Base Of Flagellum Research Articles

Chlamydomonas IFT172 Is Encoded by FLA11, Interacts with CrEB1, and Regulates IFT at the Flagellar Tip

The transport of flagellar precursors and removal of turnover products from the flagellar tip is mediated by intraflagellar transport (IFT) , which is essential for both flagellar assembly and maintenance . Large groups of IFT particles are moved from the flagellar base to the tip by kinesin-2, and smaller groups are returned to the base by cytoplasmic dynein 1b. The IFT particles are composed of two protein complexes, A and B, comprising approximately 16-18 polypeptides. How cargo is unloaded from IFT particles, turnover products loaded, and active IFT motors exchanged at the tip is unknown. We previously showed that the Chlamydomonas microtubule end binding protein 1 (CrEB1) localizes to the flagellar tip and is depleted from the tips of the temperature-sensitive (ts) mutant fla11ts . We demonstrate here that FLA11 encodes IFT protein 172, a component of IFT complex B, and show that IFT172 interacts with CrEB1. Because fla11ts cells are defective in IFT particle turnaround at the tip, our results indicate that IFT172 is involved in regulating the transition between anterograde and retrograde IFT at the tip, perhaps by a mechanism involving CrEB1. Therefore, IFT172 is involved in the control of flagellar assembly/disassembly at the tip.

Behavior of Flagella and Eyespot during Fertilization of Anisogametes in Caulerpa racemosa (Forsskal) J. Agardh var. laetevirens (Montagne) (Ulvophyceae, Chlorophyta) Revealed by Field Emission Scanning Electron Microscopy

Respective fates of male and female flagella and the eyespot during fertilization of ulvophycean alga Caulerpa racemosa (Forsskal) J. Agardh var. laetevirens (Montagne) were studied using field emission scanning electron microscopy (FE-SEM). FE-SEM allowed visualization of the eyespot of the biflagellate gamete. The female gamete has one eyespot on a posterior position of the cell body; the male gamete does not. The female gamete eyespot was oriented to the direction of the plane of the flagellar beat. The female gamete is larger than the male one and has the only eyespot. For those reasons, the author followed the fate of the flagella and eyespot during fertilization. When both gametes were mixed, the initial cytoplasmic contact and cell fusion between them usually took place at the anterior end slightly below the flagellar base. In most mating pairs, the female gamete fuses at the left side of the eyespot and male gamete at a cell surface that is perpendicular to the plane of the flagellar beat. As fusion proceeds, the gamete pair is transformed into a quadriflagellate planozygote in which two flagella each from the male and female gametes point in nearly the same direction. In particular, the no. 2 flagellum of the female gamete and one male flagellum were directed to the eyespot. These observations indicated that cell fusion at the particular area of the gamete is important for proper arrangement of the flagella and eyespot in the planozygote.

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA, SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.

Visualization of the Rapid Fertilization Process of the Marine Green Alga, Ulva arasakii Chihara (Ulvophyceae, Chlorophyta) Using High-speed Video Microscopy

Flagellar movement and mating behavior of the biflagellate gametes of the marine green alga, Ulva arasakii was studied using high-speed video microscopy to clarify the rapid fertilization process of marine green algae. Discharged male and female gametes always swing their flagella backward during forward swimming. The beat pattern is flagellar beat with undulatory waves produced at the flagellar base being propagated toward the tip. One beat cycle takes 15 ms in both male and female gametes. When the male gamete encountered the female gamete, initial contact between the two gametes took place at one flagellar tip and at the anterior end of the cell body. Although attachment and detachment of flagellar tips occurred during the fertilization process, the male gamete kept in contact with the female gamete. Then male and female gametes adhered respectively at the anterior side of the cell body and at the flagellar tips. Nevertheless, they continued to move their flagella with a flagellar beat and oscillated their cell bodies. Finally, the gamete pair lay side by side with their longitudinal axes nearly parallel to one another within 30–100 ms after initial contact of the two gametes.

Self-Assembly and Type III Protein Export of the Bacterial Flagellum

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process.

FLAGELLAR REGENERATION IN SPERMATOZOPSIS SIMILIS (CHLOROPHYTA)

The unicellular green alga Spermatozopsis similis Preisig et Melkonian bears two flagella of unequal length. After deflagellation, cells first regenerated the longer flagellum to about one third of its original length, before the shorter flagellum started to develop. Growth rates were similar for both flagella. Thus, the length difference between both flagella was restored by a lag‐phase during regeneration of the shorter flagellum. To explain the lag‐phase, we have considered a gating mechanism near the flagellar base that controls the entry of precursors into the flagellum. This would allow cells to restrict the time of effective flagellar growth and thereby control flagellar length. Our data indicated that cells are capable of individually regulating flagellar assembly onto basal bodies. We discuss a recent model of flagellar length regulation based on a balance of assembly and disassembly and conclude that flagellar length is controlled by additional factors, including the availability of flagellar proteins and the developmental status of basal bodies.

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

SUMMARY Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE-SEM). FE-SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side-by-side with their longitudinal axes nearly parallel, while the rest are oriented anti-parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side-by-side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.

Ultrastructural study of the type species of Peridiniopsis, Peridiniopsis borgei (Dinophyceae), with special reference to the peduncle and flagellar apparatus

The general ultrastructure and flagellar apparatus of Peridiniopsis borgei were studied by serial section electron microscopy. The cells contained a conspicuous multiple-stalked pyrenoid in an anterior-dorsal position, relative to a large, central sac pusule (SP), and were surrounded by an unusual mucilage layer, located between the thecal plates and the outer amphiesmal membranes. The central SP was linked to the longitudinal flagellar canal by a narrow, 1.5 μm long tubular extension. An apical fibrous complex with three radiating fibres was associated with the apical pore complex. A flat peduncle emerged near the flagellar bases and was supported by a single row of 75–80 microtubules, which extended for nearly 50 μm along an elaborate path inside the cell. The flagellar apparatus resembled that of Peridinium cinctum but differences were noted. The layered connective was smaller and had fewer thin layers than that of P. cinctum. The microtubular root associated with the anterior surface of the transverse basal body nucleated a novel type of transverse microtubular root extension, in which 23 microtubules surrounding a fibrouscore extended along the surface of the SP. Two distinct fibres connected triplets of the transverse basal body to the longitudinal microtubular root (LMR). A large eyespot, located within chloroplast lobes, was found in the sulcal area. It differed from previously described dinoflagellate eyespots by having up to six layers of electron-opaque globules; the inner layers were intercalated among thylakoid lamellae. A vesicle containing a layer of crystal-like bodies was present between the ventral surface of the eyespot and the LMR. In all cells sectioned, a double unit membrane was located between the thecal plates and the cytoplasmic membrane.

Prorocentrum nux sp. nov. (Dinophyceae), a small planktonic dinoflagellate from the Mediterranean Sea, and discussion of P. nanum and P. pusillum

A new Prorocentrum species was discovered in the Gulf of Naples (Mediterranean Sea) and is described in the present study as P. nux Puigserver & Zingone sp. nov. Prorocentrum nux is small (6.3–9.0 μm long, 5.3–10.0 μm wide, and 5.3–10.7 μm deep), irregularly oval in valve view, and lacks an apical spine. Unlike most Prorocentrum species, its cells are not laterally compressed, owing to the marked convexity of the valves and to the overgrowth of the intercalary bands. The thecal plates are thin and smooth, with a few pores of two different sizes near the valve margins. Seven platelets are arranged around the flagellar pore. Two parietal chloroplasts are present, each having a pyrenoid without a visible starch shield. The nucleus is located posteriorly. A pusule is present close to the flagellar bases. Numerous trichocysts were observed both within the cell and discharged outside it. In the genus Prorocentrum, two other small-celled species have been described, viz. P. nanum and P. pusilium; the latter has sometimes been considered a synonym of the former. However, based on their original descriptions and on other published information, P. nanum and P. pusillum are quite distinct from each other and from P. nux, with respect to cell shape, and the size and location of the thecal pores. Unlike P. nux, both P. nanum and P. pusillum are described as having a thick theca and a laterally flattened cell body. We reexamined the only existing culture material classified as P. nanum, viz. UTEX LB 1008, derived from the Plymouth 184 strain and originally identified as Exuviaella pusilla. This material has consistently been misidentifiedand has the same distinctive characters as P. nux.

Morphology of the antennal sensilla in four Australian spittlebug species (Hemiptera: Cercopidae) with implications for phylogeny

AbstractThe antennal sense organs of four Australian spittlebugs, Aufidus trifasciatus Stål, Euryaulax carnifex (Fabricius), Petyllis deprivata (Walker) and Tonnoiria tasmaniae Lallemand (Hemiptera: Cercopidae) are described for the first time, based on scanning electron microscope observations. Four major types of sense organs were found: one basiconic sensillum and 8–9 coeloconic sensilla on the expanded flagellar base, and several trichoid sensilla and one campaniform sensillum on the pedicel. The antennal sensilla found in the four species show very similar patterns in their shape, distribution and arrangement and this suggests that the genera Aufidus Stål, Petyllis Kirkaldy and Tonnoiria Lallemand, all currently placed in the Afrotropical and Oriental tribe Rhinaulacini, may belong to the Australian tribe Euryaulacini. In addition, the history of the nomenclature of two Australian species previously assigned to the genus Eoscarta is reviewed, and the following two resurrected combinations established: Euryaulax carnifex (Fabricius) comb. rev. and Euryaulax vacuola (Jacobi) comb. rev.

Scanning Electron Microscopy of Antennal Sense Organs in Prosapia bicincta (Say) (Hemiptera: Cercopidae)

The external morphology of the sensilla on the antennae of the adult spittlebug, Prosapia bicincta (Say) (Hemiptera: Cercopidae), is described for the first time, using scanning electron microscope observations. The sensilla include one peg-like basiconic sensillum (about 65.05 to 65.90 μ in length and 13.33 to 14.08 μ in width at base), 18 to 23 porous coeloconic sensilla (mostly 7.38 to 8.94 μ in diam), one campaniform sensillum and a cluster of trichoid sensilla (about 45 to 55 (μ long). The first two types of sensilla are located on the expanded flagellar base, while the latter two sensilla are on the pedicel.

Measurement of the Force Produced by an Intact Bull Sperm Flagellum in Isometric Arrest and Estimation of the Dynein Stall Force

The force generated by a detergent-extracted reactivated bull sperm flagellum during an isometric stall was measured with a force-calibrated glass microprobe. The average isometric stall force from 48 individual measurements was 2.5 ± 0.7 × 10 −5 dyne (2.5 ± 0.7 × 10 −10 N). The force measurements were obtained by positioning a calibrated microprobe in the beat path of sperm cells that were stuck by their heads to a glass microscope slide. The average position of the contact point of the flagellum with the probe was 15 μm from the head-tail junction. This average lever arm length multiplied by the measured force yields an estimate of the active bending moment (torque) of 3.9 × 10 −8 dyne × cm (3.9 × 10 −15 N × m). The force was sustained and was for the most part uniform, despite the fact that the flagellum beyond the point of contact with the probe usually continued beating. It appears that the dynein motors in the basal portion of the flagellum continue to pull in an isometric stall for as long as the motion of the flagellum is blocked. If dynein motors in the flagellum distal to the contact point with the probe were contributing force to the displacement of the probe, then the flagellar segment immediately past the point of contact would have to show a net curvature in the direction of the probe displacement. No such curvature bias was observed in the R-bend arrests, and only a small positive curvature bias was measured in the P-bend arrests. Our analysis of the data suggests that more than 90% of the sustained force component is generated by the part of the flagellum between the probe and the flagellar base. Based on this premise, the isometric stall force per dynein head is estimated to be 5.0 × 10 −7 dyne (5 pN). This equals ∼1.0 × 10 −6 dyne (10 pN) per intact dynein arm. These values are close to the isometric stall force of isolated dynein. This suggests that all of the dynein heads between the base and the probe, on the active side of the axoneme, are contributing to the force exerted against the probe.

Overproduced Salmonella typhimurium flagellar motor switch complexes

Three Salmonella typhimurium flagellar motor proteins, FliG, FliM and FliN, are required for the switching of rotation sense. The proteins have been localized to the cytoplasmic module of the flagellar base. Structures, which were morphologically indistinguishable from the native transmembrane MS-ring and cytoplasmic C-ring basal body modules, formed in Escherichia coli upon plasmid-encoded synthesis of these proteins together with FliF. The structures localized to the cell membrane and contained all three motor proteins, as determined by immuno-electron microscopy. This result supports the deduction, based on earlier biochemical analysis, that the C-ring is composed entirely of these proteins and, therefore, functions as a dedicated motor component. In addition, it demonstrates that the morphologically correct assembly of the C-ring onto the MS-ring proceeds independently of other structural components of these modules.

Centrin protein and genes in Trichomonas vaginalis and close relatives.

Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined. By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).

Bacterial flagellation and cell division.

The peritrichous flagella of Salmonella are synthesized and function through many cell generations. There are two different aspects in the relationship between flagellar biogenesis and cell division. Filament growth is independent from the cell cycle and the length of filaments appear to be locally controlled at each flagellar base, whereas the number of filaments (or flagellar basal bodies) is dependent on cell cycle. We present a model to explain how the number of filaments is maintained through generations. We will also introduce a new direction for research that might directly connect flagellation and cell division; the global communication between flagellar genes and external factors of a complex regulatory network in a cell.

Ultrastructure of the colourless flagellate Hyperamoeba flagellata with special reference to the flagellar apparatus

Summary Hyperamoeba flagellata is a fresh-water protist of uncertain taxonomic position. It exists in three living forms: as an amoeba, a flagellate and a cyst. The ultrastructure of the flagellated stage of H. flagellata is investigated here by TEM for the first time. Special attention was paid to the structure of the flagellar apparatus. There is one long smooth flagellum only which is directed anteriorly. It emerges from the flagellar basal body (kinetosome) located at the apical end of the cell. Two cones of microtubules extend from the flagellar base towards the nucleus. The outer cone originates from the lateral side of the flagellar kinetosome, while the inner cone arises from an MTOC associated with the flagellar kinetosome by a short fibrillar rootlet. The second (barren) kinetosome lies ventrally and is directed to the right side of the cell. It has two microtubular rootlets which pass laterally in the same direction. The microtubular cones overlap the anterior end of the nucleus. A dictyosome lies between the basal bodies and the nucleus. Mitochondria with tubular cristae have an electron dense core which is orientated in a longitudinal axis. The structure of the flagellated stage of H. flagellata is compared with flagellated cells of protostelids and cercomonads. It is proposed that this organism may represent a link between protostelids and cercomonads.

Cell and nuclear division in a freshwater choanoflagellate, Monosiga ovata Kent

Summary Monosiga ovata undergoes a longitudinal cell division not constrained in any way by a cell covering. The first stage in cell division involves the withdrawal of the single anterior flagellum. The flagellar axoneme, from the distal end of the transition region to the tip, is withdrawn deep into the cell where it is depolymerised. Replication of the two parent basal bodies follows to produce two pairs of basal bodies. The transition fibres and the immediate ring of radiating cytoskeletal microtubules around the basal body of the previously emergent flagellum remain intact throughout division. The basal body of the other future emergent flagellum quickly acquires transition fibres and a ring of radiating cytoskeletal microtubules. During prophase the collar becomes ovoid in cross section and the number of tentacles increases from about 26 to 40. Within the nucleus aggregates of heterochromatin and groups of microtubules become apparent. During metaphase the nucleus becomes elongated, spindle microtubules appear and densely staining chromatin becomes aligned at the median metaphase plate. The nuclear envelope remains intact throughout prophase and early metaphase and there is no indication that the polar basal bodies make contact with the spindle microtubules within the nucleus. The antiparallel polar basal bodies begin to regenerate flagella. At this stage the two flagellar basal bodies are at the extreme ends of the elongated collar and the two emergent flagella project through the sides of the collar. As metaphase proceeds, fenestrae appear in the nuclear envelope at the poles. Eventually the nuclear envelope disintegrates and nuclear division continues through anaphase and telophase. As cell division occurs the flagellar bases move towards the central anterior region of the two daughter cells. The number of collar tentacles continues to increase to approximately 50. As cytokinesis progresses the two daughter cells become more distinct and eventually separate each with a fully developed flagellum and a surrounding collar.

The fine structure of the male gamete of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae). I. General structure of the cell

The ultrastructure of male gametes of Ectocarpus siliculosus is described and discussed in relation to the information available on other brown algal zoids. In general, the fine structure and the organelle associations agree with those of other brown algal cells. In particular, the male gametes typically contain a single chloroplast with a pyrenoid and a prominent concave eyespot, into which the swelling of the posterior flagellum (the photoreceptor) fits. The continuity of the chloroplast endoplasmic reticulum and nuclear membrane, as well as the presence of a perinuclear Golgi consisting of several dictyosomes, is shown. The fine structure of microbodies, lipid bodies and different types of vesicles, including physodes and coated vesicles, is also discussed. Several specializations of cellular elements and their interactions are unique and have not been reported before. These features include the connection between the nuclear envelope, the Golgi and the flagellar bases mediated by fibres resembling intermediate filaments, and the organization of the Golgi as well as the association of mitochondria and a microtubular flagellar root.

The fine structure of the male gamete of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae). I. General structure of the cell

The ultrastructure of male gametes of Ectocarpus siliculosus is described and discussed in relation to the information available on other brown algal zoids. In general, the fine structure and the organelle associations agree with those of other brown algal cells. In particular, the male gametes typically contain a single chloroplast with a pyrenoid and a prominent concave eyespot, into which the swelling of the posterior flagellum (the photoreceptor) fits. The continuity of the chloroplast endoplasmic reticulum and nuclear membrane, as well as the presence of a perinuclear Golgi consisting of several dictyosomes, is shown. The fine structure of microbodies, lipid bodies and different types of vesicles, including physodes and coated vesicles, is also discussed. Several specializations of cellular elements and their interactions are unique and have not been reported before. These features include the connection between the nuclear envelope, the Golgi and the flagellar bases mediated by fibres resembling intermediate filaments, and the organization of the Golgi as well as the association of mitochondria and a microtubular flagellar root.

Assembly and function of Chlamydomonas flagellar mastigonemes as probed with a monoclonal antibody.

Mastigonemes are hair-like projections on the flagella of various kinds of lower eukaryotes. We obtained a monoclonal antibody (mAb-MAST1) to mastigonemes of Chlamydomonas reinhardtii, and found that it reacts with a single flagellar glycoprotein of about 230 kDa. Interestingly, immunofluorescence microscopy demonstrated that mAb-MAST1 recognizes not only the flagellar mastigonemes but also a ring composed of 10 or more particles located in the anterior end of the cell body close to the flagellar bases. The ring structure may be the pool of the mastigoneme protein. When the flagella are amputated, they regenerate to their original length in 90-120 minutes. We found that mastigonemes appear on the new flagellar surface as early as 15 minutes after deflagellation, and that new mastigonemes are mostly assembled onto the distal region of the flagellar surface. Mastigonemes thus appear to be inserted into the membrane only in the distal region of the flagellum. Alternatively, mastigonemes may be inserted at the base and transported very rapidly to the distal portion where they are trapped. When live cells are treated with mAb-MAST1, mastigonemes disappear from the flagellar surface. In these mAb-MAST1 treated cells, the swimming velocity decreases to 70-80% of the normal value, although the flagellar beat frequency increases to approximately 110% of the control. These findings demonstrate vectorial transport of mastigonemes to their assembly sites, and show that mastigonemes function to increase flagellar propulsive force by increasing the effective surface of the flagellum.