Flagellar Transition Region Research Articles

Plocamiomonas psychrophila gen. et sp. nov. (Pelagophyceae, Heterokontophyta), an Arctic marine nanoflagellate characterized by microscopy, pigments and molecular phylogeny

ABSTRACT During a field campaign in Baffin Bay (June, 1998), a sample dominated by small yellow brown biflagellates was collected from a small pocket of liquid water on the sea ice and established into a unialgal culture. Later it was given strain number CCMP2097 and deposited at the NCMA collection. Growth experiments over a range of temperatures and salinities indicated adaptation to variable Arctic conditions. This strain was studied here using light and transmission electron microscopy, HPLC for characterization of photosynthetic pigments and the relationship to related taxa was elucidated from molecular phylogeny. This integrative approach resulted in suggesting Plocamiomonas psychrophila gen. et sp. nov. the first systematically named Arctic species of the Pelagophyceae. Plocamiomonas had two unequally long flagella and the spherical cells measured 7.5 µm in diameter. Novel morphological characters included (1) a swelling on the mature flagellum, (2) a bi-layered theca, thin elongated arm-like structures emanating from the cell surface and (3) a thin filament above the flagellar transition region. This set of morphological characters represented a novel combination for the Pelagophyceae and together with evidence from the phylogenetic position of Plocamiomonas and eight uncultured cold-water pelagophytes supported the proposal of a new order Plocamiomonadales and a new family Plocamiomonadaceae.

A New Heterotrophic Cryptomonad: Hemiarma marina n. g., n. sp.

We report a new heterotrophic cryptomonad Hemiarma marina n. g., n. sp. that was collected from a seaweed sample from the Republic of Palau. In our molecular phylogenetic analyses using the small subunit ribosomal RNA gene, H.marina formed a clade with two marine environmental sequences, and the clade was placed as a sister lineage of the freshwater cryptomonad environmental clade CRY1. Alternatively, in the concatenated large and small subunit ribosomal RNA gene phylogeny, H.marina was placed as a sister lineage of Goniomonas. Light and electron microscopic observations showed that H.marina shares several ultrastructural features with cryptomonads, such as flattened mitochondrial cristae, a periplast cell covering, and ejectisomes that consist of two coiled ribbon structures. On the other hand, H.marina exhibited unique behaviors, such as attaching to substrates with its posterior flagellum and displaying a jumping motion. H.marina also had unique periplast arrangement and flagellar transitional region. On the basis of both molecular and morphological information, we concluded that H.marina should be treated as new genus and species of cryptomonads.

Suigetsumonas clinomigrationis gen. et sp. nov., a Novel Facultative Anaerobic Nanoflagellate Isolated from the Meromictic Lake Suigetsu, Japan

A novel facultative anaerobic bacterivorous nanoflagellate was isolated from the water just below the permanent oxic-anoxic interface of the meromictic Lake Suigetsu, Japan. We characterized the isolate using light and transmission electron microscopy and molecular phylogenetic analyses inferred from 18S rDNA sequences. The phylogenetic analyses showed that the isolate belonged to class Placididea (stramenopiles). The isolate showed key ultrastructural features of the Placididea, such as flagellar hairs with two unequal terminal filaments, microtubular root 2 changing in shape from an arced to an acute-angled shape, and a lack of an x-fiber in root 2. However, the isolate had a single helix in the flagellar transition region, which is a double helix in the two known placidid nanoflagellates Placidia cafeteriopsis and Wobblia lunata. Moreover, the isolate had different intracellular features compared with these two genera, such as the arrangement of basal bodies, the components of the flagellar apparatus, the number of mitochondria, and the absence (or presence) of paranuclear bodies. The 18S rDNA sequence was also phylogenetically distant from the clades of the known Placididae W. lunata and P. cafeteriopsis. Consequently, the newly isolated nanoflagellate was described as Suigetsumonas clinomigrationis gen. et sp. nov.

Ultrastructure of the Algivorous Amoeboflagellate Viridiraptor invadens (Glissomonadida, Cercozoa)

The family Viridiraptoridae represents a morphologically and ecologically distinct lineage of glissomonad flagellates (Cercozoa, Rhizaria). It currently comprises two highly specialised, algivorous genera inhabiting freshwater ecosystems, Orciraptor and Viridiraptor, for which ultrastructural data were lacking. In this study, the ultrastructure of Viridiraptor invadensHess et Melkonian, the sole described species of the viridiraptorid type genus, has been studied by transmission and scanning electron microscopy. In particular the flagellar transitional region and basal apparatus, both reconstructed from serial sections, revealed ultrastructural traits, that agree with the phylogenetic placement of the viridiraptorids within Glissomonadida: The transitional region contains a distal plate/collar complex and the basal apparatus comprises two ventral posterior roots and an anterior root, all known from other glissomonads. However, two additional small microtubular roots, two conspicuous rhizoplasts and probasal bodies present during interphase represent novel characteristics. Furthermore, an acorn/V-shaped filament system was discovered at the proximal end of the flagellar transitional region and used to establish a basal body triplet numbering system for flagellate cells of the Rhizaria. Finally, ultrastructural data on perforated algal cell walls suggest that the previously described reticulocyst of Viridiraptor represents an extrusome-derived, mesh-like coat supporting the invasion/feeding process.

Centrin Scaffold in Chlamydomonas reinhardtii Revealed by Immunoelectron Microscopy

In the flagellate green alga Chlamydomonas reinhardtii the Ca(2+)-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.

Ultrastructure of the male gametes from two centric diatoms, Chaetoceros laciniosus and Coscinodiscus wailesii (Bacillariophyceae)

The ultrastructure of the male gametes of two centric diatom species, Coscinodiscus wailesii and Chaetoceros laciniosus, is described. The species differ in the presence and configuration of certain organelles and structures, e.g. chloroplasts, Golgi bodies and microtubules (MTs), but both display a deviant configuration of the flagellar apparatus, as in other diatoms that have been examined, viz. a 9 + 0 microtubular configuration in the flagellar axoneme and a ring of nine doublets of MTs in the basal body. MTs were seen to arise from the basal body; in Coscinodiscus wailesii, the MTs formed several small bundles, which radiated in all directions from the basal body and extended down into the cell, whereas in Chaetoceros laciniosus only one bundle of MTs was observed, which was associated with the nucleus. Both species possessed two transitional plates in the flagellar transition region, one at the level of the plasmalemma, the other – less distinct – further into the basal body.

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculatawith monoclonal anti-p210.

Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3-4 microm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly.

Ultrastructure and taxonomy of a marine photosynthetic stramenopile Phaeomonas parva gen. et sp. nov. (Pinguiophyceae) with emphasis on the flagellar apparatus architecture

SUMMARY Phaeomonas parva gen. et sp. nov., a marine photosynthetic stramenopile from oceanic water near the Caroline Islands, is described. Cells are naked and spherical to ovoid. The alga is motile with two laterally inserted flagella during the light period, whereas during the dark period, it absorbs the flagella and rounds up. The anterior (immature, No. 2) long flagellum possesses tubular tripartite mastigonemes. The posterior (mature, No. 1) short flagellum is smooth and has autofluorescence at the base. The cupshaped, yellowish-brown chloroplast occupies the posterior half of the cell, and a pyrenoid occurs in the inner cavity of the cup-shaped chloroplast. The flagellar apparatus has several unusual features. Two basal plates and a two-gyred proximal helix in the flagellar transitional region may suggest that P. parva is related to the Pelagophyceae, Dictyochophyceae and Sulcochrysis biplastida, a photosynthetic stramenopile of uncertain taxonomic position. The R3 and R4 roots form a loop that resembles phagotrophic chrysophytes. However, this resemblance is superficial because Phaeomonas is not phagotrophic, its R3 root has a different number of microtubules and its R3 root does not split to form a food-uptake mouth. Phaeomonas has a ‘bypassing root’, which is found only with the Phaeophyceae, Giraudyopsis stellifera (Chrysomerophyceae), and Ankylochrysis lutea (probably a member of the Pelagophyceae). The taxonomic position of P. parva could not be determined solely from ultrastructural features. However, molecular phylogeny and biochemical analyses (published separately) strongly supported a relationship between P. parva and four other monotypic strameno-piles, Glossomastix, Pinguiochrysis, Pinguiococcus and Polypodochrysis. Although these algae are morphologically distinct, they have unusually high percentages of polyunsaturated fatty acids, especially eicosapentoic acid. This unusual assemblage of stramenopiles is classified in a new class, the Pinguiophyceae (published separately), and P. parva is its only biflagellate member.

Basal body replication in green algae – when and where does it start?

Basal body duplication in the green alga Spermatozopsis similis was reinvestigated using GT335, an antibody binding to polyglutamylated tubulins, and antibodies directed to p210, a component of the flagellar transition region which represents the distal border of the basal body. p210 was also detected in small spots at the base of each basal body which increased in size prior to mitosis. The presence of p210 on one of the microtubular flagellar roots suggested a transport of basal body material along these tracks. Immunogold electron microscopy confirmed the presence of p210 in the probasal bodies. Further, small probasal bodies are apparently connected to the mature basal bodies by centrin fibers as observed after artificially induced basal body separation in Xenopus egg extract. While basal bodies grew, most of the p210 remained at the tip of elongating basal bodies, but two or four additional spots were observed in distinct patterns near the base of the basal bodies. In cytokinesis, basal body pairs separated and p210 was observed in a strong signal at the tip and a weaker one in the vicinity of the proximal end of each basal body. We interpret the data as indicating that a new p210-containing structure forms near the proximal end of the basal bodies during basal body elongation, representing the precursor of the next generation of basal bodies. Thus, basal bodies appear to seed the succeeding generation already during their own development, a mechanism which could ensure the correct number and position of basal bodies.

Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates.

Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.

Symbiomonas scintillans gen. et sp. nov. and Picophagus flagellatus gen. et sp. nov. (Heterokonta): Two New Heterotrophic Flagellates of Picoplanktonic Size

Two new oceanic free-living heterotrophic Heterokonta species with picoplanktonic size (< 2 microm) are described. Symbiomonas scintillans Guillou et Chrétiennot-Dinet gen. et sp. nov. was isolated from samples collected both in the equatorial Pacific Ocean and the Mediterranean Sea. This new species possesses ultrastructural features of the bicosoecids, such as the absence of a helix in the flagellar transitional region (found in Cafeteria roenbergensis and in a few bicosoecids), and a flagellar root system very similar to that of C. roenbergensis, Acronema sippewissettensis, and Bicosoeca maris. This new species is characterized by a single flagellum with mastigonemes, the presence of endosymbiotic bacteria located close to the nucleus, the absence of a lorica and a R3 root composed of a 6+3+x microtubular structure. Phylogenetical analyses of nuclear-encoded SSU rDNA gene sequences indicate that this species is close to the bicosoecids C. roenbergensis and Siluania monomastiga. Picophagus flagellatus Guillou et Chrétiennot-Dinet gen. et sp. nov. was collected in the equatorial Pacific Ocean. Cells are naked and possess two flagella. This species is characterized by the lack of a transitional helix and lateral filaments on the flagellar tubular hairs, the absence of siliceous scales, two unequal flagella, R1 + R3 roots, and the absence of a rhizoplast. SSU rDNA analyses place this strain at the base of the Chrysophyceae/Synurophyceae lineages.

A 210 kDa protein is located in a membrane-microtubule linker at the distal end of mature and nascent basal bodies.

A monoclonal antibody raised against purified flagellar basal apparatuses from the green flagellate Spermatozopsis similis reacted with a protein of 210 kDa (p210) in western blots. The protein was partially cloned by immunoscreening of a cDNA library. The sequence encoded a novel protein rich in alanine (25%) and proline (20%), which contained regions similar to proteins of comparable amino acid composition such as extracellular matrix components or the membrane-cytoskeletal linker synapsin. Using a polyclonal antibody (anti-p210) raised against the C-terminal part of p210, it was shown that the protein was highly enriched in the basal apparatuses. Immunogold electron microscopy of isolated cytoskeletons or whole cells revealed that p210 was located in the flagellar transition region. The protein was part of the Y-shaped fibrous linkers between the doublet microtubules and the flagellar membrane, as indicated by statistical analysis of post-labeled sections using anti-centrin and anti-tubulin as controls. In premitotic cells p210 was located in a fibrous layer at the distal end of nascent basal bodies, which was perforated by the outgrowing axoneme. During deflagellation the protein remained at the basal body but we observed changes in its distribution, indicating that p210 partially moved to the tip of the basal body. p210 can be used as a marker to determine basal body position, orientation (parallel or antiparallel) and number in S. similis by indirect immunofluorescence. We suppose that p210 is involved in linking basal bodies to the plasma membrane, which is an important step during ciliogenesis.

γ‐tubulin in Chlamydomonas: Characterization of the gene and localization of the gene product in cells

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding γ-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with γ-tubulin from higher plants and humans, respectively. To examine the distribution of γ-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in γ-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that γ-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for γ-tubulin in assembly of the central pair microtubules of the axoneme. Cell Motil. Cytoskeleton 42:285–297, 1999. © 1999 Wiley-Liss, Inc.

Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells.

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.

Purification and characterization of basal apparatuses from a flagellate green alga.

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis similis by detergent extraction and mechanical disintegration. Sucrose density centrifugation yielded highly enriched basal apparatuses as shown by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated basal apparatuses. A mass spectrometric analysis of the carboxyterminal peptides of alpha tubulin documented detyrosination and glutamylation as posttranslational modifications and showed that some 5% of the alpha tubulin carries a polyglutamyl side chain which can reach at least 17 residues in length. Monoclonal antibodies raised against the purified basal apparatuses were used to characterize novel components in the basal apparatus. A 210-kD component identified by mAB BAS (basal apparatus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted with two high molecular weight bands (approximately 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end of the basal bodies. Immunofluorescence staining of isolated cytoskeletons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardtii and Dunahella bioculata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus.

Fine structural studies on an ultraplanktonic species ofPyramimonas, P. virginica(Prasinophyceae), with a discussion of subgenera within the genusPyramimonas

The ultrastructure of Pyramimonas virginica, the only known ultraplanktonic species of Pyramimonas, has been examined in detail. This species differs from other species of Pyramimonas in the structure of the flagellar apparatus, the scaly periplast, the pyrenoid, the eyespot, location of the eyespot in the cell, and mitosis. The flagellar apparatus is simple and shows signs of reduction. A 2-2-2-2 microtubular root system is present but a rhizoplast was not found. A very short transitional helix (perhaps two rings) is present in the flagellar transition region, together with a short one-piece stellate structure. Numbering of individual flagella in different species of Pyramimonas has shown a surprising variation in the orientation of the flagellar apparatus relative to the rest of the cell. Thus the eyespot in the various species is located differently relative to the flagellar apparatus and to other organelles such as the nucleus (in Pyramimonas there is no connection between the flagellar apparatus and ...

Ultrastructure and reconstruction of the flagellar apparatus architecture in Ankylochrysis lutea (Chrysophyceae, Sarcinochrysidales)

The ultrastructure of the sarcinochrysidalean alga Ankylochrysis lutea (van der Veer) Billard comb. nov. (Chrysophyceae) is investigated, with particular reference to the three-dimensional structure of the flagellar apparatus. This shows characteristics of heterokont algae, but has a number of unusual features: the R2 root is absent; the R3 root is straight; the ‘bypassing root’ consists of only one microtubule; two transitional plates are present in the flagellar transition region. The homologies of the flagellar apparatus components of A. lutea and two additional sarcinochrysidalean algae, Giraudyopsis stellifera Dangeard and Sarcinochrysis marina Geitler, are discussed. Because the flagellar apparatus of A. lutea differs from both G. stellifera and S. marina, the flagellar apparatus is not a usable character to delimit the order Sarcinochrysidales, which is probably a paraphyletic taxon. The external cell covering and the scale-like structures are unique features of A. lutea and may be diagnostic features of the Sarcinochrysidales sensu stricto sensu O'Kelly. The taxonomic significance of the external cell covering is discussed.

Sulcochrysis biplastida gen. et sp. nov.: Cell structure and absolute configuration of the flagellar apparatus of an enigmatic chromophyte alga

SUMMARYSulcochrysis biplastida gen. et sp. nov., a golden, marine, mixotrophic flagellate is described. Cells resemble Ochromonas in light microscopic features, but they are distinct at the electron microscopic level from Ochromonas or any other typical chrysophyte.Ultrastructural features that discriminate Sulcochrysis from the Chrysophyceae are: (i) a proximal helix in the flagellar transition region; (ii) basal bodies situated in the anterior depression of the nucleus; (iii) the lack of the rhizoplast; and (iv) simple flagellar hairs lacking lateral filaments. These features suggest that Sulcochrysis is a relative of the Pedinellophyceae, Dictyochophyceae and Pelagophyceae. However, Sulcochrysis has a flagellar root system similar to that of the Ochromonas‐type cell and it may use the R3 root for prey capture, as do ochromonadalean algae. The R3 root and the phagotrophic mechanism using the R3 root are interpreted as a plesiomorphy, because these are also distributed in primitive heterokonts such as the bicosoecids. Sulcochrysis has one microtubule probably homologous with the x‐fibre of Bicosoeca maris Picken. Based on these features it is suggested that Sulcochrysis is an organism that links bicosoecids (Bicosoeco‐phyceae), Pedinellophyceae, Dictyochophyceae and Pelagophyceae.

Nucleus Basal Body Connector of Dunaliella: Threshold Concentration of Calcium Necessary for in vitro Contraction*

AbstractDetergent‐isolated flagellar apparatuses of the flagellate green alga Dunaliella bioculata retain remnants of the nucleus (the karyoskeleton) which are linked to the basal bodies by the centrin‐containing nucleus basal body connectors (NBBC). Such complexes were subjected to different calcium concentrations (1 × 10−9 M − 5 × 10−4 M Ca2+) and the distance between the basal bodies and the karyoskeleton was measured by light microscopy. The threshold concentration of Ca2+ for NBBC contraction was determined to be around 5 × 10−8 M Ca2+. At &gt; 10−7 M Ca2+ NBBC were maximally contracted and the distance between the basal bodies and the karyoskeleton was only about 50% of the initial distance. Using a polyclonal antibody generated against centrin (Salisbury et al., 1984), the NBBC were visualized by indirect immunofluorescence in both the extended and contracted state. Our results demonstrate that in vitro contraction of centrin‐containing filaments in green algae is initiated at about the same free Ca2+ concentration in three different centrin‐containing basal apparatus components (i.e. the distal connecting fibre, the flagellar transitional region and the NBBC).

IMMUNOLOCALIZATION OF CENTER IN THE FLAGELLAR APPARATUS OF MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) AND OTHER BROWN ALGAL MOTILE CELLS1

ABSTRACTCentrin or a centrin homologue was localized using immunofluorescence in the flagellar basal body region in zoids of five brown algal species: Ectocarpus siliculosus (Dillw.) Lyngb., Scytosiphon lomentaria (Lyngb.) Link, Laminaria digitata (Huds.) Lamour., Sphacelaria rigidula (Kütz.) Prud'homme van Reine, and Fucus serratus L. The antigen is restricted to short rods extending along the basal body(ies) and towards the nucleus, which always remains firmly linked to the flagellar apparatus in isolated cytoskeletons. To identify these antigenic sites, pre‐ and postembedding immunogold electron microscopy was applied to male gametes of E. siliculosus. At least three different structures associated with the basal bodies were antigenic: a fibrous structure connecting the proximal end of the posterior basal body to the nucleus (nucleus‐basal body connector), a striated band that links the two basal bodies to each other and is located in the angel formed by them, and amorphous material interconnecting the basal bodies in their most proximal regions. In addiction, specific labeling occurs along the external surface and within the lumen of both basal bodies and in the flagellar transitional region. The possible function of these centrin‐containing structures is discussed.