Immunocytochemical Identification of Spore Proteins in Two Microsporidia, with Emphasis on Extrusion Apparatus

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera ( Glugea atherinae and Encephalitozoon cuniculi ) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea , 35, 55 and 150 kDa in Encephalitozoon ). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.     

α‐ and β‐Tubulin Phylogenies Support a Close Relationship Between the Microsporidia Brachiola algerae and Antonospora locustae

ABSTRACT. Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced α‐ and β‐tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein‐based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.     

Assembly and Function of the Actin Cytoskeleton of Yeast: Relationships between Cables and Patches

Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found.To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function.Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created.     

Infection by microsporidia disrupts the host cell cycle

Microsporidia of the genus Encephalitozoon infect mammalian cells and have become a source of morbidity and mortality in immunocompromised humans. Encephalitozoon microsporidia develop and mature within parasitophorous vacuoles, enlarging the vacuole over time until it eventually occupies most of the cytoplasm of the host cell. The ability of the host cell to accommodate such a large burden for several days suggests that the parasite subverts normal host cell processes to ensure optimal environmental conditions for its growth and development. Since this environment would be threatened if cell division of the host cell occurred, we have formulated the hypothesis that infection with Encephalitozoon microsporidia induces an arrest in the cell cycle of the host cell. In support of this hypothesis, we have found that mitotic index and DNA duplication are reduced in infected cells as compared to uninfected cells. The number of host cell nuclei in S phase is increased. The levels of cyclin D1 and the percentage of cells in G1 are reduced; however, the levels of cyclin B1 are elevated even though the percentage of cells in G2/M is decreased. These results suggest that host cells infected with Encephalitozoon microsporidia are blocked at multiple points in the cell cycle.     

EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall

Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.     

Biofilms for Monitoring Presence of Microsporidia in Environmental Water

The development of molecular methodologies for targeting pathogens such as the Microsporidia has greatly improved our monitoring capabilities and initiatives. This study analyzed samples collected from five locations in Pensacola, Florida, USA for the presence of Microsporidian pathogens. To circumvent various impediments associated with water collection and filtration, we utilized biofilms as sentinels for detection of Microsporidia. We implemented membrane‐dissolution and sample purification in a single confined step followed by real‐time PCR to confirm pathogen presence. The results of this study demonstrate that microsporidia are present in environmental water sites in the Florida panhandle and that biofilms may serve as another alternative mode to circumvent filtration methods for their detection.     

Design of a Fluorescent Electrophoretic Mobility Shift Assay Improved for the Quantitative and Multiple Analysis of Protein-DNA Complexes

We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.     

Preliminary report on two new plasma protein polymorphisms in the goose (Anser anser)

Two-dimensional agarose gel (pH 8.6)-horizontal poly acrylamide gel (pH 9.0) electrophoresis of plasma proteins in the goose was done. This method resulted in improved separation of proteins in the pre-transferrin and transferrin regions. Preliminary evidence was presented for the existence of two new plasma protein poly morphisrns.     

Actin in the acrosome of the spermatozoa of the crab,Cancer pagurus L. (decapoda,crustacea)

The cytoskeletal protein actin was identified in the mature spermatozoon of the European edible crab, Cancer pagurus Linnaeus, by indirect immunofluoresce with monoclonal and polyclonal anti-actin antibodies, fluorescent phalloidin, and DNAase I. The actin was localized in two distinct concentric rings within the acrosome vesicle of the spermatozoon and appeared to correlate with the internal zonation of the vehicle. Modifications of the fluorescent pattern for actin were observed in sperm cells which were undergoing changes associated with the acrosome reaction. In these cases, fluorescent staining was observed in the nucleocytoplasm immediately subjacent to the perforatorial column and sometimes in the perforatorial column within the acrosome vesicle. Equally intense fluorescence was observed in an apical perforatorial projection. SDS-PAGE of C. pagurus sperm confirmed the presence of actin in the cells. A single band of actin (approximately 43 kDa) comigrated with rabbit muscle actin when immunoblotted onto nitrocellulose with mouse monoclonal anti-actin. The actin-associated cytoskeletal proteins α-actinin, tropomyosin, and spectrin were also identified within the spermatozoon of C. pagurus using specific polyclonal antibodies, but their presence was not confirmed by SDS-PAGE and immunoblotting. © 1994 Wiley-Liss, Inc.     

Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.     

Actin Out: Regulation of the Synaptic Cytoskeleton

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.     

Fine Structure of Encephalitozoon cuniculi from Rabbits,Mice and Hamsters*

SYNOPSIS. Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is ∼ 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.     

A Morphogenesis Checkpoint Monitors the Actin Cytoskeleton in Yeast

A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I. Reed. 1995. J. Cell Biol. 129:739– 749). The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Mol. Biol. Cell. 7:1657– 1666). In this report, we have investigated the nature of the defect(s) that trigger this checkpoint. A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact. Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation. In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle. However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.     

Microsporidian mitochondrial proteins: expression in Antonospora locustae spores and identification of genes coding for two further proteins

Microsporidia are obligate intracellular parasites, phylogenetically allied to the fungi. Once considered amitochondriate, now a number of mitochondrion-derived genes have been described from various species, and the relict organelle was recently identified in Trachipleistophora hominis. We have investigated the expression of potential mitochondrial targeted proteins in the spore stage to determine whether the organelle is likely to have a role in the spore or early infection stage. To investigate whether the Antonospora locustae genome codes for a different complement of mitochondrial proteins than Encephalitozoon cuniculi an EST library was searched for putative mitochondrial genes that have not been identified in the E. cuniculi genome project. The spore is the infectious stage of microsporidia, but is generally considered to be metabolically dormant. Fourteen genes for putatively mitochondrion-targeted proteins were shown to be present in purified spore mRNA by 3'-rapid amplification of cDNA ends and EST sequencing. Pyruvate dehydrogenase E1alpha and mitochondrial glycerol-3-phosphate dehydrogenase proteins were also shown to be present in A. locustae and E. cuniculi spores, respectively, suggesting a role for these proteins in the early stages of infection, or within the spore itself. EST sequencing also revealed two mitochondrial protein-encoding genes in A. locustae that are not found in the genome of E. cuniculi. One encodes a possible pyruvate transporter, the other a subunit of the mitochondrial inner membrane peptidase. In yeast mitochondria, this protein is part of a trimeric complex that processes proteins targeted to the inner membrane and the intermembrane space, and its substrate in A. locustae is presently unknown.     

The Polymeric State of Actin in the Human Erythrocyte Cytoskeleton

Reports on the polymeric state of actin in the red cell have been diverse. We have used phalloidin to stabilize the actin in erythrocyte ghosts prior to extraction in low ionic strength media. A mild proteolytic digestion and Sepharose 4B gel filtration enable an F-actin polymer to be isolated in pure form [1]. Detailed size analysis of this polymer in a range of experiments suggests that actin exists in the erythrocyte principally as a polymer of 100 nm length composed of 30 monomers in a double helical chain 15 monomers long with an estimated molecular weight of 1.3 × 10⁶ daltons.