Ultrastructural Study of Cryptococcus neoformans Surface During Budding Events

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. It is surrounded by three concentric structures that separate the cell from the extracellular space: the plasma membrane, the cell wall and the polysaccharide (PS) capsule. Although several studies have revealed the chemical composition of these structures, little is known about their ultrastructural organization and remodeling during C. neoformans budding events. Here, by combining the latest and most accurate light and electron microscopy techniques, we describe the morphological remodeling that occurs among the capsule, cell wall and plasma membrane during budding in C. neoformans. Our results show that the cell wall deforms to generate a specialized region at one of the cell’s poles. This region subsequently begins to break into layers that are slightly separated from each other and with thick tips. We also observe a reorganization of the capsular PS around the specialized regions. While daughter cells present their PS fibers aligned in the direction of budding, mother cells show a similar pattern but in the opposite direction. Also, daughter cells form multilamellar membrane structures covering the continuous opening between both cells. Together, our findings provide compelling ultrastructural evidence for C. neoformans surface remodeling during budding, which may have important implications for future studies exploring these remodeled specialized regions as drug-targets against cryptococcosis.

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Let it bud: an ultrastructural study of Cryptococcus neoformans surface during budding events

10.1101/2020.05.15.098913 ◽

2020 ◽

Author(s):

Glauber R. de S. Araújo ◽

Carolina de L. Alcantara ◽

Noêmia Rodrigues ◽

Wanderley de Souza ◽

Bruno Pontes ◽

...

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cryptococcus Neoformans ◽

Drug Targets ◽

Spatial Organization ◽

Capsular Polysaccharide ◽

Light And Electron Microscopy ◽

Ultrastructural Organization ◽

Membrane Structures ◽

Daughter Cells

AbstractCryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. It is surrounded by three concentric structures that separate the cell from the extracellular space: the plasma membrane, the cell wall and the polysaccharide capsule. Although several studies have revealed the chemical composition of these structures, little is known about their ultrastructural organization and remodeling during C. neoformans budding event. Here, by combining the state-of-the-art in light and electron microscopy techniques we describe the morphological remodeling that occurs synergistically among the capsule, cell wall and plasma membrane during budding in C. neoformans. Our results show that the cell wall deforms to generate a specialized budding region at one of the cell’s poles. This region subsequently begins to break into layers that are slightly separated from each other and with thick tips. We also observe a reduction in density of the capsular polysaccharide around these specialized regions. Daughter cells present a distinct spatial organization, with polysaccharide fibers aligned in the direction of budding. In addition, to control the continuous openings between mother and daughter cells, the latter developed a strategy to shield themselves by forming multilamellar membrane structures in conjunction with their capsules. Together, our findings provide compelling ultrastructural evidence for a dynamic C. neoformans surface remodeling during budding and may have important implications for future studies exploring these remodeled specialized regions as drug-targets against cryptococcosis.

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Budding of melanized Cryptococcus neoformans in the presence or absence of l-dopa

Microbiology ◽

10.1099/mic.0.26333-0 ◽

2003 ◽

Vol 149 (7) ◽

pp. 1945-1951 ◽

Cited By ~ 27

Author(s):

Joshua D. Nosanchuk ◽

Arturo Casadevall

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

De Novo ◽

Pathogenic Fungus ◽

Parent Cell ◽

Daughter Cells ◽

Substantial Progress ◽

Mother Cells ◽

Bud Site ◽

Dopa Melanin

Cryptococcus neoformans is a pathogenic fungus that produces melanin when incubated in the presence of certain phenolic substrates such as l-3,4-dihydroxyphenylalanine (l-dopa). Melanin is an enigmatic polymer that is deposited in the cell wall and contributes to virulence. Substantial progress has been made in understanding the synthesis of melanin and the mechanisms by which it contributes to virulence, but relatively little is known about how melanin is rearranged during growth and budding. In this study we used transmission and scanning electron microscopy and immunofluorescence of melanized cells and melanin ‘ghosts' to study the process of melanization during replication. Budding in melanized C. neoformans results in focal disruption of cell-wall melanin at the bud site. In the presence of l-dopa, bud-related melanin defects are repaired and daughter cells are melanized. However, in the absence of substrate, mother cells cannot repair their melanin defects and daughter cells are non-melanized. Hence, melanin in the parent cell is not carried to the daughter cells, but rather is synthesized de novo in buds. These results imply that melanin remodelling occurs during cell growth in a process that involves degradation and synthesis at sites of budding.

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Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

Eukaryotic Cell ◽

10.1128/ec.00111-14 ◽

2014 ◽

Vol 13 (12) ◽

pp. 1484-1493 ◽

Cited By ~ 50

Author(s):

Julie M. Wolf ◽

Javier Espadas-Moreno ◽

Jose L. Luque-Garcia ◽

Arturo Casadevall

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cryptococcus Neoformans ◽

Extracellular Vesicles ◽

Protein Composition ◽

Multivesicular Body ◽

Dense Matrix ◽

Fungal Cell ◽

Content Type ◽

Pass Through

ABSTRACTCryptococcus neoformansproduces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.

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The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle.

The Journal of Cell Biology ◽

10.1083/jcb.114.1.111 ◽

1991 ◽

Vol 114 (1) ◽

pp. 111-123 ◽

Cited By ~ 288

Author(s):

J A Shaw ◽

P C Mol ◽

B Bowers ◽

S J Silverman ◽

M H Valdivieso ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Light And Electron Microscopy ◽

Yeast Cells ◽

Chitin Synthesis ◽

Triple Mutant ◽

Chitin Synthases ◽

Bud Emergence ◽

Primary Septum ◽

Mother Cells

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.

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Cell wall structure and deposition in Glaucocystis

The Journal of Cell Biology ◽

10.1083/jcb.77.1.103 ◽

1978 ◽

Vol 77 (1) ◽

pp. 103-119 ◽

Cited By ~ 50

Author(s):

JH Willison ◽

RM Brown

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Plasma Membranes ◽

Wall Structure ◽

Unicellular Alga ◽

Wall Deposition ◽

Cellulose Deposition ◽

Terminal Complexes ◽

Mother Cells ◽

In Transit

Events leading to cell wall formation in the ellipsoidal unicellular alga Glaucocystis are described. The wall is deposited in three phases: (a) a thin nonfibrillar layer, (b) cellulosic microfibrils arranged in helically crossed polylamellate fashion, and (c) matrix substances. At poles of cells, microfibrils do not terminate but pass around three equilaterally arranged points, resulting in microfibril continuity between the twelve helically wound wall layers. These findings were demonstrated in walls of both mother cells and freeze-fractured growing cells, and models of the wall structure are presented. Cellular extension results in spreading apart, and in rupture, of microfibrils. On freeze-fractured plasma membranes, there were 35 nm X 550 nm structures associated with the ends of microfibrils. These are interpreted as representing microfibril-synthesizing centers (terminal complexes) in transit upon the membrane. These terminal complexes are localized in a zone, or zones. The plasma membrane is subtended by flattened sacs, termed shields, which become cross-linked to the plasma membrane after completion of wall deposition. During wall deposition, microtubules lie beneath the shields, and polarized filaments lie between shields and plasma membrane. The significance of these findings in relation to understanding the process of cellulose deposition is discussed, and comparisons are made with the alga Oocystis.

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Cell wall development of Micrasterias americana, especially in isotonic and hypertonic solutions

Journal of Cell Science ◽

10.1242/jcs.21.3.617 ◽

1976 ◽

Vol 21 (3) ◽

pp. 617-631

Author(s):

K. Ueda ◽

S. Yoshioka

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Close Contact ◽

Light And Electron Microscopy ◽

Wall Layer ◽

Wall Material ◽

Cell Wall Development ◽

Wall Growth ◽

Wall Materials ◽

Main Component

The cell wall development of Micrasterias americana was investigated by light and electron microscopy. From digestion experiments with pectinase and cellulase, and from fluorescence spectra in Calcofluor and Coriphosphin solution, it was concluded that pectin substances were the main component of the young developing cell wall and that cellulose was synthesized after the daughter hemicell was well developed. In 0-16 M mannitol, wall materials accumulated and were incompletely incorporated into the wall at the region where wall growth would be expected. The plasma membrane was in close contact with the cell wall at the sinus, and this contact was assumed to prevent penetration of wall material at this region, resulting in the accumulation of wall material at regions other than the sinus. The cellulosic wall layer was formed after the production of pectic substances in the 0-16 M mannitol. In 0-3 M mannitol neither a definite wall layer of cellulose nor a pectic wall was produced, presumably due to extensive dilution of the wall materials in the plasmolysed space between the cell wall and the plasma membrane. Under normal circumstances, the shape of the daughter cell is assumed to be determined by the shape of the developed primary wall, which is induced by precocious differentiation of the wall at the sinus.

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Mother-daughter asymmetry of pH underlies aging and rejuvenation in yeast

eLife ◽

10.7554/elife.03504 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 75

Author(s):

Kiersten A Henderson ◽

Adam L Hughes ◽

Daniel E Gottschling

Keyword(s):

Plasma Membrane ◽

Mother Cell ◽

Daughter Cell ◽

Asymmetric Distribution ◽

Replicative Aging ◽

Cytosolic Ph ◽

Daughter Cells ◽

Proton Atpase ◽

Mother And Daughter ◽

Mother Cells

Replicative aging in yeast is asymmetric–mother cells age but their daughter cells are rejuvenated. Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation. Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells. This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1). Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells. We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify. We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons. However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation.

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Microanatomy of the Periplasm of Bacillus Licheniformis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065365 ◽

1971 ◽

Vol 29 ◽

pp. 262-263

Author(s):

B.K. Ghosh

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Bacillus Licheniformis ◽

External Environment ◽

Permeability Barrier ◽

Periplasmic Space ◽

Modified Technique ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

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