Desmosomes and hemidesmosomes in the flagellate Crithidia fasciculata

Summary The flagellum of the trypanosomatid flagellate Crithidia fasciculata expands asymmetrically as it emerges from the reservoir. Where the flagellar memhrane approaches the membrane lining the reservoir, desmosomes are found. These structures are arranged in several slightly curved lines and have many features in common with vertebrate desmosomes.In cultures, the flagellates stick to each other by their flagella and form rosettes. In these bundles of cells, probable sites of adhesion between flagella, or between flagella and pieces of debris, are marked by a dense filamentous tract which passes posteriorly along the flagellum and by a thick band lying just below the flagellar membrane. It is suggested that similar adhesions are found in the insect host where the flagellate attaches itself to the gut wall.     

ULTRASTRUCTURAL OBSERVATIONS OF A COLACIUM PALMELLA: THE RESERVOIR,EYESPOT, AND FLAGELLA1

A recently described euglenoid of the genus Colacium inhabits the rectums of damselfly larvae during the winter as a nonflagellated, stalkless palmella stage. Contrary to previous reports, which were based on light microscope observations, ultrastructural observations establish that the reservoir and canal with its nonemergent flagella remain structurally intact in the palmella condition. The 2 non-emergent flagella are structurally distinct. The larger flagellum, which probably gives rise to the single, emergent flagellant of the swimming euglenoid, has the typical 9 + 2 microtubular arrangement, together with a paraflagellar rod throughout most of its length. The crystalline paraflagellar body, typical of swimming euglenoids, is absent. The smaller flagellum has a 9 + 0 arrangement of the axonemal microtubules. This flagellum appears to terminate within the reservoir with a successive loss of number and arrangement of microtubules near its distal end. Hair-like structures lining the reservoir membrane may represent preformed nontubular mastigonemes. The eyespot granules are clustered around the cytostome and do not display the compact organization or position typical of the flagellated stage or the stalked stage of Colacium.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

The flagellar developmental cycle in algae: flagellar transformation inCyanophora paradoxa (Glaucocystophyceae)

Summary Flagellar development during cell division was studied inCyanophora paradoxa using agarose-embedded cells, Nomarski optics and electronic flash photography. The cells bear two heterodynamic and differently oriented (anterior and posterior) flagella. Prior to cell division, cells produce two new anterior flagella while the parental anterior flagellum transforms into a posterior flagellum. The parental posterior flagellum remains a posterior flagellum throughout this and subsequent cell divisions. The development of a single flagellum thus extends through at least two cell cycles and flagellar heterogeneity is achieved by semiconservative distribution of the flagella during cell division. Based on these principles a universal numbering system for basal bodies and flagella of eukaryotic cells is proposed.     

Effect of divalent cations on flagellar scales in the green flagellateTetraselmis cordiformis

Summary The structure and topography of flagellar scales (underlayer scales, rodshaped scales, hair-scales) in the green flagellateTetraselmis cordiformis has been studied in detail and the effect of divalent cations and fixation conditions on scale structure and topography was followed quantitatively. Hair-scales occur in two rows on opposite sides of a flagellum and are linked to the flagellar membrane and to two axonemal doublets by B-tubule-flagellar membrane connectives. Underlayer scales form about 24 longitudinal rows along the flagellum and occur in two distinctive shapes (pentagonal and square). The square shaped underlayer scales are related in position to the attachment sites of the hair-scales. Rod-shaped scales occur in about 20 longitudinal rows along the flagellum and are characteristically positioned as double scales. Calcium in the culture medium is necessary to retain rod-shaped scales on the flagellum, absence of calcium or chelation with EGTA or pyrophosphate leads to disappearance of rod-shaped scales from the flagellum. Other divalent cations can only partially substitute for calcium. It is suggested that calcium provides the linkage between underlayer scales and rod-shaped scales inTetraselmis. Flagellar scales inTetraselmis apparently fall into two categories: a) hair-scales (not affected by fixation or absence of divalent cations, firmly bound to axonemal microtubules via the flagellar membrane), b) underlayer scales and rod-shaped scales (affected by fixation and absence of divalent cations, kept on the flagellum mainly by electrostatic forces). The function of flagellar scales inTetraselmis is discussed.     

Flagellar transformation in the heterokontEpipyxis pulchra (Chrysophyceae): Direct observations using image enhanced light microscopy

Summary Cells ofEpipyxis pulchra possess two heteromorphic flagella that differ markedly in function, particularly during motility and prey capture. Flagellar heterogeneity is achieved during the course of at least three cell cycles. Prior to cell division, cells produce two new long, hairy flagella while the parental long flagellum is transformed into a new short, smooth flagellum. The parental short flagellum remains a short flagellum for this and subsequent cell division cycles. Although flagellar transformation requires only two cell cycles, developmental differences exist between daughter cells and the maturation of a flagellum/basal body requires at least three cycles.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

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.     

Turnover and transport of agglutinins in conjugatingChlamydomonas gametes

Summary During gamete-gamete adhesion in the unicellular green algaChlamydomonas eugametos, the sexual adhesion molecules or agglutinins that are located on the flagella are subject to tip-oriented migration and rapid inactivation. It is demonstrated that sexual adhesiveness is maintained by incorporation of additional agglutinins, recruited from a cellular pool. The location of this reservoir is unknown but, as indicated by its insensitivity to the chaotropic agent guanidine thiocyanate, it appears to be distinct from the large amount of agglutinins on the plasma membrane of the cell body. By viewing flagella of conjugating gametes in a confocal scanning laser microscope after immuno-labelling of the agglutinins, evidence was obtained for a linear arrangement of the agglutinins in two rows on the flagellar surface. This suggests that after insertion at the base of the flagellum, the agglutinins follow linear tracks to the tip and that the transport system is confined to two longitudinal domains. It is estimated that the half-life of flagellar agglutinins drops from 1–2 h in nonconjugating gametes to 1 min during conjugation, which suggests that after incorporation at the flagellar base, the agglutinins migrate to the tip with a velocity of 100 nm/s. Presumably after arrival at the tip, the molecules are inactivated. It is postulated that rapid turnover and transport of agglutinins are required for optimal signalling between partner gametes.Abbreviations BSA bovine serum albumine - CHI cycloheximide - CSLM confocal scanning laser microscope - GA glutaraldehyde - GTC guanidine thiocyanate - GAM-IgG goat-anti-mouse immuno-globuline - mAb monoclonal antibody - mt mating type - PBS phosphate-buffered saline - SDS sodiumdodecyl sulphate - TRIS tris-(hydroxymethyl)-aminomethane     

Twist (and rotation?) of central-pair microtubules in flagella ofPoterioochromonas

Summary The chrysoflagellate algaPoterioochromonas bears two unequal flagella. There is a short naked one and a long flagellum with mastigonemes. Ultrastructural investigation reveals that the centralpair microtubules in both flagella have no fixed position with respect to the flagellar base and root system, or the mastigoneme rows in the long flagellum. The central-pair microtubules are twisted several times along the length of the flagellum. This might indicate active or passive rotation of the central-pair microtubules during flagellar beat.     

Localization of calmodulin in flagella of zoospores ofPhytophthora cinnamomi

Summary Calmodulin distribution in the tinsel and whiplash flagella of zoospores ofPhytophthora cinnamomi has been studied by immunofluorescence microscopy and immunogold labelling. In whole zoospores labelled with a monoclonal antibody raised against pea calmodulin, followed by a second antibody-FITC, both flagella appear to be weakly stained except for a region at the base of the tinsel flagellum which was stained intensely. A similar staining pattern was also detected in isolated flagella labelled with anti-calmodulin. To identify the calmodulin rich region of the tinsel flagellum, we labelled sections of zoospores embedded in Lowicryl K4M with anti-calmodulin followed by a second antibody gold probe. In the tinsel flagellum, the gold labelling was restricted to a paraxonemal swelling close to the base. Very little gold labelling was detected elsewhere. The swelling extends for 1.5–2.0 n from the base of the tinsel flagellum and is hook shaped in cross section. Immunoblot analysis confirmed that the staining was specific for calmodulin.     

Flagella-dependent gliding motility inChlamydomonas

Summary Flagella are generally recognized as organelles of motility responsible for the ability ofChlamydomonas to swim through its environment. However, the same flagella are also responsible for an alternative form of whole cell locomotion, termed gliding. Use of paralyzed flagella mutants demonstrates that gliding is independent of axonemal bend propagation. Gliding motility results from an interaction of the flagellar surface with a solid substrate. Gliding is characterized by bidirectional movements at 1.6±0.3 m/second and occurs when the cell is in a characteristic gliding configuration, where the two flagella are oriented at 180° to one another. A variety of observations suggest that the leading flagellum is responsible for the force transduction resulting in cell locomotion, although both flagella have the capacity to function as the active flagellum. The characteristics of gliding motility have been compared with theChlamydomonas flagellar surface motility phenomenon defined as surface translocation of polystyrene microspheres.     

Unusual distribution of a glycolipid antigen in the flagella ofChlamydomonas

Summary Mouse hybridomas were obtained that secrete monoclonal antibodies recognizing glycolipid antigens located in the flagellar membrane of the biflagellate alga,Chlamydomonas reinhardtii. The antigen is an acidic lipid that migrates slightly slower than a GM1 ganglioside on thin layer chromotography. The binding of the antibodies to the thin layer plate was inhibited by periodate oxidation suggesting that the antibodies are recognizing a carbohydrate epitope. In a variety ofChlamydomonas strains, these antibodies were found to stain the flagella of only a sub-set of the cells in the population, generally varying from 50% to 75% of the cells. Even after cloning, the population of cells continued to express this variability in staining, and presumably, expression of the glycolipid epitope. Although most cells showed either strong staining of both flagella or no detectable staining of both flagella, a subset of the cells in the culture exhibited differential antibody labeling of the two flagella, suggesting that an individualChlamydomonas can exhibit a different glycolipid composition in each of its two flagellar membranes and even differential expression along the length of an individual flagellum.     

Observations on the microanatomy of the type species of Pyramimonas (P. tetrarhynchus Schmarda)

Salient features of microanatomy of Pyramimonas tetrarhynchus , type species of the genus, are described and illustrated. The species is shown to possess a flagellar scale reservoir comparable to that previously described for a marine species (P. amylifera) though somewhat simpler. The body scales are shown to arise directly from Golgi eisternae as in P. amylifera. Other features discussed include the flagellar roots and certain aspects of the mode of liberation of scales to the exterior. Participation by endoplasmic reticulum in the latter process is shown to be probable but there is no precise explanation forthcoming for the elaborate scale arrangement encountered both on the flagella and on the cell. Participation by cytoplasmic tubules is nevertheless effectively excluded for the latter.     

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or , -dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.Abbreviations and symbols CHI cycloheximide - GTC guaniline thiocyanate - mt ⁺/mt ^- mating type plus or minus - PAS Periodic-acid-Schiff reagent - SDS sodium dodecyl sulphate     

ULTRASTRUCTURAL VARIATIONS IN CRYPTOMONAD FLAGELLA1

The arrangement of flagellar appendages in 19 cryptomonad species was examined and four new flagellar types are described. The first new type has a single row of mastigonemes on both flagella and hairs on the side opposite the mastigonemes. The second type, which is common, has unilateral rows of mastigonemes on both flagella, but no hairs. A third type has an acronematic short flagellum and a single row of mastigonemes on the long flagellum. A fourth type lacks mastigonemes but has a unilateral row of curved “spikes” on the short flagellum and hairs on both flagella. These additional flagellar variations may contribute to a more natural system of classification for cryptomonads.     

FLAGELLAR DEVELOPMENT AND REGENERATION IN VOLVOX CARTERI (CHLOROPHYTA)1

The biflagellate somatic cells of Volvox carteri f. nagariensis lyengar exhibit an asymmetric pattern of flagellar development. Initiallt each somatic cell has two short (4 μm) flagella but after several hours one flagellum on each cell elongates unitl it reaches a length of 12 μm. Due to the regular arrangement of somatic cells in the Volvox spheroid it is apparent that the same flagellum on each somatic is the first to elongale. The asymmetric flagellar length is maintained for about 8 h after which the second flagellum on each somatic cell elongates. When the second flagellum attains the same length (12 μm) as the first flagellum, both flagella elongale at the same rate until reaching a final length of 22 μm. Experimental removal of somatic cell flagella results in their regeneration. Somatis cells regenerate both flagella simultaneously and full length flagella are produced in about 2 h. The intial rate of flagellar regeneration is about ten times faster than the intial rate of flagllar growth in development. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial rate of flagellar regeneration but the flagella produced in the presence of the drug are half the length of flagella produced in its absence. Somatic cells are able to regenerate flagella up to the time of α and β tubulin, the major structural proteins of the flagellar axoneme, and other cellular proteins.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.     

Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoeba,Metamonada and Parabasala

Summary The hypothesis that protists without mitochondria, the so-called Archezoa of Cavalier-Smith, are primitive has received some support from rRNA sequence studies on Microsporidia and Diplomonadida. In spite of the lack of mitochondria the archezoan groups of protists show considerable differences in their organization: mastigont and cytoskeletal system, mitosis, Golgi apparatus, hydrogenosomes. This paper examines the characters of the flagellar apparatus and its associated cytoskeleton to obtain clues used for phylogenetic consideration on the three cited groups of flagellates. Archamoebae of the Pelobiontida order comprising families such as Pelomyxidae and Mastigamoebidae share common features: a rudimentary mastigont system composed of only one basal body giving rise to a poorly motile flagellum and a basal body associated microtubular cone capping the nucleus. No Golgi apparatus has been detected.Metamonada, comprising three orders: Retortamonadida, Diplomonadida, and Oxymonadida, have been tentatively assembled on the basis of the absence of mitochondria, Golgi apparatus, and basal body arrangement. They all have four basal bodies arranged in two pairs with always one recurrent flagellum generally included in a cytostomal depression. The recurrent basal body/flagellum is in relation to recurrent microtubular fibers. However, they display marked differences in their cytoskeletal system and fiber ultrastructure indicating a distant evolutionary relationship. The presence of a corset of microtubules in retortamonads and three microtubular fibers are distinguished in diplomonads, as well as a paracrystalline preaxostyle and axostyle in oxymonads are features that lend support to these groups being highly divergent.Parabasala, comprising the orders Trichomonadida and Hypermastigida, is a monophyletic group with a set of homologous features such as the presence of the same arrangement of four basic basal bodies, the parabasal apparatus (striated fibre supporting Golgi), the microtubular pelta-axostyle complex, the external mitotic apparatus (crypto-pleuro-mitosis), the hydrogenosomes. These three phyla appear distantly related, the Parabasala being a homogeneous group, perhaps also the Pelobiontida, while the Metamonada is heterogeneous and composed of three evolutionary lineages. Additional information such as rRNA and protein sequence data could contribute to a better understanding of the phylogenetic relationships among these groups.Abbreviations EM electron microscopy - MTOC microtubule organizing centre - PF parabasal fibre     

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.