Transverse Flagellum Research Articles

Flagellar apparatus transformation and development in Prorocentrum micans and P. minimum (Dinophyceae)

Flagellar transformation was studied in Prorocentrum minimum (Pavillard) Schiller on free-swimming cells using low-illumination light microscopy. Cells bear two ventrally inserted, heterodynamic flagella. Prior to cell division, two new transverse flagella are produced. The parental longitudinal flagellum (1) remains unaltered during cell division, while the parental transverse flagellum (2) is either retracted or abscissed, and regrows as a longitudinal flagellum (1(2)). The basal apparatuses of both P. micans Ehrenberg and P. minimum show the typical complement of dinophycean roots at interphase. Two new daughter basal bodies (2(1) and 2(2)) are formed prior to any alteration of the parental basal apparatus. The parental longitudinal microtubular root (LMR) and basal body connective (BBC) appear to be maintained unaltered and associated with the parental longitudinal basal body (1). A new longitudinal microtubular root (Imr), basal body connective (bbc) and fibrous connectives (fc's) are produced between the transforming transverse basal body (2) and its daughter (2(2)). New striated root connectives (src's), transverse striated roots (tsr's) and their associated microtubules (tsrm's) are formed next to the new, transverse daughter basal bodies (2(1) and 2(2)). Basal body 2(1) forms its own transverse microtubular root (tmr) while the same root on basal body 2(2) might be derived from the parental transverse striated root microtubule (TSRM) as a result of root transformation and transfer. The parental transverse striated root (TSR) and transverse microtubular root (TMR) are disassembled during cell division. The pattern of flagellar root development is compared to other known root developmental processes for which we introduce a system of classification. We also introduce a flagellar root nomenclature similar to the flagellar nomenclature to indicate unaltered parental roots, newly formed roots, transferred roots and transformed parental roots that constitute another root type in the new daughter cell.

Chapter 47 Centrin-Based Contractile Fibers: Chromatographic Purification of Centrin

Publisher Summary Centrin is a component of an assortment of calcium-modulated cytoskeletal fiber systems found associated with flagellar basal apparatus, centrosomes, and mitotic spindle poles of eukaryotic cells. It is an acidic, low-molecular-weight, calcium-binding phosphoprotein that belongs to the EF-hand superfamily of calcium-binding proteins centrin-based systems include striated flagellar roots, transition zone fibers, fibrous bundles, and pericentriolar lattice. Centrin-based fibers play a role in anchoring the flagellar basal apparatus to the nucleus, in positioning of basal bodies and centrioles during the cell cycle, in severing microtubules at the time of flagellar excision, and in coiling the paraflagellar rod of transverse flagella. In this chapter is presented detailed methods for generating milligram quantities of pure centrin. These methods involve expression of a cDNA clone encoding Chlamydomonas centrin in Escherichia coli, and chromatographic purification of the expressed protein. The chromatographic method described works well for the purification of centrin from isolated Chlamydomonas flagella. The chapter discusses the construction of E. coli BL21 pT7-5Cen, induction and lysis of E. coli BL21 pT7-5Cen, phenyl-sepharose CLdB chromatography, superose-12 gel filtration chromatography, mono Q anion-exchange chromatography, and the troubleshooting methods. Problems encountered include inactive ampicillin, which may result in the selection and growth of bacteria that have lost the pT7-Zen plasmid; failure of transcription induction because of inactive Isopropylthio- β -glactoside (IPTG) or T7 promoter; induction of protein products that are larger or smaller than centrin because of improper bacterial transcription and/or translation; inefficient digestion of the bacterial cell wall with lysozyme; and inefficient binding of centrin to a fouled phenylsepharose column. To troubleshoot and assess the efficiency of the purification procedure, it is advised to prepare SDS-PAGE samples of E. coli BL21 pT7-5Cen before and after IPTG induction, whole-cell lysate following sonication, P1, S1, P2, S2, column voids, and column elution fractions.

IMMUNOLOCALIZATION OF CENTRIN IN OXYRRHIS MARINA (DINOPHYCEAE)1

ABSTRACTA new polyclonal antibody was raised against centrin isolated from the flagellate green alga Spermatozopsis similis (Chlorophyta; anti‐SSC). It stains by immunofluorescence and immunoelectron microscopy well‐known reference systems for centrin like the nucleus–basal body connectors in Chlamydomonas reinhardtii (Chlorophyta) and the system II fibers (rhizoplasts) of Scherffelia dubia (Chlorophyta). In addition, it recognizes in immunoblots a single 20‐kDa protein in isolated cytoskeletons of Spermatozopsis similis and Tetraselmis striata (Chlorophyta) as well as purified centrin isolated from Tetraselmis striata. Using this antibody, centrin was localized in whole cells and isolated cytoskeletons of Oxyrrhis marina Dujardin (Dinophyceae) by immunofluorescence and immunogold electron microscopy. In the flagellar apparatus of O. marina, five different structures were antigenic. Four short fibers (connectives 1–4) link the basal bodies to the four major fibrous flagellar roots, which do not cross‐react with anti‐centrin. The most prominent of the labeled structures (connective 5), a crescent‐shaped fiber, extends from the flagellar canal of the transverse flagellum along the base of the tentacle to the flagellar canal of the longitudinal flagellum, interconnecting the distal parts of the microtubular roots/bands in the basal apparatus. For most of its length, it underlies and is connected to a transversely oriented subamphiesmal microtubular band. In immunoblot analyses, anti‐SSC recognizes only a single 20‐kDa protein in cytoskeletons of O. marina. Functional and phylogenetic aspects of centrin‐containing structures in dinoflagellates are discussed.

Effects of calcium on the longituindal flagellum of Oxyrrhis marina

2–4 nm filaments represent a new class of cytoskeletal components. They are found in ciliary and flagellar roots and centrosomes of all eucaryotes. They are also the major components of paraflagellar rods (PFR) in Euglena, trypanosomes and dinoflagellates. Oxyrrhis marina, a marine dinoflagellate, possesses a transverse and a longitudinal flagellum. Only the longitudinal flagellum carries the PFR along the proximal two-thirds of its length. This flagellum is not only capable of the classic flagellar beat but is also able to retract and bend, a property mediated by external calcium. To determine if calcium has a direct role in the bending, experimental conditions were established to permeabilization and reactivation. Our conditions to reactivate the axoneme function (wave propagation) appear similar to those observed in the case of the sea urchin sperm. The results show that in vitro, an increase in calcium concentration induces a conformational change of the longitudinal flagellum in the absence of ATP with a half maximum effect at 0.1 μM. In the presence of ATP, this morphology modification causes a total inhibition of the wave propagation which is replaced by non-propulsive contractions of low amplitude. As these properties are not shared by reactivated sea urchin sperm flagella or the transverse flagellum of O marina devoid of PFR, we propose that PFR are responsible for the bending phenomenon. A calcium shock also induces flagellar excision with a half maximum effect at 0.3 μM, and immunofluorescence results suggest that a centrin-like protein is present in O marina and is responsible for this excision.

Dinoflagellate flagella adopt various conformations in response to different needs

Summary— The two flagella of Dinoflagellates have, up to now, been poorly described. They display different structures and different patterns of behaviour compared with other organisms. In addition, the two flagella are different from each other: the transverse flagellum is ribbon‐shaped and beats with a spiral undulation inside a furrow located around the cell body while the longitudinal flagellum has a larger diameter than simple flagella because it contains structures in addition to the axoneme and propagates essentially sinusoidal waves to push the cell. Ceratium flagella are particularly interesting to study because they both show different types of movements and have complex structures in addition to the axoneme. We propose that the additional structures are responsible for the particular movements of Dinoflagellate flagella. The presence of food particles in vacuoles in the vicinity of the flagella pocket suggests that their flagellar apparatus may not only be a propulsive organelle but could also be involved in prey capture.

LEPIDODINIUM VIRIDE GEN. ET SP. NOV. (GYMNODINAIALES, DINOPHYTA), A GREEN DINOFLAGELLATE WITH A CHLOROPHYLL A‐ AND B‐CONTAINING ENDOSYMBIONT1,2

ABSTRACTA further description of the green dinoflagellate, strain Y‐100, with a chlorophyll a‐ and b‐containing endosymbiont is given with special emphasis on the morphology of the host cell. The host dinoflagellate cell is unarmored and has a gymnodinoid overall appearance. The theca or amphiesma basically consists of the outer membrane and flattened thecal vesicles in which no thecal plates are developed. Unusual hand basket‐shaped scales cover the entire cell surface together with a layer of mucilaginous material. These findings suggest that the organism is a new member of the Gymnodiniaceae (Gymnodiniales). We propose the name Lepidodinium viride gen. et sp. nov. The ultrasturcture of the host cell is typical of the dinoflagellates; however, the organism has 1) an unusual cytoplasmic projection that may be a homologue of the peduncle, 2) a single membrane‐bounded body containing membranous sheets, closely situated next to the endosymbiont, and 3) an electron opaque network‐forming appendage surrounding the transverse flagellum. Name of these features have been found in other dinoflagellates.

Gamete mating and fusion in the marine dinoflagellate Scrippsiella sp.

Abstract Gamete mating and fusion of Scrippsiella sp. have been studied by light and scanning electron microscopy. When two gametes mate, the transverse flagellum of one gamete migrates out of the girdle and grasps the longitudinal flagellum of the other gamete and then returns to the girdle. Two gametes move together by helical movement of this transverse flagellum over the longitudinal flagellum of the other gamete. Fusion starts when the sulcal regions near the flagellar pores of the gametes contact each other, and then it extends first to the hypothecal regions. The epicones of the gametes fuse later. As fusion proceeds, one of the transverse flagella is lost and the other elongates to surround both gametes. When plasmogamy is completed, a motile zygote (planozygote) is formed which retains two longitudinal flagella and one transverse flagellum. Comparison of this process with that of cell division shows clearly the important differences between them which should help to avoid confusion in the future....

An ultrastructural study of planozygotes and encystment of a marine dinoflagellate,Scrippsiellasp

The fine structure of the planozygotes and encystment of Scrippsiella sp. have been studied by scanning and transmission electron microscopy. The planozygote is armoured and has two longitudinal flagella and one transverse flagellum. Unlike the vegetative cell, the planozygote has two pairs of basal bodies, large numbers of starch granules and many vesicles containing electron-dense particles. The build-up of storage materials and the reduction in size and number of the organelles in the cytoplasm, which are necessary processes for encystment, take place in the planozygote. The main feature of encystment is the formation of the thick cyst wall. The theca is shed at a very early stage of encystment before wall formation. During encystment the cell is surrounded by three layers c membranes. The cyst wall is constructed between the middle and the inner membrane and thickens due to the deposition of wall material on the inward side of the middle membrane. Mucofibrous material is seen between the outer and the middle membranes. Bundles of the mucofibrous material are raised to form protrusions which are calcified as encystment proceeds. The mucofibrous material may play an important part in the formation of the protrusions, perhaps acting as a skeleton, while the outer membrane may provide protection for the formation and calcification of these protrusions.

ULTRASTRUCTURE OF A NEW SAND‐DWELLING DINOFLAGELLATE, SCRIPPSIELLA ARENICOLA SP. NOV1

ABSTRACTA new dinoflagellate, Scrippsiella arenicola Horiguchi et Pienaar sp. nov., is described from tidal pools with sandy substrates along the east coast of South Africa. S. arenicola exhibits a vertical migratory rhythm which is in synchrony with the tidal cycle. It is a medium‐sized armoured dinoflagellate with many rod‐shaped chloroplasts. Thecal plate arrangement is pp, x, 4′, 3a, 7′, 6c, 5′, 2″ and 4s. The 2a and 3a plates are separated from each other. S. arenicola has several unique ultrastructural features. Electron‐dense fibres are found on the protruded part of the thecal plates, such as on the ornamental projections or extremities of the lists. In addition to the 9 + 2 axoneme, additional fibres are found in the free moving part of the longitudinal flagellum. The portion of the transverse flagellum covered by the left sulcal list possesses a dense array of mastigonemes which connect the flagellum and the cell. The flagellar pore platelets differ from ordinary thecal plates in their thickness and fibrous nature. The ultrastructure of the apical stalk and its associated structures is described. The vertical migration and mode of cell division is also described.

Contractile eukaryotic flagella: Centrin is involved

Isolated transverse flagella ofPeridinium inconspicuum (Dinophyceae) undergo a rapid Ca2+-induced (50μM Ca2+) contraction in the absence of exogenous ATP. Longitudinal flagella from the same species do not contract under these conditions. Contraction leads to a supercoiling of the axoneme and a shortening of the paraxonemal fiber that accompanies the axoneme over most of its length. Using a polyclonal antibody generated against centrin, a 20 kDa Ca2+-modulated contractile protein of striated flagellar roots of the green flagellateTetraselmis striata, we have found that the paraxonemal fiber in transverse flagella of three taxa ofDinophyceae is immunoreactive by indirect immunofluorescence. The localization of the antigen in the paraxonemal fiber of transverse flagella was confirmed by two-colour double immunofluorescence using monoclonal mouse-anti-β-tubulin for identification of the axoneme. No structure was immunoreactive to anticentrin in the longitudinal flagella of all taxa. Electrophoretic and immunoblot analysis of isolated flagella ofP. inconspicuum show that the antigen is a 21 kDa protein, indicating that it is either centrin or a closely related protein. We conclude that centrin confers contractility to the transverse flagellum of dinoflagellates and possibly to other contractile eukaryotic flagella.

Ultrastructure of the flagellar apparatus of Oxyrrhis marina

Summary— Oxyrrhis marina, like all dinoflagellates, possesses one transverse and one longitudinal flagellum, which show structural differences.The transverse flagellum contains a small fibre, 20 nm in diameter, associated with doublet no.7, whereas the longitudinal flagellum is substantially by a large (200–300 nm) hollows structure closely resembling the paraflagellar rod described by several authors in kinetoplastidae and in euglenoids. This structure is made up of a hemicylindrical network of filaments which are often linked on one side to the outer doublet no. 4, and on the other side to a dense plate. Another thinner filamentous network closes this hemicyclinder. In cross‐section, the wall of this structure is made up of 8 filaments 2–4 nm in diameter that show a thicker periodic structure. In longitudinal section the same filaments appear arranged in periodic rhombus meshes or a helicoidal pattern, depending on the orientation of the section relative to the axoneme.

Swimming behaviour of the unicellular biflagellate Oxyrrhis marina: in vivo and in vitro movement of the two flagella.

The movement of the 2 flagella of Oxyrrhis marina was examined with respect to their individual waveforms and the swimming behavior of the organism. The longitudinal flagella propagated helicoidal waves whose amplitude decreased toward the tip of th flagellum. Their beat frequencies were 50-60 Hz. The transverse flagella beat helicoidally within a furrow. Sudden changes in the direction of the cell trajectories were generated by transient arrests of the longitudinal flagellum beat, which were accompanied by a switch from the backward orientation to a forward one. This sweeping motion generated the rotation of the cell body. Ca2+ ions highly stimulated the frequencies of this arrest response, which compared to the "walking-stick" behavior of sea urchin spermatozoa. Isolated flagella were ATA-reactivated after detergent treatment. They exhibited 2 types of motion within the same experimental conditions. A progressive helicoidal motion was generated upon longitudinal flagellum reactivation, whereas a rolling motion with little progression characterized transverse flagellum reactivation. The differences in motile behavior reflect regulations of flagellar movement which were not destroyed by the isolation procedure and may be indicative of regulation by accessory structures.

Form and Function of the Dinoflagellate Transverse Flagellum1

ABSTRACTA reexamination of the dinoflagellate transverse flagellum in relation to swimming in more than 50 species, using a television recording system, has revealed the following new facts: the flagellar beat always proceeds counterclockwise when seen from the cell apex; the cell always rotates in the direction of the flagellar beat, and fluid is propelled in the opposite direction. These observations can be explained by the actions of flagellar mastigonemes not included in previous models. The shape of the flagellar wave is not isotropic. New explanations are offered for other morphological features of the cell as they relate to swimming.

Thoracosphaera heimii (Lohmann) Kamptner is a dinophyte: Observations on its morphology and life cycle

Thirty clones of Thoracosphaera heimii (Lohmann) Kamptner have been isolated and cultured from oceanic waters of the western North Atlantic and Gulf of Mexico. Observations on the life cycle and morphology of one clone isolated from the Sargasso Sea are reported herein. The calcareous cell wall is present in the coccoid, vegetative life phase. Reproduction is accomplished by the formation of transitory aplanospore or planospore stages or occasionally by binary fission of weakly calcified cells. The planospore is non-thecate and Gymnodinium-like with an undulating transverse flagellum and a whip-like longitudinal flagellum. All life stages possess chloroplasts and a nucleus with continually condensed chromosomes. The planospore morphology and the dinocaryotic nucleus demonstrate that T. heimii is a dinophyte and not a coccolithophorid. The taxonomic affinity and classification of T. heimii within the Dinophyceae is discussed and a new order Thoracosphaerales Tangen, ord. nov. is proposed for primarily coccoid marine dinoflagellates that possess a calcified cell wall in the vegetative life phase.

THE DINOFLAGELLATE TRANSVERSE FLAGELLUM: THREE‐DIMENSIONAL RECONSTRUCTIONS FROM SERIAL SECTIONS1

ABSTRACTSerial sections through in situ transverse flagella of the dinoflagellate Peridinium cinctum f. irregulatum (Lindem.) Lefévre are presented. Three‐dimensional reconstructions based upon tangential and radial series show a helically coiled axoneme lying external to and distinct from an accessory strand. Hitherto undescribed vesicles within the expanded flagellar sheath are suggested to provide a decoupling effect between axoneme and strand. The flagellar axis bears two types of hair but anchoring threads between cingulum and flagellum have not been found. Functional and taxonomic implications of these observations are briefly discussed.

The flagella of Peridinium cinctum fa. westii: in situ fixation and observation by scanning electron microscopy

Abstract For SEM studies, Parducz fixative followed by critical point drying effectively preserved the flagella and external membrane of the armoured dinoflagellate, Peridinium cinctum fa. westii from Lake Kinneret, Israel. The longitudinal flagellum had an external folded or ridged appearance for much of its length and a twin lobed structure at its base. The transverse flagellum in these specimens corresponded closely to the recent descriptions for a related strain of P. cinctum.

Growth cycle of free-livingGymnodinium microadriaticumfromTridacna maxima

The growth cycle of free-living Gymnodinium microadriaticum consisted of the following stages. Vegetative cells on the periphery of a group of cells gave rise to a zoosporangium. The zoospore broke out of the surrounding membrane as a motile gymnodinioid cell possessing longitudinal and transverse flagella and released a refractile body. After a period of free swimming the zoospore came to rest, shed its flagella and rounded up to become a vegetative cell again, which eventually underwent binary fission within the parent cell membrane to form two daughter cells. These daughter cells eventually became gymnodinioid zoospores again. Vegetative cells within a group of cells formed two non-motile autospores by binary fission.

IN SITU PRESERVATION OF THE TRANSVERSE FLAGELLUM OF PERIDINIUM CINCTUM (DINOPHYCEAE) FOR SCANNING ELECTRON MICROSCOPY1

ABSTRACTThree‐dimensional structure of the transverse flagellum was studied by means of scanning electron microscopy (SEM) in Peridinium cinctum (O.F.M.) Ehrenberg. Several fixation and dehydration procedures were compared. Cells fixed, rapidly in 1% osmium tetroxide and critical‐point dried showed accurate preservation of structural features seen in living cells; in particular, both transverse and posterior flagella were retained in the furrows on the cell surface. Osmium‐fixed and freeze‐dried, samples were prone to cellular collapse and loss of flagella. Glutar‐aldehyde‐fixed and critical‐point dried cells showed a layer of material covering the thecal plates, most conspicuously over the girdle region; flagella were absent. Stereo‐pair micrographs illustrate the 3‐dimensional configuration of the transverse flagellum. Modifications of previous structural models are proposed.

The propulsive mechanism of the dinoflagellate transveese flagellum reconsidered

As a result of the recent discovery that the transverse flagellum of dinoflagellates does not beat helically within the cinguium it has been necessary to formulate a new model for the manner in which this flagellum can impart a forward motion to the cell. It is theorised that the contribution to forward propulsion by the transverse flagellum can only arise directly from an asymmetry of the flagellar wave. In addition we have drawn attention to topographic features of the cell, and particularly of the left sulcal list, which may augment this thrust by redirecting cingular flow down the sulcus.

MICROMORPHOLOGY OF A SMALL DINOFLAGELLATE PROROCENTRUM MARIAE‐LEBOURIAE (PARKE & BALLATINE) COMB. NOV.1,2,3

SUMMARYThe surface structures of the bivalvate dinoflagellate Prorocentrum mariae‐lebouriae are described in detail. It has an almost spheroidal shape in face‐view, a compressed saucer‐shape in side view, with a distinct striated band at the edge of the cell. Its surface is covered with small spines in a regular pattern, with 450 nm distance between pairs. The spines are 100–120 nm wide and 200–300 nm long. There are 600–700 spines on each valve. At the anterior cell end, one of the values has a V‐shaped depression which contains a specialized structure accommodating the 2 flagellar pores. The flagellar pores are‐enclosed by 8 small, thick plates held together and to the values by sutures. The flagellar pore area consists of 2 distinct structures: an apical collar possessing a curved forked plate and a larger structure composed of an unbranched, plate. There are 2 flagellar canals located between the flagellar pore plates. Beneath each flagellar canal lies a row of 11 microtubules. A row of microtubules forming a microtubular cylinder is situated adjacent to the oblong flagellar canal near a simple pusule. The microtubular cylinder encircles electron dense bodies. The bases of the longitudinal and transverse flagella appear to lie at an angle to each other. The above features are illustrated with transmission and scanning electron micrographs.