COMPARATIVE ULTRASTRUCTURE OF ZOOSPORES WITH PARALLEL BASAL BODIES FROM THE GREEN ALGAE DICTYOCHLORIS FRAGRANS AND BRACTEACOCCUS SP.

The chlorococcalean algae Dictyochloris fragrans and Bracteacoccus sp. produce naked zoospores with two unequal flagella and parallel basal bodies. Ultrastructural features of the flagellar apparatus of these zoospores are basically identical and include a banded distal fiber, two proximal fibers, and four cruciately arranged microtubular rootlets with only one microtubule in each dexter rootlet. In D. fragrans, each proximal fiber is composed of two subfibers, one striated and one nonstriated, and each sinister rootlet is composed of five microtubules (4/1), decreasing to four away from the basal bodies. In Bracteacoccus sp., each proximal fiber is a single unit, the sinister rootlets are four (3/1) or rarely five (4/1) microtubules, and each basal body is associated with an unusual curved structure. The basic features of the flagellar apparatus of the zoospores of these two algae resemble those of Heterochlamydomonas rather than most other chlorococcalean algae that have equal length flagella, basal bodies in the V-shape arrangement, and clockwise absolute orientation. It is proposed that these algae with unequal flagella and parallel basal bodies have a shared common ancestry within the green algae.     

STRUCTURE AND ABSOLUTE CONFIGURATION OF THE FLAGELLAR APPARATUS IN THE ISOGAMETES OF BATOPHORA (DASYCLADALES,CHLOROPHYTA)1

The overall appearance of the flagellar apparatus in the isogametes of Batophora oerstedii. J. Ag. is most like that which occurs in motile cells of the Ulvophyceae. Like other Ulvophyceae, the basal bodies overlap and are arranged in the 11/5 configuration, microtubular roots are arranged in a cruciate pattern and system II striated fibers are present. The basal body connective which generally lacks striation in the Ulvophyceae is clearly different in Batophora, being composed of two large non-striated halves which connect to the anterior surface of each basal body and are then connected to one another by a distinctly fibrous centrally striated region. This variation in the basal body connective and the presence of two posteriorly directed system II striated fibers is clearly different from homologous structures reported in siphonous green algae of the Caulerpales. Based upon these variations and similarities among flagellar apparatus components in siphonous green algae, it is suggested that the Dasycladales and Siphonodadales are more closely related to one another than to the Caulerpales.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSZS SIMILIS (CHLOROPHYTA): ANALYSIS OF ISOLATED BASAL APPARATUSES IN THE PARALLEL CONFIGURATION1,2

The biflagellate green alga Spermatozopsis similis is demonstrated to be a model organism for the biochemical and functional analysis of the basal apparatus. Basal apparatuses were isolated in the presence of 10⁻⁶ M Ca²⁺, which induces the reorientation of the basal bodies into the parallel state. Serial thin sectioning of enriched basal apparatuses stained with tannic acid reveals several novel details of the structure of the basal bodies, the distal connecting fiber, and the striated microtubule-associated fibers. We observed a pronounced difference in size of a striated fiber connecting the basal bodies to the five-stranded microtubular roots depending on its association with the developmentally older or younger basal body. Instead of a proximal connecting fiber, the proximal end of each basal body is associated with a striated triangular plate; these plates appear to serve as spacers for the basal bodies in the parallel and antiparallel configurations. We suggest that the plates play a role in maintaining basal body orientation during forward and backward swimming. The results are summarized in representative drawings of the basal apparatus.     

THE MORPHOGENESIS OF BASAL BODIES AND ACCESSORY STRUCTURES OF THE CORTEX OF THE CILIATED PROTOZOAN TETRAHYMENA PYRIFORMIS

Dividing cells of Tetrahymena pyriformis were observed by transmission electron microscopy for signs of morphogenesis of cortical structures. The earliest stage of basal body development observed was of a short cylinder of nine single tubules connected by an internal cartwheel structure. This is set perpendicular to the mature basal body at its anterior proximal surface under the transverse microtubules and next to the basal microtubules. Sequential stages show that the single tubules become triplet tubules and that the "probasal bodies" then elongate and tilt toward the organism's surface while maintaining a constant distance of 75–100 mµ with the "parent." The new basal body after it is fully extended contacts the pellicle, and then assumes a parallel orientation with and moves anterior to the parent basal body. The electron-opaque core in the lumen of the basal body and accessory structures around its outer proximal surface appear after the developing basal body has elongated. These accessory structures associating with their counterparts from other basal bodies and with the longitudinal microtubules may play a role in the final positioning of basal bodies and thus in the maintenance of cortical patterns. Observations on a second sequence of basal body formation suggest that the oral anlage arises by multiple duplication of somatic basal bodies.     

THE THREE-DIMENSIONAL STRUCTURE OF THE BASAL BODY FROM THE RHESUS MONKEY OVIDUCT

The structure of the oviduct basal body has been reconstructed from serial, oblique, and tangential sections This composite information has been used to construct a three-dimensional scale model of the organelle The walls are composed of nine equally spaced sets of three tubules, which run from base to apex pitched to the left at a 10°–15° angle to the longitudinal axis. The transverse axis of each triplet set at its basal end intersects a tangent to the lumenal circumference of the basal body at a 40° angle (triplet angle). As the triplet set transverses from base to apex, it twists toward the lumen on the longitudinal axis of the inner A tubule; therefore, the triplet angle is 10° at the basal body-cilium junction. Strands of fibrous material extend from the basal end of each triplet to form a striated rootlet. A pyramidal basal foot projects at right angles from the midregion of the basal body. In the apex, a 175 mµ long trapezoidal sheet is attached to each triplet set. The smaller of the two parallel sides is attached to all three tubules while the longitudinal edge (one of the equidistant anti-parallel sides) is attached to the C tubule. The sheet faces counterclockwise (apex to base view) and gradually unfolds from base to apex; the outside corner merges with the cell membrane.     

THE FLAGELLAR APPARATUS OF THE ZOOSPORE OF UROSPORA PENICILLIFORMIS(CHLOROPHYTA)1

The flagellar apparatus of Urospora penicilliformis (Roth) Aresch. is unique, or at least very unusual among green algae. The flagellar axonemes are rigid, and contain wing-like projections. There are no central microtubules in the most proximal part of the axoneme. The transition region contains a series of electron dense transverse lamellae rather than a single septum, and lacks a stellate pattern. There is no cartwheel pattern in the proximal part of the basal bodies. The latter are associated with four different types of fibrous elements: ascending striated fibers that attach to an electron dense plate in the papillar center, lateral striated fibers that parallel microtubular roots, fibrous elements that link adjacent basal bodies, and finally two massive striated fibers that descend into the cell, passing closely along the nucleus (system II fibers, or rhizoplasts). Each of the four microtubular flagellar roots is sandwiched between two system I striated structures. The roots are probably equal; they contain proximally four, and distally up to eight microtubules. Based on the zoospore flagellar apparatus, it is concluded that the multinucleate U. penicilliformis is related to the Ulvaphyceae. Finally, a possible explanation in functional terms is given for the peculiar external morphology and behavior of the zoospore.     

ULTRASTRUCTURE OF THE BASAL BODY COMPLEX AND PUTATIVE VESTIGIAL FEEDING APPARATUS IN PHACUS PLEURONECTES (EUGLENOPHYCEAE)

Phacus pleuronectes (O. F. Müller) Dujardin is a phototrophic euglenoid with small discoid chloroplasts, a flat rigid body, and longitudinally arranged pellicular strips. The flagellar apparatus consisted of two basal bodies and three flagellar roots typical of many phototrophic euglenoids but also had a large striated fiber that connected the two basal bodies and associated with the ventral root. The three roots, in combination with the dorsal microtubular band, extended anteriorly and formed the major cytoskeletal elements supporting the reservoir membrane and ultimately the pellicle. A cytoplasmic pocket arose in the reservoir/canal transition region. It was supported by the ventral root and a C-shaped band of electron-opaque material that lined the cytoplasmic side of the pocket. A large striated fiber extended from this C-shaped band toward the reservoir membrane. The striated fibers in the basal apparatus and associated with the microtubule-reinforced pocket in P. pleuronecte s appear to be similar to those of the phagotrophic euglenoids.     

STRUCTURE OF THE ANISOGAMETES OF THE GREEN SIPHON PSEUDOBRYOPSIS SP. (CHLOROPHYTA)1

The fine structure of the male and female gametes of Pseudobryopsis, particularly that of the flagellar apparatus, is compared with that of swarmers of other green algae. There is general similarity, with differences in detail, to the Ulvales and other green siphons that have been studied. The similarities include overlapping basal bodies, the capping plate type of connective between basal bodies, terminal caps, and system II fibrous roots (rhizoplasts). The capping plate of the female gamete differs from that in other green siphons and the Ulvales in form and in the presence of a faint striation. A diagram illustrating the actual arrangement of the components of the flagellar apparatus is given, along with a discussion of the fact that the mirror image of the true arrangement has been given in some reports on ulvaphycean algae.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FLAGELLAR APPARATUS OF BRYOPSIS MAXIMA (CHLOROPHYCEAE)1

The flagellar apparatus in male gametes of the siphonaceous green alga, Bryopsis maxima Okamura, was studied and compared with that of other green biflagellate cells. The proximal portions of two basal bodies are connected by a single striated proximal band, unique among the biflagellate reproductive cells of green algae studied. Anterior to the flagellar bases is a pair of distal bands different from the single structure in other biflagellate cells. These bands which arise from the distal portion of each basal body, extend upward in the papilla and curve down toward the lower edges of the basal bodies. They seem to have no direct association with each other. Two pairs of distinct flagellar roots, one consisting of 3–5 microtubules and the other of a partially striated fiber of undetermined numbers of microtubules, diverge from the basal body region and extend towards the cell posterior. Their component microtubules are disorganized into single or smaller groups midway over the cell length. The uniqueness of the flagellar apparatus is briefly discussed.     

POLYPHYLETIC ORIGIN OF PARALLEL BASAL BODIES IN SWIMMING CELLS OF CHLOROPHYCEAN GREEN ALGAE (CHLOROPHYTA)1

The evolutionary affinities of Heterochlamydomonas Cox and Deason and Dictyochloris Vischer ex Starr were investigated using phylogenetic analyses of a combined data set of 18S and 28S rDNA sequences with those from 38 additional green algae. Previous ultrastructural studies have shown that motile cells of Heterochlamydomonas and Dictyochloris have an unusual flagellar apparatus organization in that the two flagella are of unequal length and the basal bodies are persistently parallel. Because of this similarity these taxa, along with Bracteacoccus Tereg, a third taxon with this same flagellar apparatus arrangement, are hypothesized to be closely related. We show, with maximum parsimony and Bayesian analyses, that the parallel basal bodies are not homologous in the three genera. Rather, Heterochlamydomonas is most closely related to Chlamydomonas baca in the clockwise flagellar apparatus clade, and Dictyochloris and Bracteacoccus are nested within the Sphaeropleales, which has the directly opposite flagellar absolute orientation. Surprisingly, Dictyochloris and Bracteacoccus are not supported as closest relatives. These relationships are supported by morphological features such as the presence or absence of a walled motile cell but not by the orientation of the basal bodies. In addition, our data are derived from multiple isolates of each study genera, and the analyses show that Heterochlamydomonas and Dictyochloris are each monophyletic.     

A Novel 95-kD Protein Is Located in a Linker between Cytoplasmic Microtubules and Basal Bodies in a Green Flagellate and Forms Striated Filaments In Vitro

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH₂- and COOH-terminal domains flanking a central domain of ~650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.     

THE FLAGELLAR APPARATUS OF ENTOCLADIA VIRIDIS MOTILE CELLS,AND THE TAXONOMIC POSITION OF THE RESURRECTED FAMILY ULVELLACEAE (ULVALES,CHLOROPHYTA)1

The flagellar apparatuses of the quadriflagellate zoo-spores and biflagellate female gametes of the marine chaetophoracean alga Entocladia viridis Reinke are significantly different from those of algae belonging to Chaetophoraceae sensu stricto, but closely resemble those of ulvacean genera. These differences permit the taxonomic reassignment of certain marine chaetophoracean genera and an evaluation of the flagellar apparatus features used to characterize the class Ulvophyceae. Critical features of the zoospore include arrangement of the four basal bodies into an upper and a lower pair with the proximal ends of the upper basal bodies overlapping, terminal caps, proximal sheaths connected to one another by striated bands, and a cruciate microtubular rootlet system having a 3-2–3-2 alternation pattern and striated microtubule-associated components that accompany the two-membered rootlets. An indistinct distal fiber occurs just anterior to the basal bodies, and is closely associated with the insertion into the flagellar apparatus of the three-membered rootlets. The flagellar apparatus demonstrates 180° rotational symmetry, and its components show counterclockwise absolute orientation when viewed from above. Newly described features include the prominently bilobed structure of the terminal caps on the upper basal body pair, and the presence of both a granular zone and an additional single microtubule anterior to each of the four rootlets, an arrangement termed the “stacked rootlet configuration.” Rhizoplasts were not observed and are presumed to be absent. The gamete is identical, except for the absence of the lower basal body pair and the presence of an electron-dense membrane associated structure that resembles the mating structure found in Ulva gametes. These findings, correlated with life history data, sporangial and gametangial structure and developmental patterns, chloroplast pigment arrays, and vegetative cell ultrastructural features, compel the removal of Entocladia viridis and similar members of the marine Chaetophoraceae to a separate family, the Ulvellaceae. The latter is referred to the order Ulvales of the Ulvophyceae. The counterclockwise absolute orientation of components, and terminal caps, may be the most consistent flagellar apparatus features of ulvophycean green algae, while variations in other features previously considered diagnostic for the Ulvophyceae may serve instead to identify discrete lineages within this class.     

Tetrahymena Poc1 ensures proper intertriplet microtubule linkages to maintain basal body integrity

Basal bodies comprise nine symmetric triplet microtubules that anchor forces produced by the asymmetric beat pattern of motile cilia. The ciliopathy protein Poc1 stabilizes basal bodies through an unknown mechanism. In poc1∆ cells, electron tomography reveals subtle defects in the organization of intertriplet linkers (A-C linkers) that connect adjacent triplet microtubules. Complete triplet microtubules are lost preferentially near the posterior face of the basal body. Basal bodies that are missing triplets likely remain competent to assemble new basal bodies with nine triplet microtubules, suggesting that the mother basal body microtubule structure does not template the daughter. Our data indicate that Poc1 stabilizes basal body triplet microtubules through linkers between neighboring triplets. Without this stabilization, specific triplet microtubules within the basal body are more susceptible to loss, probably due to force distribution within the basal body during ciliary beating. This work provides insights into how the ciliopathy protein Poc1 maintains basal body integrity.     

Comparative cytology and taxonomy of theUlvaphyceae II. Ulvalean characteristics of the stephanokont flagellar apparatus ofDerbesia tenuissima

Summary The stephanokont flagellar apparatus of the zoospores ofDerbesia tenuissima (De Not.) Crouan is examined and compared to the flagellar apparatuses of other green algae. The flagella ofDerbesia are attached to two of three bands which lie at the junction of the body and papilla. Serial longitudinal and cross sections reveal that the basal bodies are attached to the bands along their sides and at their proximal ends. The bands are not striated in any plane. The lack of striation in the bands and the partial covering of the proximal end of the basal bodies by one of the bands closely resemble the type of flagellar connection system described as the Bryopsis-type byMelkonian (1980). Zoospores of ulvalean green algae also possess these features, suggesting that green siphons are phylogenetically related to theUlvales. It is proposed that green siphons be tentatively classified in theUlvaphyceae rather than in theChlorophyceae orCharophyceae.This work supported by NSF Grant DEB 78-03554.     

Development of the flagellar apparatus during the cell cycle in unicellular algae

Summary Recent evidence has shown that algal cells acquire different flagella and a heterogeneous basal apparatus through the prolonged development of these structures over more than one cell cycle. A system for numbering algal flagella and basal bodies, which is based on developmental studies, is discussed along with the various means by which the flagellar/basal body developmental cycle can be determined. We review the information now available on development of the separate components of the flagellar apparatus-this comes particulary from the Chlorophyta and the Chromophyta-and attempt to elucidate any information which may help in phylogenetic comparisons. New data is provided on developmental changes in the cartwheel part of the basal body and basal body-associated connecting fibrils in green algae.Abbreviations Bb basal body - d right (dexter) root - df right fibrils connecting Bb triplets to microtubular and/or fibrous roots - EM electron microscopy - F flagellum - IMF immunofluorescence microscopy - LM light microscopy - NBBC nucleus-basal body connector - s left (sinister) root - sf 3left fibrils connecting Bb triplets to microtubular and/or fibrous roots. See Nomenclature section of Introduction for the numbering of basal bodies and their flagella; the same numbers apply to Bb-associated d and s roots, and df and sf fibrils     

FINE STRUCTURE OF THE SPERMATOZOID OF ZAMIA WITH SPECIAL REFERENCE TO THE FLAGELLAR APPARATUS

The locomotor apparatus of the spermatozoid of Zamia integrifolia consists of numerous flagella having the typical 9 + 2 substructure connected through basal bodies to a spiral band of complex structure. Basal bodies have a fine structure somewhat resembling that found in algae, mosses, and ferns, but they are much longer. They are composed of a circle of 9 double fibers just beneath the plasma membrane, changing to 9 doublets interconnected by fibrils in a star-pattern, giving over to a centriolar type of 9 triplet fibers embedded in an electron-dense layer of the spiral band, and ending in a “cartwheel” configuration. A system of microtubules arranged in a spiral, secondary to the flagellated spiral, is thought to underlie the plasma membrane in flagellated regions. It is suggested that this system accounts for “euglenoid” movements of the sperm. Other details of cellular fine structure are described.     

A light and electron microscopic study of the quadriflagellate green algaSpermatozopsis exsultans

The green flagellateSpermatozopsis exsultans Korshikov has been studied in culture by light and electron microscopy. The organism is naked, bears four flagella and is conspicuously spirally twisted. The ultrastructure and location of cell organelles (except the flagellar apparatus) has been investigated in detail using an absolute configuration analysis. With the exception of a doubling of the flagella and of the secondary cytoskeletal microtubule system,S. exsultans has the exact same complement of organelles occupying the same relative positions as has been described forS. similis. The two species are therefore correctly placed in the same genus. The usefulness of absolute orientations of cell organelles for green algal taxonomy and phylogeny is stressed.Dedicated to Prof.M. Mix on the occasion of her 60th birthday.     

A STUDY OF THE SPERMATOZOIDS OF DICHOTOMOSIPHON TUBEROSUS (CHLOROPHYCEAE)1

Spermatozoids of the siphonous green alga Dichotomosiphon tuberosus (A. Br.) Ernst are specialized gametes which differ in many respects from other green algal motile cells, but whose microanatomy nevertheless indicates its chlorophycean affinities. Each cell is anteriorly biflagellate and contains an irregularly shaped nucleus attached to the flagellar bases by a complex support apparatus. There is a single reduced chloroplast in each spermatozoid and numerous (50–100) minute spherical mitochondria, only 0.3 μm diam. These move vigorously in the living cell and when viewed with the light microscope they bear a striking resemblance to bacteria. Rather unexpectedly, no contractile vacuoles could be detected, even though the gametes are naked freshwater cells. Daring spermatogenesis the nucleoli of the vegetative cells disperse and are replaced by a large dense body presumably formed from either nucleolar material or condensed chromatin. The flagellar apparatus includes a cruciate flagellar root system, a feature now known to be characteristic of most green algae, exceptions being those putative ancestors of the higher plants and bryophytes. Discharge of spermatozoids from the antheridia is extremely rapid and the whole process may be finished in 30 sec. The antheridium lacks a pore apparatus, but at maturity bursts open explosively at the apex. Phyletic affinities are discussed and it is concluded that the ultrastructure of the motile cells does not, at this time, support the separation of the siphonous green algae from other green algae into a separate class.     

ABSOLUTE CONFIGURATION ANALYSIS OF THE FLAGELLAR APPARATUS OF PTEROSPERMA CRISTATUM (PRASINOPHYCEAE) AND CONSIDERATION OF ITS PHYLOGENETIC POSITION1

Pterosperma cristatum Schiller, a member of the Pra-sinophyceae, was examined with light and electron microscopy with special attention on the absolute configuration of flagellar apparatus components and associated structures. This alga is characterized by asymmetrically arranged basal bodies, connecting fibers and microtubular roots. The microtubular root system is homologous with the cruciate root system, the so-called X-2-X-2 root system typical of non-charophycean green algae. Two ducts are associated with microtubular roots. A similar flagellar apparatus and duct system was found in two other prasinophyte genera, Pyramimonas and Halosphaera. The close phylogenetic affinity of these three genera is discussed.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.