scholarly journals Three stages in the development of the cyst wall of the eye pathogen Acanthamoeba castellanii

2019 ◽  
Author(s):  
Pamela Magistrado-Coxen ◽  
Yousuf Aqeel ◽  
A Lopez ◽  
John Samuelson
Keyword(s):  
Cyst Wall ◽  
Acanthamoeba Castellanii ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Eye Infections ◽  
Second Stage ◽  
Sequence Of Events ◽  
Starting Point ◽  
Third Stage ◽  
Mature Cyst

AbstractWhen deprived of nutrients, trophozoites of the eye pathogen Acanthamoeba castellanii make a cyst wall, which contains cellulose and has two layers connected by cone-shaped ostioles. We recently showed chitin is also present and identified three sets of lectins, which localize to the ectocyst layer (Jonah lectin) or the endocyst layer and ostioles (Luke and Leo lectins). To determine how the cyst wall is made, we examined encysting protists using structured illumination microscopy, probes for glycopolymers, and tags for lectins. In the first stage (3 to 9 hr), cellulose, chitin, and a Jonah lectin were each made in dozens of encystation-specific vesicles. In the second stage (12 to 18 hr), a primordial wall contained both glycopolymers and Jonah lectin, while small, flat ostioles were outlined by a Luke lectin. In the third stage (24 to 36 hr), an ectocyst layer enriched in Jonah lectin was connected to an endocyst layer enriched in Luke and Leo lectins by large, conical ostioles. Jonah and Luke lectins localized to the same places in mature cyst walls (72 hr) independent of the timing of expression. The Jonah lectin and the glycopolymer bound by the lectin were accessible in the ectocyst layer of mature walls. In contrast, Luke and Leo lectins and glycopolymers bound by the lectins were mostly inaccessible in the endocyst layer and ostioles. These results show that cyst wall formation is a tightly choreographed event, in which glycopolymers and lectins combine to form a mature wall with a protected endocyst layer.ImportanceWhile the cyst wall of Acanthamoeba castellanii, cause of eye infections, contains cellulose like plants and chitin like fungi, it is a temporary, protective structure, analogous to spore coats of bacteria. We showed here that, unlike plants and fungi, A. castellanii makes cellulose and chitin in encystation-specific vesicles. The outer and inner layers of cyst walls, which resemble the primary and secondary walls of plant cells, respectively, are connected by unique structures (ostioles) that synchronously develop from small, flat circles to large, conical structures. Cyst wall proteins, which are lectins that bind cellulose and chitin, localize to inner or outer layers independent of the timing of expression. Because of its abundance and accessibility in the outer layer, the Jonah lectin is an excellent target for diagnostic antibodies. A description of the sequence of events during cyst wall development is a starting point for mechanistic studies of its assembly.

scholarly journals The most abundant cyst wall proteins ofAcanthamoeba castellaniiare cellulose-binding lectins from three gene families that localize to distinct structures in cyst walls

2018 ◽  
Author(s):  
Pamela Magistrado-Coxen ◽  
Yousuf Aqeel ◽  
Angelo Lopez ◽  
John R. Haserick ◽  
Breeanna R. Urbanowicz ◽  
...  
Keyword(s):  
Cyst Wall ◽  
Fluorescent Protein ◽  
Acanthamoeba Castellanii ◽  
Gene Families ◽  
Carbohydrate Binding ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Carbohydrate Binding Modules ◽  
Green Fluorescent ◽  
Cellulose Binding

AbstractAcanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls ofA. castellaniiretain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundantA. castellaniiCWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).Author summaryFifty years ago, the cyst wall ofAcanthamoeba castellaniiwas shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. WhileA. castellaniicyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

scholarly journals THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII (NEFF STRAIN)

1969 ◽  
Vol 41 (3) ◽  
pp. 786-805 ◽  
Author(s):  
Blair Bowers ◽  
Edward D. Korn
Keyword(s):  
Golgi Complex ◽  
Cyst Wall ◽  
Matrix Material ◽  
Acanthamoeba Castellanii ◽  
Dry Weight ◽  
Amorphous Layer ◽  
Wall Structure ◽  
Weight Protein ◽  
Granular Matrix ◽  
Mature Cyst

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.

Ultrastructural Aspects of Encystation and Cyst-Germination in Phytophthora Parasitica

1971 ◽  
Vol 9 (1) ◽  
pp. 175-191
Author(s):  
D. E. HEMMES ◽  
H. R. HOHL
Keyword(s):  
Cyst Wall ◽  
Germ Tube ◽  
Average Length ◽  
Cell Periphery ◽  
Phytophthora Parasitica ◽  
Cell Organelles ◽  
Wall Formation ◽  
Third Stage ◽  
Germ Tubes ◽  
Mature Cyst

Encystation in Phytophthora parasitica can be divided into 3 stages. In the first, the zoospores line their peripheries with flattened vesicles and fibrillar vacuoles in preparation for encystation. In the second stage, as the zoospores round up and shed their flagella, an initial wall is produced which takes the form of the mature cyst wall in thickness, but not in density. The participation of the flattened vesicles and fibrillar vacuoles in the formation of this initial wall is suggested by the disappearance of these organelles concomitant with wall formation. The third stage involves the maturation of the cyst wall and occurs only after dictyosomes produce vesicles which move to the cyst periphery and fuse to the plasmalemma. Germ tubes are formed in direct and indirect germination and involve the evagination of the plasmalemma and cyst wall proximal to an accumulation of dictyosome-derived vesicles. These vesicles remain at the germ-tube tip as it extends. In indirect germination the germ tube stops after having attained an average length of 6 µm and the vesicles appear to fuse at the hyphal apex, thus forming a cap. Lomasomes do not appear to be cell organelles with a specific function such as well synthesis, but rather seem to represent aggregations of excess membranous material that have formed as a result of the discharge of vesicles at the cell periphery during wall formation. When dictyosome vesicles are inhibited from forming and moving toward the cell periphery, lomasomes are not formed.

scholarly journals Acidocalcisome-Mitochondrion Membrane Contact Sites in Trypanosoma brucei

Pathogens ◽  
2018 ◽  
Vol 7 (2) ◽  
pp. 33 ◽  
Author(s):  
Srinivasan Ramakrishnan ◽  
Beejan Asady ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Close Association ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Starting Point ◽  
Membrane Contact

Membrane contact sites are regions of close apposition between two organelles, typically less than 30 nanometers apart, that facilitate transfer of biomolecules. The presence of contact sites has been demonstrated in yeast, plants, and mammalian cells. Here, we investigated the presence of such contact sites in Trypanosoma brucei. In mammalian cells, endoplasmic reticulum-mitochondria contact sites facilitate mitochondrial uptake of Ca2+ released by the ER-located inositol 1,4,5-trisphosphate receptor (InsP3R). However, the InsP3R in trypanosomes localizes to acidocalcisomes, which serve as major Ca2+ stores in these parasites. In this work, we have used super-resolution structured illumination microscopy and electron microscopy to identify membrane contact sites that exist between acidocalcisomes and mitochondria. Furthermore, we have confirmed the close association of these organelles using proximity ligation assays. Characterization of these contact sites may be a necessary starting point towards unraveling the role of Ca2+ in regulating trypanosome bioenergetics.

scholarly journals The most abundant cyst wall proteins of Acanthamoeba castellanii are lectins that bind cellulose and localize to distinct structures in developing and mature cyst walls

2019 ◽  
Vol 13 (5) ◽  
pp. e0007352 ◽  
Author(s):  
Pamela Magistrado-Coxen ◽  
Yousuf Aqeel ◽  
Angelo Lopez ◽  
John R. Haserick ◽  
Breeanna R. Urbanowicz ◽  
...  
Keyword(s):  
Cyst Wall ◽  
Acanthamoeba Castellanii ◽  
Mature Cyst

scholarly journals A 3D Topographical Model of Parenchymal Infiltration and Perivascular Invasion in Glioblastoma

2018 ◽  
Author(s):  
Kayla J. Wolf ◽  
Stacey Lee ◽  
Sanjay Kumar
Keyword(s):  
Tumor Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Diffuse Infiltration ◽  
Sequence Of Events ◽  
Topographical Effects ◽  
Culture Models ◽  
Perivascular Invasion ◽  
Vascular Beds

Glioblastoma (GBM) is the most common and invasive primary brain cancer. GBM tumors are characterized by diffuse infiltration, with tumor cells invading slowly through the hyaluronic acid (HA)-rich parenchyma toward vascular beds and then migrating rapidly along microvasculature. Progress in understanding local infiltration, vascular homing, and perivascular invasion is limited by an absence of culture models that recapitulate these hallmark processes. Here we introduce a platform for GBM invasion consisting of a tumor-like cell reservoir and a parallel open channel “vessel” embedded in 3D HA-RGD matrix. We show that this simple paradigm is sufficient to capture multi-step invasion and transitions in cell morphology and speed reminiscent of those seen in GBM. Specifically, seeded tumor cells grow into multicellular masses that expand and invade the surrounding HA-RGD matrices while extending long (10-100 µm), thin protrusions resembling those observed for GBM in vivo. Upon encountering the channel, cells orient along the channel wall, adopt a 2D-like morphology, and migrate rapidly along the channel. Structured illumination microscopy reveals distinct cytoskeletal architectures for cells invading through the HA matrix versus those migrating along the vascular channel. Substitution of collagen I in place of HA-RGD supports the same sequence of events but with faster local invasion and a more mesenchymal morphology. These results indicate that topographical effects are generalizable across matrix formulations, but that mechanisms underlying invasion are matrix-dependent. We anticipate that our reductionist paradigm should speed the development of mechanistic hypotheses that could be tested in more complex tumor models.

The Coherence of Mary Barton: Romance, Realism, and Utopia

2015 ◽  
Vol 5 (3) ◽  
pp. 185-200
Author(s):  
Robert Z. Birdwell
Keyword(s):  
Thomas Carlyle ◽  
The Novel ◽  
Second Stage ◽  
The Third ◽  
Third Stage ◽  
Three Stages ◽  
Mode Of Production

Critics have argued that Elizabeth Gaskell's first novel, Mary Barton (1848), is split by a conflict between the modes of realism and romance. But the conflict does not render the novel incoherent, because Gaskell surpasses both modes through a utopian narrative that breaks with the conflict of form and gives coherence to the whole novel. Gaskell not only depicts what Thomas Carlyle called the ‘Condition of England’ in her work but also develops, through three stages, the utopia that will redeem this condition. The first stage is romantic nostalgia, a backward glance at Eden from the countryside surrounding Manchester. The second stage occurs in Manchester, as Gaskell mixes romance with a realistic mode, tracing a utopian drive toward death. The third stage is the utopian break with romantic and realistic accounts of the Condition of England and with the inadequate preceding conceptions of utopia. This third stage transforms narrative modes and figures a new mode of production.

How well do voice-hearing assessment measures capture the positive experiences of individuals?

2019 ◽  
Author(s):  
Lucy Armstrong ◽  
Lorna Hogg ◽  
Pamela Charlotte Jacobsen
Keyword(s):  
Systematic Review ◽  
Lived Experience ◽  
Second Stage ◽  
The Third ◽  
Third Stage ◽  
Hearing Assessment ◽  
Positive Experiences ◽  
Assessment Measures ◽  
Stage 1 ◽  
The Voice

The first stage of this project aims to identify assessment measures which include items on voice-hearing by way of a systematic review. The second stage is the development of a brief framework of categories of positive experiences of voice hearing, using a triangulated approach, drawing on views from both professionals and people with lived experience. The third stage will involve using the framework to identify any positve aspects of voice-hearing included in the voice hearing assessments identified in stage 1.

Sign in / Sign up

Export Citation Format

Share Document