Ultrastructure of the Parabasalid Protist Holomastigotoides

ABSTRACT. The ultrastructure of two species of Holomastigotoides is presented. The basic unit of organization of these large cells is the flagellar band. Each flagellar band consists of a row of flagellar basal bodies linked by three fiber systems. The number of flagellar bands is species dependent. The flagellar bands originate at the cell apex and are arranged in parallel spirals of increasing gyre, thus defining the conical shape of the cell. In the cell apex a striated root called a parabasal fiber is juxtaposed with the basal bodies of each flagellar band. Linear extensions of two parabasal fibers function as the spindle poles for the persistent extra-nuclear spindle. The nucleus is in close contact with the spindle poles and spindle microtubules. Parallel sheets of microtubules which constitute axostyles are nucleated along the underside of the parabasal fibers. The axostyles extend away from the cell apex, with many reaching the basal region of the cell. Some of the axostyles follow the spiral pattern of the flagellar bands. Numerous Golgi bodies are spaced regularly along the flagellar bands. Together the parabasal fiber, axostyles and Golgi bodies associated with a flagellar band are termed a parabasal complex.     

Microtubules and the flagellar apparatus in zoospores and cysts of the fungusPhytophthora cinnamomi

Summary A correlated immunofluorescence and ultrastructural study of the microtubular cytoskeleton has been made in zoospores and young cysts ofPhytophthora cinnamomi. Labelling of microtubules using antibodies directed towards tubulin has revealed new details of the arrangement of the flagellar rootlets in these cells, and of the variability that occurs from cell to cell. Most of the variation exists at the distal ends of the rootlets, and may be correlated with differences in cell shape in these regions. The rootlets have the same right and left configuration in all zoospores. The arrangement of the rootlet microtubules at the anterior end of the zoospores raises the possibility that the microtubules on the left hand side of the groove may not comprise an independent rootlet which arises at the basal bodies.The absolute configuration of the flagellar apparatus has been determined from ultrastructural observations of serial sections. In the vicinity of the basal bodies, there is little, if any, variation between individuals, and the structure of the flagellar apparatus is similar to that described for related species of fungi. Two ribbon-like coils surround the central pair of microtubules at the distal tip of the whiplash flagellum, and clusters of intramembranous particles, similar to ciliary plaques, have been found at the bases of both flagella. There are two arrays of microtubules associated with the nucleus in the zoospores. One array lies next to the outer surface of the nuclear envelope, and probably functions in the shaping and positioning of the apex of the nucleus. The nuclear pores in this region are aligned in rows alongside these microtubules. The second array is formed by kinetochore microtubules which extend into a collar-like arrangement of chromatin material around the narrow end of the (interphase) nucleus. During encystment, all flagellar rootlets are internalized when the flagella are detached at the terminal plate. The rootlets arrays are no longer recognizable 5–10 minutes after the commencement of encystment.     

The absolute configuration of the flagellar apparatus in zoospores from two species ofLaminariales (Phaeophyceae

Summary We examined the zoospores produced by the unilocular sporangia ofLaminaria digitata (L.) Lamour. andNereocystis luetkeana Post. & Rupr. by serial sectioning to determine the absolute configuration of their flagellar apparatuses. The basal bodies, which are interconnected by three striated bands, lie parallel to the ventral face of the zoospore, and the posterior basal body always is found to the right of the anterior basal body when the cell is viewed from the ventral face, anterior end up. The four rootlets associated with the basal bodies include a major anterior rootlet of about seven microtubules extending from the anterior basal body along the ventral face towards the apex, a five-membered bypassing rootlet that passes ventral to the basal bodies and is connected to the posterior basal body by a posterior fibrous band, and two short rootlets having a single member each, the minor anterior and posterior rootlets. We consider the configuration observed here to be typical of most phaeophycean motile cells. The flagellar apparatus features suggest a considerable phylogenetic difference between thePhaeophyceae and other classes of chlorophyll c-containing organisms.     

Reconciling the bizarre inheritance of microtubules in complex (euglenid) microeukaryotes

We introduce a hypothetical model that explains how surface microtubules in euglenids are generated, integrated and inherited with the flagellar apparatus from generation to generation. The Euglenida is a very diverse group of single-celled eukaryotes unified by a complex cell surface called the "pellicle", consisting of proteinaceous strips that run along the longitudinal axis of the cell and articulate with one another along their lateral margins. The strips are positioned beneath the plasma membrane and are reinforced with subtending microtubules. Euglenids reproduce asexually, and the two daughter cells inherit pellicle strips and associate microtubules from the parent cell in a semi-conservative pattern. In preparation for cell division, nascent pellicle strips develop from the anterior end of the cell and elongate toward the posterior end between two parent (mature) strips, so that the total number of pellicle strips and underlying microtubules is doubled in the predivisional cell. Each daughter cell inherits an alternating pattern of strips consisting of half of the nascent strips and half of the parent (mature) strips. This observation combined with the fact that the microtubules underlying the strips are linked to the flagellar apparatus created a cytoskeletal riddle: how do microtubules associated with an alternating pattern of nascent strips and mature strips maintain their physical relationship to the flagellar apparatus when the parent cell divides? The model of microtubular inheritance articulated here incorporates known patterns of cytoskeletal semi-conservatism and two new inferences: (1) a multigenerational "pellicle microtubule organizing center" (pMTOC) extends from the dorsal root of the flagellar apparatus, encircles the flagellar pocket, and underpins the microtubules of the pellicle; and (2) prior to cytokinesis, nascent pellicle microtubules fall within one of two "left/right" constellations that are linked to one of the two new dorsal basal bodies.     

The cytology and ultrastructure of zoospores of Hydrurus foetidus (Chrysophyceae)

The unusual tetrahedral shape of Hydrurus foetidus (Vill.) Trev. zoospores is associated with a complex skeletal system of microtubules extending from a broad flagellar root (up to 19 microtubules) into each of three, pointed anterior processes. The posterior end, also pointed and supported by a separate set of microtubules, contains a single large chloroplast with a prominent posterior furrow containing mitochondrial elements. A large immersed pyrenoid is penetrated by paired thylakoids. There is no eyespot. Numerous large Golgi bodies occur immediately anterior to the nucleus and up to 5–6 contractile vacuoles lie near the cell surface at the anterior end. Two terminally inserted flagella extend from the cell surface, a long one serving for cell locomotion, and the other vestigial with an axonemal pattern of 9+0. The flagellar root system consists of: (1) a thin, striated rhizoplast extending from the basal body of the long flagellum and ramifying over the surface of a conspicuous, anteriorly directed, conical projection of the nucleus; (2) a broad microtubular root which emanates from near the basal body of the long flagellum and appears to function as a MTOC; (3) a compound root, consisting of a striated fiber and two associated microtubules, which runs alongside the basal body of the stubby flagellum before terminating at the cell surface; and (4) a short two-membered microtubular root, also associated with the basal body of the stubby flagellum. Other components of the flagellar apparatus include a large dense body near the proximal end of the basal body of the short flagellum, and a small, dense, core-like structure closely associated with one of its triplet fibers. The flagellar apparatus of H. foetidus is remarkably similar in ultrastructure to that of Chrysonebula holmesii Lund.     

OBSERVATIONS ON SPERMIOGENESIS IN THE FUNGUS GNAT SCIARA COPROPHILA

Although 9-membered centrioles are found in somatic tissues of Sciara, the centriole which lies at the spindle pole of the second meiotic division in male Sciara is composed of a row of approximately 70 short tubules in an oval array. Shortly after telophase of this unequal division, in the daughter cell destined to undergo spermiogenesis, microtubules become confluent with the tubules of the centriole. These tubules have the same density as other cytoplasmic microtubules after glutaraldehyde-OsO₄ fixation and, like them, are not preserved with OsO₄ fixation. As the centriole, now with approximately 70 attached, posteriorly directed, doublet tubules, migrates from the polar to the apolar end of the nucleus to take a final position in an oval groove which forms in the nuclear envelope, the tubules lengthen and become demonstrable after OsO₄ fixation and more electron opaque than other cytoplasmic microtubules following glutaraldehyde-OsO₄ fixation. Later, a singlet tubule appears peripherally to each doublet of the oval and 4 "arms" develop at specific sites on the tubules. Posteriorly, where the oval of tubules becomes discontinuous and forms a spiral, the arrangement of arms is different and the singlet tubules are lacking. Dense solid bodies develop inside this odd flagellum and become enclosed by a smooth double membrane. A single mitochondrial derivative has three components: a central area of homogeneous, moderately electron-opaque, proteinaceous material; a peripheral ring of cristae; and a crystalloid which is specifically oriented with respect to the flagellar tubules.     

FLAGELLAR MOTION AND FINE STRUCTURE OF THE FLAGELLAR APPARATUS IN CHLAMYDOMONAS

The biflagellate alga Chlamydomonas reinhardi was studied with the light and electron microscopes to determine the behavior of flagella in the living cell and the structure of the basal apparatus of the flagella. During normal forward swimming the flagella beat synchronously in the same plane, as in the human swimmer's breast stroke. The form of beat is like that of cilia. Occasionally cells swim backward with the flagella undulating and trailing the cell. Thus the same flagellar apparatus produces two types of motion. The central pair of fibers of both flagella appear to lie in the same plane, which coincides with the plane of beat. The two basal bodies lie in a V configuration and are joined at the top by a striated fiber and at the bottom by two smaller fibers. From the area between the basal bodies four bands of microtubules, each containing four tubules, radiate in an X-shaped pattern, diverge, and pass under the cell membrane. Details of the complex arrangement of tubules near the basal bodies are described. It seems probable that the connecting fibers and the microtubules play structural roles and thereby maintain the alignment of the flagellar apparatus. The relation of striated fibers and microtubules to cilia and flagella is reviewed, particularly in phytoflagellates and protozoa. Structures observed in the transitional region between the basal body and flagellar shaft are described and their occurrence is reviewed. Details of structure of the flagellar shaft and flagellar tip are described, and the latter is reviewed in detail.     

THE FLAGELLAR APPARATUS OF CHRYSOLEPIDOMONAS DENDROLEPIDOTA (CHRYSOPHYCEAE), A SINGLE-CELLED MONAD COVED WITH ORGANIC SCALES1

The flagellar apparatus of Chrysolepidomonas dedrolepidota Peters et Andersen is similar to that of other members of the Ochromonadales, Chrysophyceae. there are four microtubular roots (R_1-4) and a system II fiber (= rhizoplast). the R₁ root consists of three microtubules that nucleate many cytoplasmic microtubules. One compressed band of 10 or more cytoplasmic microtubules is directed black along the R1 root in an anti-parallel direction. The R₂ root consists of one to two microtubules, and it extends toward the distal end of the R₁ root. The R₃ root consists of six (?seven) microtubules near its proximal end. The “a” and “f” microtubules of the R₃ root are under the short flagellum, and the “f” microtubule loops back and under the basal body, extending down to the nucleus. The R₄ root consists of one to two microtubules extending along the left side of the shot flagellum and curving under the short flagellum where it terminates near the “a” microtubule of R₃ Both flagella have a transitional plate and a transitional helix with five gyres. There is a thin, second plate in the basal body at the level of the distal end of the “c” tubules of the basal body triplets. The tripartite flagellar hairs have long lateral filaments but lack short lateral filaments. We compare the flagellar apparatus with that of other members of the Ochromonadales and members of the Hydrurales and Hibberdiales.     

The flagellar apparatus ofDunaliella: Isolation of basal body-flagellar root complex

Summary InDunaliella bioculata, a biflagellate wall-less unicellular alga, the cytoskeleton is organized around the two basal bodies. Each basal body is associated with two dissymetric flagellar roots and numerous micro tubules which constitue a regular frame around the cell. We isolated the basal body-flagellar-root apparatus and studied its ultrastructure after negative staining. The two different flagellar roots are formed of microtubules and bundles of twisted filaments 3,5–4 nm in diameter. The proximal end of each root fans out and envelopes the basal body. We have shown preliminary results on the protein composition of basal body-flagellar roots fraction.     

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.     

COMPARATIVE ANALYSES OF THE DINOFLAGELLATE FLAGELLAR APPARATUS. III. FREEZE SUBSTITUTION OF AMPHIDINIUM RHYNCHOCEPHALUM1

The flagellar apparatus of the marine dinoflagellate Amphidinium rhynchocephalum Anissimowa was examined using the techniques of rapid freezing/freeze substitution and serial thin section three dimensional reconstruction. The flagellar apparatus is composed of two basal bodies that are offset from one another and lie at an angle of approximately 150° The transverse basal body is associated with two individual microtubules that extend from the proximal end of the basal body toward the flagellar opening. One of these microtubules is closely appressed to a striated fibrous root that also extends from the proximal base of the transverse basal body. The longitudinal basal body is associated with a nine member microtubular root that extends from the proximal end of the basal body toward the posterior of the cell. The longitudinal microtubular root and the transverse striated fiber are connected by a striated connective fiber. In addition to the microtubules associated with the transverse and longitudinal basal bodies, a group of microtubules originates adjacent to one of the transverse flagellar roots and extends into the cytoplasm. Vesicular channels extend from the flagellar openings to the region of the basal bodies where they expand to encompass the various connective structures of the flagellar apparatus. The possible function and evolutionary importance of these structures is discussed.     

Cell shape and microtubules in zoospores of the green algaChlorosarcinopsis gelatinosa (Chlorosarcinales): Effects of low temperature

Summary The effect of low temperature (2 °C) on cell shape and microtubules in zoospores of the green algaChlorosarcinopsis gelatinosa has been investigated. The zoospores are 4–6 times longer than wide with a mean length of 12,5 m and can be kept in the dark for several hours without changes in cell shape. Cell shape changes have been evaluated quantitatively by measuring changes in cell length. Low temperature induces a decrease in cell length which exhibits a two-step kinetic: during the first 30 minutes a rapid rate of decrease in cell length was measured, while during the next 4 hours a slow rate of decrease in cell length was observed. Complete regeneration of zoospore length occurs when cold-treated cells are subjected to the original zoospore induction temperature (30 °C) for two hours. Observation of numbers, disposition and types of microtubules in the zoospore during decrease in cell length has shown that within 30 minutes after cold application the secondary cytoskeletal microtubules (scmt) disappear, while flagellar root microtubules are unaffected. During this period most cells develop a prominent posterior appendage (tail). Sections demonstrate the presence of several microtubules in these tails. Flagellar root microtubules probably extend into the tails and disappearance of scmt starts at the posterior pole of the cell. Regeneration of zoospores to original cell length is coupled with reappearance of scmt starting at the anterior pole of the cell. It is concluded that secondary cytoskeletal microtubules constitute the main cytoskeleton inChlorosarcinopsis zoospores and that flagellar root microtubules contribute to only a minor extent to the cytoskeleton, because they cannot retain the cell shape. The results are discussed with respect to the functional significance of flagellar root microtubules in green algae.     

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana-telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.     

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS : II. The Role of Nucleating Sites in Shape Development

The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.     

Morphology of Mastigamoeba aspera Schulze, 1875 (Archamoebae, Pelobiontida)

The morphology of Mastigamoeba aspera, a type species of the genus Mastigamoeba Schulze, 1875, has been investigated at the light- and electron-microscopical level. Motile individuals are oval or peach-shaped. Motile flagella is situated at the anterior end of uninucleate cells. During locomotion, the surface of mastigamoebes forms many conical or finger-shaped hyaline pseudopodia, wereas bulbous uroid is often formed at the posterior end of the cell. Micropopulations of M. aspera consist of uninucleate flagellate forms as well as multinucleate aflagellate ones. There is a thick layer ofglycocalix on the cell surface where many rod-shaped bacterial ectobionts live. The nucleus is vesicular with spherical central nucleolus. The flagellar apparatus of M. aspera is connected with nucleus to form so called kariomastigont. A single kinetosome is associated with many radial microtubules and a lateral root. A distinct microtubule organization centre (MTOC) is situated at the basal part of the kinetosome. Microtubules of the nuclear cone are connected with the MTOC. This microtubules take part in the formation of kariomastigont. The axoneme has a standart set of microtubules 9(2)+2. Digestive vacuoles are the main component of the cytoplasm of M. aspera. Beside, many light-difracted granules and glycogen bodies were found in the cells. Mitochondria, dictyosomes of the Golgi apparatus and microbodies were not revealed in the cytoplasm of M. aspera.     

Ultrastructure of the flagellar apparatus inPleurochrysis (classPrymnesiophyceae)

Summary The ultrastructure of the flagellar apparatus of aPleurochrysis, a coccolithophorid was studied in detail. Three major fibrous connecting bands and several accessory fibrous bands link the basal bodies, haptonema and microtubular flagellar roots. The asymmetrical flagellar root system is composed of three different microtubular roots (referred to here as roots 1,2, and 3) and a fibrous root. Root 1, associated with one of the basal bodies, is of the compound type, constructed of two sets of microtubules,viz. a broad sheet consisting of up to twenty closely aligned microtubules, and a secondary bundle made up of 100–200 microtubules which arises at right angles to the former. A thin electron-dense plate occurs on the surface of the microtubular sheet opposite the secondary bundle. The fibrous root arises from the same basal body and passes along the plasmalemma together with the microtubular sheet of root 1. Root 2 is also of the compound type and arises from one of the major connecting bands (called a distal band) as a four-stranded microtubular root and extends in the opposite direction to the haptonema. From this stranded root a secondary bundle of microtubules arises at approximately right angle. Root 3 is a more simple type, composed of at least six microtubules which are associated with the basal body. The flagellar transition region was found to be unusual for the classPrymnesiophyceae. The phylogenetic significance of the flagellar apparatus in thePrymnesiophyceae is discussed.     

Ultrastructure of the Flagellar Apparatus and Attachment of Herpetomonas ampelophilae in the Gut and Malpighian Tubules of Drosophila melanogaster1

Electron microscopy was used to examine the flagellar apparatus of Herpetomonas ampelophilae from the gut and malpighian tubules of Drosophila melanogaster. The flagellates attach to the microvilli either by weaving their flagella between the microvilli or by engulfing several microvilli with an external flagellar membrane. The first type predominated in the gut while the second type was limited to the malpighian tubules. Desmosomes were not involved in either type of attachment. A subpellicular collar with emerging microtubules was found to be adjacent to the desmosome of the flagellar pocket of herpetomonads in the gut.     

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.     

Form and Function of the Dinoflagellate Transverse Flagellum1

A 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.     

The Differentiation of Herpetomonas megaseliae: Ultrastructural Observations*

Ultrastructure of both undifferentiated (promastigote and paramastigote) and differentiated (opisthomastigote) forms of Herpetomonas megaseliae is described. There is a posterior migration of the kinetoplast at the end of the exponential growth phase. The posterior extension of the flagellar pocket precedes migration of the kinetoplast. Opisthomastigotes have an electron-translucent mitochondrial matrix in comparison with undifferentiated forms. The Golgi body changes from a stack of flattened sacs to an aggregation of vesicles. Several structures previously reported from Trypanosomatidae, e.g. subpellicular organelles, pellicular microtubules, membrane whorls, stored metabolic products, surface blebs, and an intraflagellar body are also present in H. megaseliae.