Distribution of actin and tropomyosin in Cryptosporidium muris

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (×2,000). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various cellular functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites'' firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.     

High-Speed Cell Sorting of Infectious Trophic and Cystic Forms of Pneumocystis carinii

ABSTRACT. The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6±0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.     

Videomicroscopic recording of Pneumocystis carinii motion.

Videomicroscopy in combination with differential-contrast optics was used to study fresh preparations of Pneumocystis carinii from immunosuppressed rats. Certain spherical intracystic bodies appeared to move freely within the cyst wall. Flexing type movement was observed in intracystic ellipsoidal forms attached at a common point in the inner margin of the cyst wall. Greater movement was seen in non-attached thinner elongated forms. Possible extracellular trophic forms and movement were also identified. The movement of the morphological forms of P. carinii has been recorded in real time onto videotape. These initial observations suggest P. carinii is capable of movement and additional studies are under way to substantiate this possibility.     

Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5'-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5'-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.     

Yeast glucan in the cyst wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of beta-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains beta-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is beta-1,3-glucan laminaripentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is beta-1,3-glucan.     

Localization of actin and tubulin in developing and adult mammalian photoreceptors

Summary In order to define cytoskeletal domains of the mammalian photoreceptor, actin and tubulin were localized in adult retinae of mouse and human. For light-microscopic localization, actin was labeled using fluorescent phalloidin or monoclonal antibodies against actin, and tubulin was labeled using monoclonal antibodies against alpha- and beta-tubulin in an immunocytochemical method. Actin and tubulin were also localized by ultrastructural immunocytochemistry in the mouse. Filamentous actin was present in the retina at the outer limiting membrane and in synaptic terminals, especially of the cones, while globular actin was observed additionally in the inner segments. Müller cell cytoplasm and apical microvilli at the outer limiting membrane were also labeled for filamentous actin. Alpha- and beta-tubulin were evident throughout the photoreceptors, including the inner segments, but not in the synaptic terminals or at the outer limiting membrane. In the early postnatal retina of mouse, actin and tubulin were present at the ventricular surface. This pattern changed as photoreceptors fully elongated and as synaptogenesis occurred in the outer plexiform layer.     

Yeast Glucan in the Cyst Wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae , which consists of an outer dense layer of mannan, a middle lucent layer of β−1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall P. carinii , as well as the cell wall of S. cerevisiae , can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains β-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β,3-gIucan laminari-pentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae . These observations indicate that a major component of the cyst wall of P. carinii is β-1,3-glucan.     

Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.     

Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii

Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe. P. carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies. Although the life cycle of P. carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P. carinii alternates between numerous small trophic forms and fewer large cystic forms. To understand further the molecular mechanisms that regulate progression of the cell cycle of P. carinii, we have sought to identify and characterize genes in P. carinii that are important regulators of eukaryotic cell cycle progression. In this study, we have isolated a cDNA from P. carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was shown to rescue growth of the temperature-sensitive S. pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P. carinii life cycle. Although P. carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint. The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.     

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristac that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytcs of the lung alveolus.     

Ultrastructural observations on life cycle stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristae that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytes of the lung alveolus.     

Embryogenesis in <Emphasis Type="Italic">Sedum acre</Emphasis> L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.     

Is Pneumocystis carinii vertically transmitted to neonatal rats?

Pneumocystis carinii is a pulmonary pathogen of immunocompromised humans or other mammals. Its infection results from activation of organisms involved in latent infection or from new infection through the air. Almost all children are known to be infected within 2 to 4 years of birth, though prenatal transplacental transmission has not yet been demonstrated. In this study we observed experimental P. carinii infection in neonatal rats, thus investigating the possibility of transplacental vertical transmission by Diff-Quik staining of the lung impression smears and in-situ hybridization for lung sections. The positive rate of P. carinii infection in immunosuppressed maternal rats was 100%, but that in normal maternal rats was 0%. Cystic forms of P. carinii were observed in three of six 1-week old neonatal rats born of heavily infected mothers, but none of them was positive by in-situ hybridization. Five weeks after birth, cystic forms were detected in four neonatal rats. In the lobes of the lungs, no predilection site of P. carinii was recognized. Counts of cystic forms on smears and the reactivity of in-situ hybridization in the lungs of neonatal rats were significantly lower than in maternal rats. The present findings suggest that P. carinii is rarely transmitted through the placenta and proliferates less successfully in the lungs of neonatal rats than in mothers.     

ULTRASTRUCTURAL STUDY ON MICROSPORE WALL MORPHOGENESIS IN ISOETES JAPONICA (ISOETACEAE)

Spore wall morphogenesis of the microspore of Isoetes japonica was studied by transmission electron microscopy. The microspore wall consists of four layers: the perispore, outer exospore, inner exospore, and endospore. The perispore consists of electron-dense materials. The exospore is divided into outer and inner sections, with a large gap between the two. The outer exospore appears as an undulating plate consisting of tripartite lamellae with homogeneous sporopollenin. The inner exospore consists of an accumulation of tripartite lamellae on the microspore cell membrane. Immediately after meiosis, the tripartite lamellae of the outer exospore forms around the microspore. The lamellated inner exospore forms next, which adheres to the cell membrane of the microspore. The deposition of homogeneous sporopollenin material on the tripartite lamellae causes the plates of the outer exospore to thicken. Some homogeneous material may also be deposited on the inner exospore. Lastly, the electron-dense perispore is deposited on the outer exospore, and the electron-lucent endospore forms beneath the inner exospore. We conclude that the lamellae of the outer exospore, inner exospore, and endospore are formed and derived, in that order, from the gametophytic microspore cytoplasm. The homogeneous sporopollenin material of the outer exospore and perispore may be derived from the sporophytic tapetal cytoplasm.     

Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

Ultrastructure of developing basidiospores in <Emphasis Type="Italic">Rhizopogon roseolus</Emphasis> (= <Emphasis Type="Italic">R. rubescens</Emphasis>)

The ultrastructure of developing basidiospores in Rhizopogon roseolus is described. When viewed in the fruiting body chamber using scanning electron microscopy, basidiospores appear narrowly ellipsoid and have smooth walls. Eight basidiospores are usually produced on the apex of each sterigma on the basidium. Transmission electron micrographs showed that basidiospores formed by movement of cytoplasm (including the nuclei) via the sterigmata, and then each basidiospore eventually became separated from its sterigma by an electron-lucent septum. The sterigma and basidium subsequently collapsed, resulting in spore release. Freshly released spores retained the sterigmal appendage connected to the collapsed basidium. After spore release, the major ultrastructural changes in the spore concerned the lipid bodies and the spore wall. During maturation, lipid bodies formed and then expanded. Before release, the spore wall was homogeneous and electronlucent, but after release the spore wall comprised two distinct layers with electron-dense depositions at the inner wall, and the dense depositions formed an electron-dense third layer. The mature spore wall complex comprised at least four distinct layers: the outer electron-lucent thin double layers, the mottled electron-dense third layer, and the electron-lucent fourth layer in which electron-lucent granular substances were dispersed.     

Freeze-fracture studies on Pneumocystis carinii. I. Structural alteration of the pellicle during the development from trophozoite to cyst

Pneumocystis carinii has generally been distinguished in three developmental stages, namely, trophozoite, precyst and cyst. The fine structure of the pellicle--the plasma membrane and the outer layer existing outside this plasma membrane--of each stage was studied by freeze-fracture technique. By this technique, P. carinii was cleaved through the cytoplasm or through the hydrophobic region of the plasma membrane, and the cross-fractured face of the outer layer was revealed on the replicas. The outer layer, which is electron-dense in the thin section, consisted of numerous fine granules about 15 nm in diameter in freeze-fracture images, whereas the electron-lucent middle layer which appeared in the precyst and cyst was less granular. Measurement of the intramembranous particles (IMP) also was carried out. The number of IMP per square micrometer of the plasma membrane of the trophozoite was 1,512 +/- 125 on the P face and 417 +/- 44 on the E face. In the precyst, the IMP density decreased, and 1,037 +/- 56 on the P face and 262 +/- 22 on the E face. In the cyst, it further decreased, nd 875 +/- 59 and 150 +/- 20 respectively. It is generally assumed that the density of IMP is related to the physiological activity of the cell membrane, so that the present results obtained in P. carinii suggest that the trophozoite is the most active stage, and that metabolic activity of the pellicle gradually decreases with the progress of development to the precyst then to the cyst.     

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

Tropomyosin co-localizes with actin microfilaments and microtubules within supporting cells of the inner ear

Summary The distribution of tropomyosin, actin and tubulin in the supporting cells of the organ of Corti was studied by immunofluorescent localization of antibodies to these proteins. Tropomyosin colocalizes with actin and tubulin in the regions of the tunnel pillar and Deiters cells where actin microfilaments and microtubules had previously been observed ultrastructurally. Despite the implications of the presence of antiparallel actin filaments in the supporting cells, the presence of tropomyosin and the absence of myosin suggest that the role of tropomyosin may be to confer rigidity to the actin filaments. Thus the primary function of the cytoskeletal proteins in the supporting cells may be structural.