Ultrastructural study of encystation and excystation in Acanthamoeba castellanii

Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron-dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron-dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection

Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.     

Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

Surface Domains in the Pathogenic Protozoan Tritrichomonas foetus

ABSTRACT The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus . The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.     

A current assessment of photosystem II structure

This review covers the recent progress in the elucidation of the structure of photosystem II (PSII). Because much of the structural information for this membrane protein complex has been revealed by electron microscopy (EM), the review will also consider the specific technical and interpretation problems that arise with EM where they are of particular relevance to the structural data. Most recent reviews of photosystem II structure have concentrated on molecular studies of the PSII genes and on the likely roles of the subunits that they encode or they were mainly concerned with the biophysical data and fast absorption spectroscopy largely relating to electron transfer in various purified PSII preparations. In this review, we will focus on the approaches to the three-dimensional architecture of the complex and the lipid bilayer in which it is located (the thylakoid membrane) with special emphasis placed upon electron microscopical studies of PSII-containing thylakoid membranes. There are a few reports of 3D crystals of PSII and of associated X-ray diffraction measurements and although little structural information has so far been obtained from such studies (because of the lack of 3D crystals of sufficient quality), the prospects for such studies are also assessed.Abbreviations ATP adenosine triphosphate - Chl chlorophyll - CP chlorophyll-binding protein - EM electron microscopy - LHC light harvesting complex - NADP nicotinamide adenine dinucleotide phosphate - OEC oxygen evolution enhancing complex - PS photosystem - Tris tris-hydroxymethyl aminomethane     

The changing landscape of membrane protein structural biology through developments in electron microscopy

Membrane proteins are ubiquitous in biology and are key targets for therapeutic development. Despite this, our structural understanding has lagged behind that of their soluble counterparts. This review provides an overview of this important field, focusing in particular on the recent resurgence of electron microscopy (EM) and the increasing role it has to play in the structural studies of membrane proteins, and illustrating this through several case studies. In addition, we examine some of the challenges remaining in structural determination, and what steps are underway to enhance our knowledge of these enigmatic proteins.     

The presence of microvilli and other membrane fragments in the non-fat phase of bovine milk

Summary An electron microscopic study of a lipoprotein fraction isolated by ultracentrifugation from skim milk has shown that it is composed of membrane. It is believed that the majority of this membrane arises from shed microvilli. The microvilli may be derived from excess Golgi membrane which accumulates as plasma membrane during the secretion of milk proteins from the mammary cell. These findings indicate that milk may serve as a physiological source of easily isolated membrane. Authorized for publication on July 28, 1971 as paper no. 4025 in the Journal Series of the Pennsylvania Agricultural Experiment Station.     

Penetration of the peritrophic membrane of the tick by Babesia microti

Summary A peritrophic membrane (PM) has been demonstrated in the gut of feeding larvae, nymphs, and adults of the tick Ixodes dammini. This is the first report of a PM in ticks. This temporary structure divides the lumen of the gut into two compartments, an endoperitrophic space, the lumen proper, and an ectoperitrophic space located between the PM and the epithelial cells of the gut wall. The PM is a mechanical barrier and even such small particles as ribosomes derived from ingested reticulocytes are retained in the lumen proper; they are never found in the ectoperitrophic compartment. In Ixodes dammini fed on hamsters infected with Babesia microti some of the parasites are found in the ectoperitrophic space. This passage is accomplished by a highly specialized organelle, the arrowhead, which develops in some Babesia during their metamorphosis in the gut of the vector. The arrowhead, while passing through the PM, changes its fine structure and loses its internal organization as if releasing some of its contents. Its disintegration continues and it disappears shortly after the Babesia have entered the epithelial cells. Only Babesia equipped with the arrowhead structure are able to cross the PM. This is the first documented case of a parasite traversing a solidified PM.Supported by Research Grant AI-15886 from the United States Public Health ServiceIt is a great pleasure to acknowledge the excellent technical assistance of Mrs. Ellen Lanners. Special thanks are extended to Dr. Alex S. Raikhel for the courtesy of translating from Russian to English his Chapter 3, Intestine, from the book, An Atlas of Electron Microscopical Anatomy of Ixodid Ticks. We are indebted to Mrs. Liubov Lyandvert for the translation from Russian of selected other parts of the same book     

Ultrastructure of the different zones of the tectorial membrane

The tectorial membrane (t.m.) of mammals, which lies over the organ of Corti, is made up of an agglomerate of protofibrils of varying degrees of hydration. Two types of protofibrils are clearly distinguishable in the mouse t.m. While type-A protofibrils are straight and unbranched (thickness: 110A) demonstrating a periodic structure (period = 70 A), type-B protofibrils are branched and coiled (thickness: 150-200 A). These protofibrils could be systematically ordered according to the different t.m. zones. Type-A protofibrils predominate in the basal layer and in the entire middle zone, where they are interlaced with strongly hydrated type-B protofibrils. Weakly hydrated type-B protofibrils essentially make up the marginal zone (with the marginal net), the covering net, Hensen's stripe and the immediate contact layer with the limbus.     

Trypanosoma congolense: mechanical removal of the surface coat in vitro

By shaking suspensions of Trypanosoma congolense in isotonic buffer the surface coat could be separated from the cell body. The release of radioactivity from trypanosomes, selectively labeled in the surface coat by diazoniobenzenesulfonate, was used to follow the kinetics of coat detachment. The proteins in the supernatants of shaken trypanosomes were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The shaking conditions had to be carefully controlled to avoid complete rupture of trypanosomes. Otherwise the coat protein was rapidly degraded by endogenous proteases. The influence of several parameters on the yield of coat release and the degree of degradation of the coat protein was investigated, including the ratio of trypanosome suspension volume to shaking vessel volume, vessel surface, temperature, shaking frequency, and preincubation of the trypanosomes at 0 C. By combining these parameters an optimal scheme was developed which allowed the separation of more than 90% of the coat protein from T. congolense, the detached protein showing no degradation at all. These results could be confirmed by electron microscopy of shaken and unshaken trypanosomes.     

Fine structure and cytochemical evidence for the presence of polysaccharide surface coat of Dirofilaria immitis microfilariae

Cherian P. V., Stromberg B. E., Weiner D. J. and Soulsby E. J. L. 1980. Fine structure and cytochemical evidence for the presence of polysaccharide surface coat of Dirofilaria immitis microfilariae. International Journal for Parasitology10: 227–233. Cytochemical staining techniques were employed at the fine structural level using ruthenium red, ruthenium violet and Alcian blue-lanthanum nitrate to demonstrate the polysaccharide rich surface coat of Dirofilaria immitis microfilariae. The coat matrix present at the external surface of the cuticle of microfilariae stained densely with each of the polycationic dyes. The reaction products were restricted to the outer surface of the cuticle suggesting that the polycationic dyes did not penetrate the cuticle. The junctions of the cuticular annulations lacked surface coat matrix and reaction products which might be indicative of the absence of carbohydrate residues or the masking of reactive sugar molecules in these areas. The speciflcity of the reaction was indicated by the absence of reaction products in untreated organisms. These carbohydrate moieties probably represent glycoproteins as structural constituents of the parasite surface. Ultrastructural analysis of the surface of microfilariae is of signiflcance in elucidating both the molecular dynamics of the parasite surface and its immunological function in the host.     

Fine Structure of the Oocyst Walls of Isospora serini and Isospora canaria and Excystation of Isospora serini From the Canary, Serinus canarius L.

SYNOPSIS. Oocysts of Isospora serini and Isospora canaria , from the canary Serinus canarius , were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied in the electron microscope. The oocyst wall of each species had an electronlucent inner layer, a more osmiophilic middle layer and an outer layer of electron-lucent ( I. serini ) or electron-dense material interspersed with some electron-lucent material ( I. canaria ). A few, relatively large lipid-like bodies were present in the outer or middle layer of the oocyst wall of I. canaria. As many as 9 membranes were present in the oocyst wall of I. canaria and 3 in that of I. serini. When exposed to a trypsin-sodium taurocholate fluid, sporozoites of I. serini excysted from 5-month-old sporocysts in vitro , but not from sporocysts stored for more than 6 months. No excystation occurred in 15-month-old I. canaria sporocysts. Similarities and differences in excystation between I. serini and other Isospora, Eimeria , and Sarcocystis species are discussed.     

Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli & mammalian cells in suspension culture

Preparation of isolated cells and microorganisms for ultrastructural examination always provides a challenge in terms of adequate immobilization of the cells and prevention of subsequent sample loss and damage during various steps of sample processing. Using a positively charged nylon membrane substrate we demonstrate that it is possible to easily immobilize and retain a sample of isolated cells in culture for a wide variety of microscopy-based techniques. Radiolabelled E. coli cells when immobilized on the charged membrane were seen to be highly resistant to detachment when subjected to the normal sample processing procedures associated with microscopy. In contrast cells on regular millipore membranes were rapidly lost during sample preparation. We demonstrate the utility of charged nylon membranes for a wide variety of microscopy based analysis including scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), TEM based immunogold labelling, laser confocal microscopy and SEM based elemental analysis.