THE RESERVOIR CYTOSKELETON AND A POSSIBLE CYTOSTOMAL HOMOLOGUE IN COLACIUM (EUGLENOPHYCEAE)1

The reservoir cytoskeleton of Colacium Ehrenberg is formed of three bands of microtubules. The microtubules of the dorsal band (DMT) become doublets and are continuous with the longitudinal microtubules of the canal and, therefore, of the pellicle. A band of para-reservoir microtubules (PMT) acts as a linkage between the edges of the dorsal band at the formation of the canal. The third band of microtubules (MTR), more ventral, branches away from the reservoir-canal transition region and forms a supportive band for a pocket formed from the reservoir membrane. The outer part of the pocket membrane is closely invested with a fibrillar mesh. The pocket of Colacium, a green euglenoid, resembles structurally the cytopharynx of the colorless phagotrophic euglenoids, Isonema papillatum and the bodonid flagellates. The homologies support the hypothesis of euglenoid derivation from the kinetoplastid flagellates.     

Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei

Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal.     

A cytostome/cytopharynx in green euglenoid flagellates (Euglenales) and its phylogenetic implications

The green, phototrophic euglenoid, Colacium libellae, has a vestigial cytostome and cytopharynx. The membrane forming the simple pocket is decorated with a dense, microfilamentous mesh. The mesh binds the pocket to a band of reinforcing microtubules which is homologous with the bodonid MTR. The opening of the cytostome at the reservoir-canal transition zone implies that the phototrophic euglenoid canal is formed by the invagination of the vestibulum of the phagotrophic euglenoids. Our observations support the hypothesis that the phagotrophic euglenoids arose from a bondonid ancestor and gave rise to the phototrophs by chloroplast acquisition.     

FLAGELLAR ROOTS AND THE RESERVOIR CYTOSKELETON OF COLACIUM LIBELLAE (EUGLENOPHYCEAE)1

The three flagellar roots of Colacium Ehrenberg give rise to the three microtublar bands of the reservoir cytoskeleton. The dorsal root (DR) originates at the basal body (bb1) of the emergent flagellum. It is initiated on the left side of the cell, runs toward the right side under the posterior end of the reservoir and thence anteriorly in a spiral path over the dorsal surface of the reservoir until it terminates on the left side of the eyespot. Along its length, it appears to initiate a dorsal band (DB) which forms the major dorsal portion of the reservoir cytoskeleton—the dorsal microtubules (DMT). Two roots originate at the basal body (bb2) of the non-emergent flagellum. The ventral root (VR) runs up the left side of the cell and initiates the band of microtubules which forms part of the presumptive vestigial cytopharynx. Therefore, it forms the reinforcing microtubules (MTR) of Colacium. The intermediate root (IR) forms the para-reservoir microtubules (PMT). Flagellar root correlation with the reservoir cytoskeletal bands strengthens their homologies with the bodonid bands and further supports the hypothesis that the euglenoids are derived from the kinetoplastid flagellates.     

Light and Electron Microscopic Description of a Colorless Euglenoid,Serpenomonas costata n. g., n. sp.1

ABSTRACT. A new genus of rigid, colorless, phagotrophic euglenoid is described from a standing pond on a salt marsh. The cell body measures 21–25 μm long, is about 18 μm wide, has a slight dorso-ventral flattening, and is marked by distinct pellicular ridges. The organism, described as Serpenomonas costata, has two unequal, antapically inserted, heterodynamic flagella. The shorter flagellum is anteriorly directed during swimming and the longer one trails posteriorly. Cells move along the substrate with a creeping motion. The ingestion apparatus is composed of separate ribs extending the length of the cell and is incorporated into the ventral pellicular ridge. The apparatus is independent of the canal and reservoir and is not protrusible. The taxonomic affinities of Serpenomonas with other euglenoids are discussed.     

ATPase activity in flagella from Euglena gracilis. Localization of the enzyme and effects of detergents.

The biochemical effects of some detergents on the ATPase activity of isolated flagella from Euglena gracilis are related to morphologic obliterations induced by those detergents. Enzymic activity can be localized by electron microscopy along the microtubules and also on the paraflagellar rod. The nonionic detergent digitonin solubilizes the enzyme linked to dyneinic arms, whereas the activity linked to residual structures appears enhanced. These results support the hypothesis that the paraflagellar rod may be a structure activity related to the motility of this type of flagellum.     

PHYLOGENY OF THE EUGLENOPHYTES: ANALYSIS USING SSU RDNA AND ULTRASTRUCTURAL CHARACTERS

The use of both molecular and morphological data to determine relationships among the euglenoids is vital for a complete understanding of their phylogeny, and the development of an accurate taxonomy. Analyses of the SSU (18S) rDNA from 12 euglenoid genera have resulted in tree topologies that are in agreement with many defining morphological characters. The euglenoid lineage is formed by phagotrophic euglenoids at its base, followed by the divergence of phototrophs that in-turn gave rise to osmotrophs. The photosynthetic lineage is anchored by euglenoids with two emergent flagella, Eutreptia and Eutreptiella , while the remainder of the lineage is composed of euglenoids with a single emergent flagellum. Among the photosynthetic euglenoids with a single emergent flagellum those that secrete mucilaginous stalks, Colacium , or form a lorica, Trachelomonas and Strombomonas , are closely associated. The remaining photosynthetic genera Euglena , Phacus , and Lepocinclis are intermixed with each other and the osmotrophic genera Astasia , and Khawkinea. Hence, they are not monophyletic, sensu Hennig. To reinforce molecular phylogenies, a robust morphological character database is necessary. For taxa with complex internal structures complete serial reconstruction is required. Serial reconstruction of the flagellar and feeding apparatuses in Ploeotia costata illustrate this necessity. Originally described as having both an MTR (Type I) and a Type II feeding apparatus, reconstruction has shown P. costata to have a single, Type II, feeding apparatus. Moreover, the Type II now appears to be an autapomorphy for Ploeotia species, while euglenoid feeding apparatuses, in toto, appear to form a continuum of structural types.     

ULTRASTRUCTURE OF THE BASAL APPARATUS AND PUTATIVE VESTIGIAL FEEDING APPARATUSES IN A QUADRIFLAGELLATE EUGLENOID (EUGLENOPHYTA)1

The flagellar apparatus and presumptive vestigial feeding apparatuses of a cold-water, photosynthetic, quadriflagellate euglenoid is described. The organism possesses two similar sets of flagella each consisting of one short and one long flagellum. Each pair of flagella is associated with three microtubular roots for a total of six roots in the basal apparatus. At the level of the ventral basal bodies, each intermediate root is nine-membered, while the ventral roots are composed of eight to nine microtubules. Only one of the ventral roots lines the single microtubule reinforced pocket. A four-membered dorsal root attaches to each dorsal basal body, and at the level of the reservoir each gives rise to a dorsal band. An additional bundle of microtubules, not arising from the microtubular roots of the basal apparatus, begins posterior to the basal apparatus as a small group of a few microtubules and extends anteriorly on the right ventral side of the reservoir ending at the canal. At the level of the stigma, the microtubules are organized into a multi-layered bundle that continues to increase in size and eventually splits to form two bundles at the level of the canal. We postulate that these bundles may represent the remnants of a rod-and-vane-type feeding apparatus like that found in many phagotrophic euglenoids.     

ATPase Activity in Flagella from Euglena gracilis. Localization of the Enzyme and Effects of Detergents*

SYNOPSIS. The biochemical effects of some detergents on the ATPase activity of isolated flagella from Euglena gracilis are related to morphologic obliterations induced by those detergents. Enzymic activity can be localized by electron microscopy along the microtubules and also on the paraflagellar rod. The nonionic detergent digitonin solubilizes the enzyme linked to dyneinic arms, whereas the activity linked to residual structures appears enhanced. These results support the hypothesis that the paraflagellar rod may be a structure actively related to the motility of this type of flagellum.     

CRYPTOGLENA PIGRA: A EUGLENOID WITH ONE CHLOROPLAST12

The taxonomic status of Cryptoglena pigra Ehrb., interpreted from observations based on bright-field microscopy, has been uncertain. Examination with the electron microscope of a clone of C pigra isolated by E. G. Pringsheim reveals certain features which, collectively, are distinctly euglenoid: periplast associated with muciferous bodies and subpellicular microtubules; canal and reservoir with microtubules; one flagellum with a swelling and emergent through a canal, and a second flagellum without a swelling and nonremergent; stigma (eyetpot) closely apprrssed to but not part of the chloroplast; nucleus with permanently condensed chromosomes attached to the inner nuclrar membrane; mitochondria with disc-shaped cristae constricted at the base; chloroplast with thylakoids often in triplets; and paramylon grains in the cytoplasm. Unlike most euglenoids, C. pigra possesses a single chloroplast that in transverse thin sections is U-shaped.     

Flagellar regeneration and associated scale deposition inPyramimonas gelidicola (Prasinophyceae,Chlorophyta)

Summary During regeneration of mechanically amputated flagella, flagellar scales and the subtending membrane accumulate in a villiform scale reservoir in which the scales interact to form patterns on the villi reminiscent of the arrangement they later assume on the flagellum. The reservoir membrane is continuous with the plasmalemma, and the scales, attached directly and indirectly to the membrane, leave the reservoir and migrate toward the developing flagella where they assemble into highly ordered layers. It is proposed that scale-scale interactions induce a process of auto-assembly initiating the complex arrangement of scale tiers on the flagellum and cell body.     

The cytoskeletal architecture of Trypanosoma brucei

The cytoskeleton of Trypanosoma brucei has been analyzed by the high-resolution technique of quick-freeze deep-etch rotary-shadowing electron microscopy. The study provides detailed structural information on the subpellicular array of microtubules, the flagellum, and the interaction of these 2 major structures of the trypanosomal cytoskeleton with each other. The subpellicular microtubules closely interact both with the cell membrane and with each other. At the anterior tip of the cell they converge into a tightly closed structure, whereas at the posterior end the microtubular array remains open ended. The microtubular array is involved also in forming the opening of the flagellar pocket. The microtubular array interacts with the paraflagellar rod of the flagellum through a dense meshwork of fibers that are anchored on the microtubular surface with one end and within the paraflagellar rod structure with the other. The highly ordered, 3-dimensional network of the paraflagellar rod itself is connected tightly to the microtubular axoneme of the flagellum through a regular array of fleur-de-lis-shaped linking structures.     

A PHYSICAL MAP OF THE CHLAMYDOMONAS REINHARDTII NUCLEAR GENOME

Shin, W., Zimmermann, S. & Triemer, R. E. Department of Life Science, Rutgers University, Piscataway, NJ 08854, USA In 1985, Willey and Wibel described the existence of a cytoplasmic pocket formed from the reservoir membrane in Colacium. A band of microtubules derived from the ventral flagellar root (MTR) lined the pocket and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid cytostomes, colorless euglenoid cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids including Euglena, Eutreptia, Eutreptiella, Cryptoglena, and Tetreutreptia. We now report on MTR/pockets in Lepocinclis, Trachelomonas, Strombomonas and Phacus thereby demonstrating the presence of this complex in representatives of all of the major photosynthetic genera. A comparison of the MTR/complex across genera indicates a reduction in structural complexity that is consistent with recent phylogenetic schemes based on molecular characters.     

Khawkinea quartana, a Colorless Euglenoid Flagellate. I. Ultrastructure

Khawkinea quartana, a naturally occurring colorless homologue of Euglena, was examined with the electron microscope. The organism is biflagellate though only one of the 2 flagella emerges from the anterior reservoir. The pellicular strips covering the body of the organism are supported by microtubules which are continuous in part with microtubules bordering the reservoir. Additional rows of microtubules are found associated with the kinetosomes. An eyespot is located in the wall of the reservoir and, adjacent to it, the contractile vacuole. The nucleus, mitochondria, and Golgi complexes are similar to those described in other euglenoid flagellates. The food reserve is paramylon. The study supports the phylogenetic origin of Khawkinea from pigmented Euglena through the loss of chloroplasts.     

Surface organization and composition of Euglena. II. Flagellar mastigonemes

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

Reconstruction of the flagellar apparatus in Ploeotia costata (Euglenozoa) and its relationship to other euglenoid flagellar apparatuses

The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate-ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule-reinforced pocket in P. costata. Comparisons with Ploeotia vilrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.     

TRACHELOMONAS HISPIDA VAR. CORONATA (EUGLENOPHYCEAE). I. ULTRASTRUCTURE OF CYTOSKELETAL AND FLAGELLAR SYSTEMS1,2

Trachelomonas hispida var. coronata Lemm. has a fibrous, mucilaginous, ovoid, mineralized envelope (lorica), the ornamentation and coloration of which are capricious in culture. Cells exhibit a radial distribution of most organelles: (i) A cortical endoplasmic reticulum, (ii) parietal chloroplasts, and (iii) a median vacuolar region surrounded by several Golgi bodies and diverse vesicles. Associated with the emergent flagellum is a “paraflagellar complex” that consists of dense globules, cross-striated ribbon-like structures, a paraflagellar body, and an array of parallel striated filaments. The stigma consists of a single layer of pigmented granules that partially surrounds the canal/reservoir transition zone where microtubular bands intersect. A microtubular cytoskeleton consists of pellicular microtubules, peri-canal microtubules, stigma-associated microtubules and para-reservoir microtubules. The thickenings on the posterior, concave margins of the pellicular strips suggest that this pellicle is of intermediate complexity between those of Euglena spirogyra (Ehrenb. and Trachelomonas volvocina (Ehrenb.).     

SPERMIOGENESIS IN THE PULMONATE SNAIL, EUHADRA HICKONIS IV. EFFECT OF X-RAYS ON THE SPERMIOGENESIS

When animals were irradiated early in spermiogenesis, i. e. , at the spherical nucleus stage (7), two or more flagella were observed to be formed in many cells at the subsequent stages. Other irradiation effects included the absence of cytoplasmic microtubules at an early stage followed by their excessive production later during formation of the flagellum, the abnormalities in structure of the mitochondrial sheath around the axoneme, and the extremely disordered arrangement of flagellar components. However, the basal body and axoneme showed a normal structure at all stages, and acrosome development appeared to be unaffected by the irradiation.     

Three-dimensional structure of the Trypanosome flagellum suggests that the paraflagellar rod functions as a biomechanical spring

Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57 nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.     

Flagellum retraction and axoneme depolymerisation during the transformation of flagellates to amoebae inPhysarum

Summary The transformation ofPhysarum polycephalum flagellates to myxamoebae is characterised by disappearance of the flagellum. This transition, from the flagellate to the myxamoeba was observed by phase contrast light microscopy and recorded by time lapse video photography to determine whether flagellates shed their flagella or they are absorbed within the cell. In addition, the kinetics of flagellum disappearance were also studied. Our observations indicate that the flagellum was absorbed within the cell; the process occurred within seconds. Flagellum resorbtion was preceded by typical morphological cell changes. The shape of the nucleus altered and its mobility within the cell decreased. It was not possible to observe the flagellum within the cell with phase contrast video recordings. Thin section electron microscopy was used to study this intracellular phenomenon. Several stages of flagellum dissolution could be identified within the cell. The two most important stages were: an axoneme surrounded by the flagellar membrane within a plasma membrane lined pocket or vacuole and the naked axoneme without its membrane, free within the cell cytoplasm. The existence of cytoplasmic microtubules prevented identification of any further dissolution stages of the flagellum. A group of microtubules adjacent to the flagellum but within the cytoplasm was observed in flagellates and also in those cells which possesed enveloped axonemes. The flagellum did not dissociate from the kinetosomes before resorbtion.Immunofluorescence studies with the 6-11-B-1 monoclonal antibody indicated that acetylated microtubules exist in myxamoebae after transformation from flagellates for up to 40 min. Acetylated tubulin is not limited to the centrioles in these cells.