Light and Electron Microscopic Observations on the Intraerythrocytic Development of Babesiosoma stableri (Apicomplexa,Dactylosomatidae) in Frogs from Algonquin Park,Ontario1

ABSTRACT. The intraerythrocytic development and ultrastructure of Babesiosoma stableri Schmittner & McGhee, 1961 are described from Rana catesbeiana and Rana septentrionalis from Algonquin Park, Ontario. Morphometric and chronological observations on B. stableri in an experimentally infected Rana pipiens support the hypothesis that two successive types of merogonic cycles occur within the erythrocytes of infected frogs; the first cycle gives rise to the second and the second cycle produces merozoites destined to become gamonts. Merozoites, meronts, and gamonts are described by light and electron microscopy. Merozoites are typically coccidian and have a trilaminate pellicle with micropores, approximately 40 sub-pellicular microtubules, an apical and posterior polar ring, a conoid with two accessory rings and a pair of intra-conoidal microtubules, three rhoptries and numerous micronemes, and a nucleus with a nucleolus and a paranuclear Golgi body. The gamonts are larger than merogonic stages and are isogamous. They have approximately 55 sub-pellicular microtubules and large stores of amylopectin. These observations indicate that the genus Babesiosoma should be transferred from the Family Haemohormidiidae (Piroplasmida, Piroplasmia) to the Family Dactylosomatidae (Eucoccidiida, Coccidia).     

A subpopulation of trypanosome microtubules recognized by a monoclonal antibody to tubulin

Two hybridoma cell lines were selected after the fusion of the myeloma cell line X-63 Ag8-653 with spleen cells from mice immunized with bovine brain microtubules. These lines, clones 3F3 and 16D3, secrete IgM antibodies both staining a fibrillar network in fibroblasts. Autoradiography of immunoblots of SDS gels showed that the antigenic determinants defined by these antibodies are present on tubulin and also on several other polypeptides in mammalian cells. In contrast, they were found to react only with tubulin in Trypanosoma brucei, parasitic protozoan which are the causative agent of sleeping sickness. By immunofluorescence microscopy, 3F3 bound only to a subpopulation of microtubules associated with the flagellum of these cells when, under the same conditions, 16D3 stained other microtubule populations including sub-pellicular microtubules. These results show that flagellar tubulin differs from tubulin of other locations in the same cell by at least one antigenic determinant which could be involved in microtubule specialization.     

From the cell biology to the development of new chemotherapeutic approaches against trypanosomatids: dreams and reality

Members of the Trypanosomatidae family comprise a large number of species that are causative agents of important diseases such as sleeping sickness, Chagas' disease and Leishmaniasis. These organisms are also of biological interest since they are able to change the morphology according to the environment where they live, through a process of reversible cell transformation, and possess structures and organelles that are not found in mammalian cells. This review analyses the process of transformation, which takes place during the life cycle of Trypanosoma cruzi in the vertebrate and invertebrate hosts. Special attention is given to the interaction of the parasite with vertebrate cells. In addition, the present knowledge of structures and organelles such as the nucleus, the plasma membrane, the sub-pellicular microtubules, the flagellum, the kinetoplast-mitochondrion complex, the peroxisome (glycosome), the acidocalcisome and the structures and organelles involved in the endocytic pathway, is reviewed from a cell biology perspective. The possible use of available data for the development of new anti parasite drugs is also discussed.     

Ultrastructure of Pellicular and Ciliary Structures of Euplotes eurystomus

SYNOPSIS. Many of the sub-pellicular and infraciliary structures in protozoa have proved difficult to study with standard thin-sectioning technics. When these structures are viewed in isolated and fragmented form, many of the thin-sectioning difficulties are circumvented. Langmuir-trough isolation followed by critical-point drying, as well as thin sectioning, were used in this study to determine the patterns of sub-pellicular microtubules and fibrils interconnecting kinetosomes of membranelles and cirri of Euplotes eurystomus. The fibrillar network in the bases of these ciliary organelles is presented in some detail and apparent variations in pattern are noted. Functional aspects of some of the structures are discussed. With special preparation nearly whole Euplotes may be obtained for study in the electron microscope. Fused cilia were frequently obtained and their ultrastructure was studied.     

WCB is a C2 domain protein defining the plasma membrane - sub-pellicular microtubule corset of kinetoplastid parasites

WCB is a protein that locates between the inner face of the plasma membrane and the sub-pellicular corset of microtubules in Trypanosoma brucei. We provide the molecular identity of WCB and bioinformatic analysis suggests that it possesses a C2 domain implicated in membrane/protein interactions and a highly charged region possessing characteristics of a putative tubulin-binding domain. Functional analyses via RNA interference (RNAi) depletion show that WCB is essential for cell morphogenesis. Depletion results in gross abnormalities in cell shape, mainly at the cytoskeletal/plasma membrane dynamic posterior end of the trypanosome. Failures in cytokinesis and zoid production are also evident. Furthermore, electron microscopy reveals that RNAi-induced trypanosomes lose local plasma membrane to microtubule corset integrity.     

Distribution and Characteristics of βII Tubulin-Enriched Microtubules in Interphase Cells

We have used a polyclonal antibody (Ab196) that specifically recognizes the βII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual β isotypes. Using photoimaging analysis, we observed βII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of βII-enriched microtubules together with the reported increased levels of βII tubulin in taxol-resistant cells (M. Haberet al.,1995,J. Biol. Chem.270, 31269–31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, βII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-βII tubulin antibody but not by anti-acetylated α tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.     

Sperm Axoneme in Some Apterygote Insects Examined by Computer-aided Image Analysis

Sperm tail axonemes from three insect species, representing the three apterygote orders Diplura, Archeognatha and Zygentoma, have been examined by high resolution electron microscopy. The samples were fixed in a mixture of glutaraldehyde and tannic acid followed by uranyl acetate post-fixation and the electron micrographs were subjected to computer analysis in order better to characterize the minute details of the axonemal microtubules. The dipluran Campodea was seen to have a row of accessory microtubules composed of 13 protofilaments, the archeognath Machilinus to have two rows of accessory microtubules composed of 16 protofilaments. and the zygentoman Lepismodes to have accessory microtubules in contact with the flagellar doublets and with a wall containing 16 protofilaments and an electron-dense lumen. The axonemal structure in Zygentoma is the one most similar to those of the pterygote insects. The protofilaments of the axonemal doublets were seen to have a somewhat widened interspace between some pairs of protofilaments and these ‘gaps’ coincided with the location of the basal parts of the outer dynein arm, the inner dynein arm, the spoke, and the intertubular material. It hence seems that these microtubule-associated structures influence the architecture of the microtubular wall.     

Endosymbiosis in protozoa of the Trypanosomatidae family

A small number of trypanosomatids present bacterium endosymbionts in the cytoplasm, which divide synchronously with the host cell. Crithidia oncopleti, Crithidia deanei. Crithidia desouzai, Blastocrithidia culicis and Herpetomonas roitmani are the best characterized species. The endosymbiont is surrounded by two membranes separated from each other by an electron-lucent space. The presence of the endosymbiont led to the appearance of morphological changes which include the lack of the paraflagellar rod associated to the axoneme, the morphology of the kinetoplast and the association of the sub-pellicular microtubules with portions of the protozoan plasma membrane. Aposymbiotic strains could be obtained by antibiotic treatment, opening the possibility to make comparative analysis of endosymbiont-containing an endosymbiont-free populations of the same species. It is clear that metabolic cycles are established between the prokaryiont and the host cell. The results obtained show that endosymbiont-containing species of trypanosomatids constitute an excellent model to study basic processes on the endosymbiont-host cell relationship and the origin of new organelles.     

Cold stability of microtubules in wood-forming tissues of conifers during seasons of active and dormant cambium

The cold stability of microtubules during seasons of active and dormant cambium was analyzed in the conifers Abies firma, Abies sachalinensis and Larix leptolepis by immunofluorescence microscopy. Samples were fixed at room temperature and at a low temperature of 2–3°C to examine the effects of low temperature on the stability of microtubules. Microtubules were visible in cambium, xylem cells and phloem cells after fixation at room temperature during seasons of active and dormant cambium. By contrast, fixation at low temperature depolymerized microtubules in cambial cells, differentiating tracheids, differentiating xylem ray parenchyma and phloem ray parenchyma cells during the active season. However, similar fixation did not depolymerize microtubules during cambial dormancy in winter. Our results indicate that the stability of microtubules in cambial cells and cambial derivatives at low temperature differs between seasons of active and dormant cambium. Moreover, the change in the stability of microtubules that we observed at low temperature might be closely related to seasonal changes in the cold tolerance of conifers. In addition, low-temperature fixation depolymerized microtubules in cambial cells and differentiating cells that had thin primary cell walls, while such low-temperature fixation did not depolymerize microtubules in differentiating secondary xylem ray parenchyma cells and tracheids that had thick secondary cell walls. The stability of microtubules at low temperature appears to depend on the structure of the cell wall, namely, primary or secondary. Therefore, we propose that the secondary cell wall might be responsible for the cold stability of microtubules in differentiating secondary xylem cells of conifers.     

A Comparative electron microscopic study of cytoplasmic microtubules and axial unit tubules in a spermatozoon and a protozoan

Cytoplasmic microtubules and axial unit tubules were studied in both sectioned and negatively-stained material. Walls of microtubules of frog lung-fluke (Haematoloechus medioplexus) spermatozoa have a helical substructure, while those of the flagellate, Trypanosoma lewisi, are composed of ten longitudinally-oriented filaments. Cross-bridges occur between some filaments of trypanosome microtubules. Doublet tubules of axial units in both cell types are structurally similar to the trypanosome microtubules, which may indicate similarity of function. Microtubules of fluke spermatozoa appear to be somewhat rigid, are resistant to sonication, and are considered to be mainly supportive. Circular profiles of wall subunits are seen in transverse sections of microtubules of both cell types and in doublet tubules of the trypanosome. Comparisons are made between sectioned and negatively-stained material; while negative-staining better reveals the fundamental substructure of microtubular elements, some distortion appears to occur. In connection with this research, a brief preliminary article demonstrated the presence of subunits in the walls of cytoplasmic microtubules of fluke spermatozoa (Burton, '66). Also, it was shown that the wall of these tubular elements possesses a helical structure, and a diagrammatic representation of the wall structure was set forth.     

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.     

Structure and function of the microtubular cytoskeleton during pollen development in Gasteria verrucosa (Mill.) H. Duval

In a study of pollen development in Gasteria verrucosa, the changes in the spatial organization of microtubules were related to the processes of cell division, nuclear movement and cytomorphogenesis. Sections of polyethylene-glycol-embedded anthers of G. verrucosa were processed immunocytochemically to record the structure and succession of fluorescently labeled microtubular configurations. Using microspectrophotometric measurements the relative quantity of tubulin in microtubules per unit of cytoplasm was determined. Cell dimensions and nuclear positions were measured to relate changes in cell shape and nuclear movements to microtubular configurations. Microtubules were detected in the different cells during microsporogenesis and microgametogenesis. In microspore mother cells which are approximately isodiametric at interphase, microtubules were predominantly arranged in a criss-cross pattern. The microtubules probably function as a flexible cytoskeleton which sustains the integrity of the cytoplasm. Bundles of microtubules were observed in the microspores, in the generative cells and during nuclear division, where they functioned in establishing and maintaining cell and spindle shapes. Microtubules radiating from nuclear membranes appeared to fix the nucleus in position. In prophase of meiosis and after microspore mitosis, periods a high fluorescence intensity were distinguished indicating a variation in the quantity of microtubules.Abbreviation MT microtubule     

Microtubular cytoskeletons of the trophic cells of five eumycetozoans

Summary The trophic cells of the protostelids,Ceratiomyxella tahitiensis, Cavostelium apophysatum, Planoprotostelium aurantium, andClastostelium recurvatum, and the reduced myxomycete,Echinostelium bisporum, were examined with indirect immunofluorescence to determine the overall structure of the microtubular cytoskeletons of each type of cell. All five species have a distinct flagellar apparatus in the amoeboflagellate state, but they vary with respect to the presence of body microtubules, those microtubules which do not focus on the flagellar apparatus. The obligate amoebae ofC. tahitiensis, C. apophysatum andC. recurvatum all have extensive microtubular cytoskeletons, but each is unique to its respective species. The obligate amoeba ofC. tahitiensis has scattered microtubules which often describe curved paths. The microtubules never appear to focus on MTOCs. The microtubular cytoskeleton ofC. apophysatum consists of relatively straight, rigid appearing microtubules. Small subpopulations of these microtubules radiate from MTOCs near the nucleus. The obligate amoeba ofC. recurvatum has a cytoskeleton consisting of numerous microtubules which radiate from a perinuclear MTOC and fill the body of the cell. These results correlate well with earlier ultrastructural observations which suggest that the amoeboflagellate state is homologous in these mycetozoans and that the obligate amoebae, when present, are unique to the various lineages in which they appear.     

EFFECTS OF HIGH PRESSURE IN THE ARMORED DINOFLAGELLATE SCRIPPSIELLA HEXAPRAECINGULA (PERIDINIALES,DINOPHYCEAE): CHANGES IN THECAL PLATE PATTERN AND MICROTUBULE ASSEMBLY1

The possible role of cortical microtubules in dinoflagellates was studied using high‐pressure treatments applied to nonmotile cells (just after ecdysis) of Scrippsiella hexapraecingula T. Horig. et Chihara. Whereas considerable disorganization of cortical microtubules was observed when cells were exposed to high‐pressure treatments of 98 MPa or more for 5–15 min, they were mostly intact in cells exposed to a pressure of <98 MPa for 5 min. After nonmotile cells were exposed to high‐pressure treatments sufficient to disorganize the cortical microtubules, they produced new motile cells with thecal plate patterns that differed considerably from the pattern known for this species. Increasing the intensity of high pressure applied to nonmotile cells resulted in an increase in the number of cells that exhibited disorganized cortical microtubules as well as a change in their thecal plate pattern, suggesting that high pressure disorganizes cortical microtubules leading to a change in the thecal plate pattern.     

The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii

Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.     

FEEDING APPARATUS OF THE COLORLESS FLAGELLATE KATABLEPHARIS (CRYPTOPHYCEAE)1

The feeding apparatus of Kalablepharis ovalis (isolated from a freshwater impoundment in Colorado) and Katablepharis clone G-2 (isolated from the littoral of the Black Sea near Yalta in the Crimea) consists of inner and outer oval-shaped arrays of microtubules that begin at the anterior end of the cell and pass into the posterior of the cell. Each array of microtubules contains groups of microtubules with two to eight microtubules per group depending on the position of the array in the cell. A specialized area of the plasma membrane, the mouth, occurs at the anterior end of the cell. The mouth is oval with the long axis oriented dorsoventrally and consists of a raised ridge surrounding a central depression. The anterior end of the microtubules of the inner and outer arrays supports the raised ridge of the mouth. In freeze-fracture replicas, the protoplasmic face of the plasma membrane contains intramembrane particles on the raised ridge of the mouth. Three small membrane-cisternae occur on the protoplasmic side of the plasma membrane in the area of the mouth. Katablepharis clone G-2 also has five or six additional large membrane-cisternae associated with the inner microtubular array in the anterior portion of the cell. These larger membrane-cisternae do not occur in K. ovalis. Vesicles with electron-dense contents occur in association with the microtubular arrays. Katablepharis ovalis has a second type of vesicle containing a single-membrane profile associated with the microtubule arrays. The structure of the microtubular arrays in Katablepharis is compared with similar structures in suctorian ciliates and dinoflagellates.     

Unusual microtubular cytoskeleton of apomictic embryo sac of Chondrilla juncea L.

Summary. The mature apomictic embryo sac of Chondrilla juncea is highly vacuolated and demonstrates a polarization similar to that of the amphimictic gametophyte. The microtubule cytoskeleton of this embryo sac is uncharacteristic and relatively weak. The microtubules are positioned along cell walls and resemble cortical microtubules of somatic cells. They do not form the parallel, brushlike structures observed around the filiform apparatus of synergids in the amphimictic embryo sac. In the apomictic embryo sac, the microtubules of both the egg cell and the central cell develop a cortical-like structure, which is entirely different from the radial arrangement observed around the nuclei in the amphimictic embryo sac. Correspondence and reprints: Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Kraków, Poland.     

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

Summary During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.     

Smaller and more numerous harvesting gaps emulate natural forest disturbances: a biodiversity test case using rove beetles (Coleoptera,Staphylinidae)

Aim To evaluate changes in the abundance, species richness and community composition of rove beetles (Coleoptera, Staphylinidae) in response to three configurations of experimental gap cuts and to the effects of ground scarification in early succession yellow birch‐dominated boreal forest. In each experimental treatment, total forest removed was held constant (35% removal by partial cutting with a concomitant decrease in gap size) but the total number of gaps was increased (two, four and eight gaps, respectively), resulting in an experimental increase in the total amount of ‘edge’ within each stand. Location Early succession yellow birch‐dominated forests, Quebec, Canada. Methods Pitfall traps, ANOVA, MIXED procedure in sas ®, post hoc Tukey's adjustment, rarefaction estimates, sum‐of‐squares and distance‐based multivariate regression trees (ssMRT, dbMRT). Results Estimates of species richness using rarefaction were highest in clearcut and two‐gap treatments, decreased in smaller and more numerous gaps and were significantly higher in scarified areas than in unscarified areas. ANOVA indicated a significant impact of harvesting on the overall standardized catch. Post hoc Tukey's tests indicated that the total catch of all rove beetles was significantly higher in uncut forests than in the treated areas. Both sum‐of‐squares and distance‐based multivariate regression trees indicated that community structure of rove beetles differed among treatments. Assemblages were grouped into (a) control plots, (b) four‐ and eight‐gap treatments and (c) two‐gap and clearcut treatments. Main conclusions Rove beetle composition responded significantly to increasing gap size. Composition among intermediate and small‐sized gap treatments (four‐ and eight‐gap treatments) was more similar to uncut control forests than were larger gap treatments (two‐gap) and clearcuts. Effects of scarification were nested within the harvested treatments. When the total area of forest removed is held constant, smaller, more numerous gaps are more similar to uncut control stands than to larger gaps and falls more closely within the natural forest heterogeneity.