Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII (NEFF STRAIN) : II. Encystment

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.     

Cytochemical and Electrophoretic Studies on the Cyst Wall of a Ciliate, Histriculus muscorum Kahl

The localization of proteins and polysaccharides in the cyst wall of a ciliate, Histriculus muscorum, was examined by light and electron microscope cytochemistry and by using an enzyme digestion test on thin sections. The endocyst and ectocyst were digested by treatment with pepsin or protease VI. The endocyst was intensely PAS-positive and alcian blue-positive. The ectocyst was also PAS- and alcian blue-positive, but the reaction was weaker than that of the endocyst. At the electron microscope level, an intensely positive reaction to methenamine silver was observed in the endocyst and a weak reaction in the ectocyst of the mature cyst. In the ectocyst of the encysting organism, however, the reaction to methenamine silver was more intense than that of the mature cyst. These results demonstrate the possible presence of glycoproteins in the ectocyst and endocyst. The mesocyst was negative to all cytochemical and enzyme digestion tests examined. The cyst wall, isolated by sonication, was analyzed by SDS-polyacrylamide gel electrophoresis. Two bands, 190 and 140 kilodaltons, were specific for the cyst wall. The 190 kilodalton band was the only PAS-positive band and its localization in the cyst was was discussed.     

A Light- and Electron-Microscopical Study of Protacanthamoeba caledonica n. sp., Type-Species of Protacanthamoeba n. g. (Amoebida,Acanthamoebidae)1

In its amoeboid stage, Protacanthamoeba caledonica n. g., n. sp. closely resembles the genus Acanthamoeba, on both light- and electron-microscopical levels, including possession of a centrosphere with a plaque-shaped centriole-like body. The cyst wall differs from that of Acanthamoeba in lack of preformed exit pores and in fine structure; the occasional apparent division into exocyst and endocyst is due to irregular splitting. The strain isolated from a Scottish estuary did not grow at 37°C and did not grow normally on agar made with 25% sea water, but cysts remained viable after a week in full-strength sea water. Protacanthamoeba n. g. is distinguished from Acanthamoeba on the basis of cyst structure, but it is assigned to the family Acanthamoebidae.     

Experimental evidence of the defense role of the exocyst in the metacestode of <Emphasis Type="Italic">Microsomacanthus lari</Emphasis> Belogurov et Kulikov,in Spasskaja, 1966 (Cestoda: Hymenolepididae)

The interaction of intermediate host (Eogammarus tiuschovi) cells with the cysticercoid of Microsomacanthus lari of the cyclocercus type was examined for the case of damage of the noncellular exocyst surrounding it. Massive penetration of the host cells, supposedly haemocytes characterized by intensive secretion, into the exocyst was observed even during the first several minutes of the experiment. Large accumulations of the product of secretion (fibrous material) were concentrated in the distal areas of the endocyst glycocalix and the microvillar layer of the cysticercoid tail appendage. The results provide evidence for the defensive role of the exocyst of M. lari in the cellular response of an intermediate host organism to invasion.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea,Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater‐like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm—these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep‐branching position being moderate (i.e. maximum likelihood bootstrap support—67%; posterior probability—0.98). Cells grew at 15–150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.     

Structure of Cyst Wall Precursors and Kinetics of Their Appearance During the Encystment of Laurentiella acuminata (Hypotrichida,Oxytrichidae)1

The encystment of Laurenliella acuminata was divided into five stages: stage A (precystic semitransparent cell with dark-globules), stage B (precystic transparent cell), stage C (precystic pigmented cell), stage D (spherical shape without cyst wall) and stage E (young resting cyst), on the basis of observations of changes in morphology and pigmentation during encystment. The duration of these stages was also established. Observations by electron microscopy confirmed that the cyst wall, composed of four layers, is derived from different kinds of precursors which are synthesized “de novo.” The ectocyst precursors are composed of stacks of between 5 and 12 small thin plates or discs; these stacks are about 0.9 μm in length and 0.06 μm in height. The mesocyst precursors are fibrillar bodies of variable shapes, about 2.4 μm in maximum length and 0.12–0.16 μm in diameter. These precursors appear in the cytoplasm of the precystic cell during the first precystic stage (stage A). The endocyst precursors are rounded bodies surrounded by a fine membrane, and their contents appeared similar to the endocyst. The granular layer precursors are spherical bodies about 0.1–0.2 μm in diameter, surrounded by a double membrane presenting ribosomes adhering to its outer membrane. Both endocyst and granular layer precursors are observed in the precystic cytoplasm from stage B. On the basis of ultrastructural studies, a formation and growth model of the cyst wall of the hypotrichous ciliate Laurentiella acuminata is proposed.     

The Fine Structure of the Cyst Wall of the Ciliated Protozoon Didinium nasutum1

SYNOPSIS. Electron microscope observations of the complex cyst wall of Didinium nasutum are reported. The cyst wall is composed of 3 major coats. The outermost coat, the ectocyst, consists of short strands of filamentous material which forms a diffuse, amorphous layer approximately 8–9 μ thick. Culture debris, bacteria and unidentified inclusions have been observed adhering to the outer coat. The mesocyst, approximately 2.5 μ thick, has 2 distinct regions. The outer region is differentiated into several slightly thicker layers which in face view have a honeycomb appearance. The deeper region of the mesocyst consists of compact lamellae lacking the obvious honeycomb appearance of the layers of the outer region. Finally, the endocyst (0.3 μ thick), which arises in the mature cyst in the space that develops between the pellicle and the mesocyst, consists of delicate fibrils in a compact matrix. Both mesocyst and endocyst may be undulant and folded. The structure, origin and possible relationships of the various coats composing the cyst wall are discussed. The present study also contributes information on the role and fate of mucocysts and other cytoplasmic structures during the formation of the cyst wall.     

Paravahlkampfia francinae n. sp. Masquerading as an Agent of Primary Amoebic Meningoencephalitis

ABSTRACT. Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia , designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri . The isolate grew at 33 °C, 37 °C, 40 °C, and 42 °C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli , the usual food source of Paravahlkampfia ustiana , the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae .     

Ultrastructure,encystment and cyst wall composition of the resting cyst of the peritrich ciliate Opisthonecta henneguyi

The cyst wall of Opisthonecta henneguyi has been studied ultrastructurally and cytochemically by light and electron microscopy, as well as by chemical and electrophoretic analyses, to examine the structure of the cyst wall and its composition. The cyst wall consists of four morphologically distinct layers. The ectocyst is a thin dense layer. The mesocyst is the thickest layer and is composed of a compact material. The endocyst is a thin layer like the ectocyst, but less dense. The granular layer varies in thickness and is composed of a granular material. In the resting cyst, kinetosomes of both oral apparatus and trochal band as well as the myoneme system are maintained, and only cilia are resorbed. The sugars present in the cyst wall are predominantly N-acetylglucosamine (90%) and glucose (10%). The mesocyst is composed of chitin, and the endocyst includes glycoproteins and acid mucopolysaccharides. During secretion of the cyst wall, the endocyst and granular layer are secreted from precursors synthesized "de novo". No cytoplasmic precursors of ectocyst and mesocyst have been detected.     

Formation of the Cyst Wall of the Ciliate Colpoda steinii

After a thin membranous envelope surrounding the cell body and cilia of Colpoda steinii has been formed, the main mass of the proteinaceous cyst wall is deposited without exocytosis. It can be composed of two layers, the denser and wrinkled ectocyst and the smooth-walled endocyst; however, the ectocyst may be missing. Evidence is presented that ecto- and endocyst are formed from vesicles derived from abundant rough endoplasmic reticulum which appears at the time of wall formation. The cilia are retained and become embedded in the peripheral cytoplasm. Synthesis of RNA and protein is required as actinomycin C and cycloheximide block cyst formation. Calcium is required during a sensitive phase prior to encystment.     

The role of Sec3p in secretory vesicle targeting and exocyst complex assembly

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.     

冠突伪尾柱虫营养期和形成包囊期间细胞的超微结构

冠突伪尾柱虫营养细胞中含有轴杆样结构。细胞形成包囊期间,胞质内发生自噬作用,并产生由这聚集在一起组成的高尔基体,在高尔基体内其分泌物质聚集成高电子密度的嗜锇晶体。细胞分化的结果形成含膜粒层,内层壁和外层壁的“尾柱虫类包囊”。     

Exocyst在囊泡转运和细胞分泌过程中功能与结构的研究进展

Exocyst是由八个亚基蛋白组成的高度保守的复合物,目前被证实在细胞胞吐和囊泡转运,特别是囊泡与质膜的拴系阶段过程中发挥着十分重要的作用。本文综述了Exocyst在细胞中的定位、功能和结构方面的最新研究发现,以及复合物各亚基之间的相互作用和组装机制。当前的结晶学实验和电镜实验结果为Exocyst复合物的组装和解离机制提供了重要线索和证据。Exocyst功能与结构研究发现有助于我们进一步洞悉Exocyst复合物在囊泡转运和分泌过程中作用机制。     

Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins

Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.     

The lid forming apparatus in cysts of the green algaAcetabularia mediterranea

Summary Cross sections and cross tangential sections of 1 to 3-day-old cysts (gametangia) ofAcetabularia mediterranea were examined by electron microscopy. In a defined zone of the peripheral cytoplasm of the cysts, where the lid is to be formed, a characteristic circular band-like structure, the putative lid forming apparatus, can be identified. In 1 -day-old cysts this structure is characterized by two electron dense amorphous layers close and parallel to the plasma membrane. In 3-day-old cysts the lower layer consists of rod-like structures. The position of the circular band-like lid forming apparatus is correlated to the position of the cyst organizing secondary nucleus which occupies a non central position. Usually the center of the lid forming apparatus lies on the shortest line between the secondary nucleus and the cyst wall. This suggests that the cyst organizing secondary nucleus plays an important role in the formation of the cyst lid.     

The Fine Structure of Secretion in Hyalophysa chattoni: Formation of the Attachment Peduncle and the Chitinous Phoretic Cyst Wall

ABSTRACT. The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electron-dense secretory bodies from the cell's ventral surface.     

The differentiation of cellular structure during encystment in the soil hypotrichous ciliate Australocirrus cf. australis (Protista,Ciliophora)

Ciliates are able to form resting cysts as a survival strategy in response to stressful environmental factors. Studies on the characteristics of cellular structure during encystment may provide useful information for further understanding of the regulatory mechanism of cellular patterns and supply new clues regarding the phylogeny of ciliates. Scanning and transmission electron microscopies were used to observe the ultrastructure of cells during encystment of the soil ciliate Australocirrus cf. australis. The dedifferentiation of ciliature was revealed for the first time. Ciliary shafts first shortened, and the remaining ciliature, including basal bodies and the fibrillar cirral basket, retracted into the cytoplasm and was surrounded by the autophagic vacuoles and then gradually digested. A large number of autophagic vacuoles were observed in mature resting cysts. Autophagy might not only be necessary for the differentiation of cellular structures during encystment but might also be important to sustain the basic life activities in the resting stage. Australocirrus cf. australis formed a kinetosome-resorbing cyst and contained four layers in the cyst wall: the ectocyst, mesocyst, endocyst and granular layer. The ciliature resorbing state and the number of layers in the cyst wall were consistent with those found in other oxytrichous ciliates. However, the phenomenon wherein the two macronuclear nodules are not fused during encystment is not commonly observed among oxytrichids. Additionally, the octahedral granules in the mesocyst of this species exhibit different morphology from the congeners.     

Spermatogenesis in the black porgy,Acanthopagrus schlegeli (teleostei: Perciformes: Sparidae)

The ultrastructure of spermatogenesis and of the spermatozoon of Acanthopagrus schlegeli (Sparidae) are described. The testis is of the unrestricted type. Germ cells are surrounded by cyst cells. Spermiogenesis involves conspicuous modifications such as intracellular movements (diplosome and mitochondria migration, nuclear rotation, and depression) and structural changes (chromatin condensation, shape of mitochondria, and loss of cytoplasm). The mature spermatozoon has a spherical nucleus with a deep, axial nuclear fossa, and an unusual notch, shaped like a bow tie. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the A. schlegeli spermatozoon is of a primitive type, but that it is characterized by a unique feature which may provide a useful systematic character. © 1993 Wiley-Liss, Inc.