the Golgi apparatus. Shortly after this stage, the syncytium is again partitioned by cell membranes, restoring the existence of mononuclear spermatids. The arrayed vesicles fuse with each other to form two concentric membranes surrounding the axoneme. The inner membrane becomes the flagellar membrane and the outer one, the membrane of a flagellar sheath. These observations lead to the conclusion that the formation of the flagellar membrane is due to the fusion of vesicles surrounding the axoneme which are derived from the Golgi apparatus. In the course of spermiogenesis, no indication of an acrosomal structure is observed.