Isolation and characterization of prosthecae ofAsticcacaulis biprosthecum

The budding process of the yeast form of Mucor rouxii was examined by electron microscopy of thin sections with particular reference to wall ontogeny. In most instances the bud wall is seen as a continuation of the inner layers of the parent cell wall. As the bud emerges it ruptures the outer layers of the parent wall. The bud wall is much thinner than the parent wall and remains so while the bud grows into a sphere of about one half the diameter of the parent cell. Then a septum begins to form centripetally, at the neck, by invagination of the plasmalemma. Before the neck canal is completely occuluded, electron-dense wall material is deposited into the septum space. Two separate septum walls are deposited, one on the parent side and one on the bud side of the invaginating plasmalemma. Septum wall formation extends to the surrounding neck walls. In this manner, the parent and bud cytoplasms become fully separated and each is surrounded by a continuous wall. The two cells remain attached to each other by the original neck wall; eventually, the bud abscisses leaving a birth scar on the bud cell and a more pronounced bud scar on the parent cell. In general, the mechanism of budding in this zygomycetous fungus resembles that of an ordinary ascomycetous yeast such as Saccharomyces cerevisiae.     

Bipolar budding in yeasts — an electron microscope study

Bud formation in yeasts with bipolar budding was studied by electron microscopy of thin sections.Budding in yeasts of the speciesSaccharomycodes ludwigii, Hanseniaspora valbyensis andWickerhamia fluorescens resulted in concentric rings of scar ridges on the wall of the mother cell. The wall between the ridges consisted of the scar plug left by the former budding and opened up in the formation of the next bud. The wall of the bud arose from under the wall of the mother cell.In the yeasts of the speciesNadsonia elongata more than one bud might be formed from the same plug.InSchizoblastosporion starkeyi-henricii the scar ridges were close together and apparently not separated by the entire plug.In all species a cross wall was formed between mother cell and bud which consisted of an electron-light layer between two layers of more electron-dense material. The cells separated along the light layer.The authors wish to thank Dr J. A. Barnett for corrections of the English text, and Mr J. Cappon for drawing Fig. 1.     

Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.     

The mechanics of budding and copulation in saccharomyces

The yeast cell contains a nucleus whose rigid centrosome carries a band of Feulgen-positive chromatin (centrochromatin) on its surface. The first step in budding is the formation of the bud by an extension of the centrosome over which the cell wall persists. Next the nuclear vacuole extends a process into the bud which contains the chromosomes. Finally the centrochromatin divides directly and the cells separate; a plug either of centrosome or cytoplasm sealing the bud pore. The cytoplasm, the centrosome, the centrochromatin and the nuclear wall are autonomous non genic organelles which never originate de novo.Copulation is the reverse of budding. The centrosomes fuse first; the cytoplasms mix; the nuclear vacuoles fuse by processes which travel along the fused centrosomes; and finally the centrochromatins fuse to form a single band.Figures 1–12. Drawings of budding yeast cells fixed in Schaudinn's fluid and stained with iron alum hemotoxylin, mounted in balsam. The cell wall is not visible due to the clearing action of the balsam. Except for Figure 5, the chromosomes and the nucleolus in the nuclear vacuole have been completely destained. The bud scar described by Barton is shown clearly at the end of the cell distal from the centrosome. The nuclear vacuole is usually forced into the extrusion formed by the bud scar. Since the cell wall is not visible, the plug of material connecting bud and mother cell as shown in Figure 12, fits into the cell wall and probably corresponds to the plug in the bud scar described by Barton. The details of the budding process are described in the text.Figures 13–18. Copulating yeast cells stained with Barrett's hemotoxylin and aceto-orcein and mounted in the stain. Chromosomes are visible in the nuclear vacuoles. The centrosome is usually visible and often appears to have a core which stains differentially. Except in Figure 16, the centrochromatin is visible as darkly stained material; in some cases surrounded by a clear zone. The “thick waisted” form of the cells identifies them as derived from recent copulations and distinguishes them from budding cells. The process of copulation is discussed in the text.     

Cell surface topography of Candida and Leucosporidium yeasts as revealed by scanning electron microscopy.

The cell surface topography of the following yeast strains was examined by scanning electron microscopy: Candida slooffii, C. lipolytica, Leucosporidium frigidum, and L. nivalis. Multipolar and lateral budding were observed in the Candida yeasts in contrast to bipolar budding in the Leucosporidium species. The cell surface topography and the morphology of the bud and birth scars in these yeasts differed markedly. Apart from the bud and birth scars, the cells of C. slooffii showed a relatively smooth topography. The bud scars were seen as a circular ridge of wall material surrounding a markedly convex scar plug. Birth scars were raised, rounded structures, which appeared to distend upon cell growth. In contrast, bud scars of C. lipolytica were platelike, lacked a distinct annulus of wall material, and were much less protuberent than those of C. slooffii. Birth scars were a more permanent feature of these cells. The topography of Leucosporidium yeasts was characterized by the presence of numerous protrusions on the cell surface. In some cases, the entire cell surface was covered by these protrusions. There appeared to be some correlations between the age of the cell and the extent of surface protrusions and degree of surface convolution...     

Chitin structures of the cell walls of synchronously grown virgin cells of Saccharomyces cerevisiae.

The ability of a lytic beta-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.     

Differences in actin localization during bud and hypha formation in the yeast Candida albicans

Stationary phase cells of Candida albicans can form either a bud or a hypha, depending upon the pH of the medium into which they are released. At low pH, cells form an ellipsoidal bud and at high pH, cells form an elongated hypha. By staining cells with rhodamine-conjugated phalloidin, we have compared the dynamics of actin localization during the formation of buds and hyphae. Before evagination, actin granules were distributed throughout the cytoplasmic cortex in both budding and hypha-forming cells. Just before evagination, actin granules clustered at the site of evagination, then filled the early evagination in both budding and hypha-forming cells. With continued bud growth, the actin granules then redistributed throughout the cytoplasmic cortex. In marked contrast, with continued hyphal growth, the majority of actin granules clustered at the hyphal apex. This distinct difference in actin granule localization may be related to the distinct differences in the expansion zones of the cell wall recently demonstrated between growing buds and hyphae. The spatial and temporal dynamics of the large neck actin granules and of actin fibres are also described.     

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.     

Development and application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, "X," next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.     

Budding in the Dimorphic Fungus Phialophora dermatitidis

Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.     

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.     

Electron Micrography of Bud Formation in Metschnikowia krissii

The fine structure of bud formation of Metschnikowia krissii was studied by means of ultramicrotomy and transmission electron microscopy. Bud protrusion and development were observed by scanning electron microscopy. Bud formation in this yeast takes place by an extension of a small localized area of the existing parent wall. The parent cell and its bud are initially separated by the plasmalemma, creating an intercellular site within which the generation of new cell wall (bud and birth scar areas) occurs centripetally. When the dividing wall is complete and new cell wall material is formed, a narrow cleavage plane becomes increasingly defined. This cleavage plane apparently proceeds laterally toward the direction of the existing outer walls which rupture, resulting in the separation of the bud from the parent cell. The bud scar is prominently convex in shape; the birth scar is less conspicuous and initially concave in shape. Comparison of bud formation in M. krissii is made with that observed in Saccharomyces cerevisiae and Rhodotorula glutinis.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.     

Role of Bud3p in producing the axial budding pattern of yeast

Yeast cells can select bud sites in either of two distinct spatial patterns. a cells and alpha cells typically bud in an axial pattern, in which both mother and daughter cells form new buds adjacent to the preceding division site. In contrast, a/alpha cells typically bud in a bipolar pattern, in which new buds can form at either pole of the cell. The BUD3 gene is specifically required for the axial pattern of budding: mutations of BUD3 (including a deletion) affect the axial pattern but not the bipolar pattern. The sequence of BUD3 predicts a product (Bud3p) of 1635 amino acids with no strong or instructive similarities to previously known proteins. However, immunofluorescence localization of Bud3p has revealed that it assembles in an apparent double ring encircling the mother-bud neck shortly after the mitotic spindle forms. The Bud3p structure at the neck persists until cytokinesis, when it splits to yield a single ring of Bud3p marking the division site on each of the two progeny cells. These single rings remain for much of the ensuing unbudded phase and then disassemble. The Bud3p rings are indistinguishable from those of the neck filament- associated proteins (Cdc3p, Cdc10p, Cdc11p, and Cdc12p), except that the latter proteins assemble before bud emergence and remain in place for the duration of the cell cycle. Upon shift of a temperature- sensitive cdc12 mutant to restrictive temperature, localization of both Bud3p and the neck filament-associated proteins is rapidly lost. In addition, a haploid cdc11 mutant loses its axial-budding pattern upon shift to restrictive temperature. Taken together, the data suggest that Bud3p and the neck filaments are linked in a cycle in which each controls the position of the other's assembly: Bud3p assembles onto the neck filaments in one cell cycle to mark the site for axial budding (including assembly of the new ring of neck filaments) in the next cell cycle. As the expression and localization of Bud3p are similar in a, alpha, and a/alpha cells, additional regulation must exist such that Bud3p restricts the position of bud formation in a and alpha cells but not in a/alpha cells.     

Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.     

Paracoccin, an N-acetyl-glucosamine-binding lectin of Paracoccidioides brasiliensis, is involved in fungal growth

Paracoccin is an N-acetyl-glucosamine-binding lectin from Paracoccidioides brasiliensis, which can be obtained in small amounts either from culture supernatants or yeast cell extracts. In the present work, immunoelectron microscopy with mouse anti-paracoccin IgG localized the antigen to the cell wall of P. brasiliensis yeast forms. Paracoccin interacted with chitin, and colocalized with beta-1,4-homopolymer of GlcNAc to the budding sites of P. brasiliensis yeast cell. In order to evaluate the role of paracoccin on fungal growth, yeast cells were cultivated in the presence of anti-paracoccin antibodies. A significant reduction of both colony forming units and individual yeast cells was observed as well as morphological alterations such as smaller colonies and cells more loosely aggregated than in control cultures without the antibody. A role of paracoccin on the cell wall organization was reinforced by alterations in the labeling pattern of chitin when yeasts were treated with anti-paracoccin antibodies. Binding of specific antibodies to paracoccin may disrupt the paracoccin/chitin interactions, resulting in the inhibition of P. brasiliensis growth.     

A Safeguard Mechanism Regulates Rho GTPases to Coordinate Cytokinesis with the Establishment of Cell Polarity

The spatiotemporal control of cell polarity is crucial for the development of multicellular organisms and for reliable polarity switches during cell cycle progression in unicellular systems. A tight control of cell polarity is especially important in haploid budding yeast, where the new polarity site (bud site) is established next to the cell division site after cell separation. How cells coordinate the temporal establishment of two adjacent polarity sites remains elusive. Here, we report that the bud neck associated protein Gps1 (GTPase-mediated polarity switch 1) establishes a novel polarity cue that concomitantly sustains Rho1-dependent polarization and inhibits premature Cdc42 activation at the site of cytokinesis. Failure of Gps1 regulation leads to daughter cell death due to rebudding inside the old bud site. Our findings provide unexpected insights into the temporal control of cytokinesis and describe the importance of a Gps1-dependent mechanism for highly accurate polarity switching between two closely connected locations.