Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Cell wall assembly in Bacillus subtilis: partial conservation of polar wall material and the effect of growth conditions on the pattern of incorporation of new material at the polar caps

The use of phage SP50 as marker for cell wall containing teichoic acid in Bacillus subtilis showed clear differences in the rates at which new wall material becomes exposed at polar and cylindrical regions of the wall, though the poles were not completely conserved. Following transition from phosphate limitation to conditions that permitted synthesis of teichoic acid, old polar caps fairly rapidly incorporated enough teichoic acid to permit phage binding. Electron microscopy suggested that the new receptor material spread towards the tip of the pole from cylindrical wall so that phages bound to an increasing proportion of the pole area until only the tip lacked receptor. Eventually, receptor was present over the whole polar surface. Direct electron microscopic staining of bacteria collected during transitions between magnesium and phosphorus limitations showed that new material was incorporated at the inner surface of polar wall and later became exposed at the outer surface by removal of overlying older wall. The apparent partial conservation of the pole reflected a slower degradation of the overlying outer wall at the pole than at the cylindrical surface, the rate being graded towards the tip of the pole. The relative proportions of the new wall material incorporated into polar and cylindrical regions differed in bacteria undergoing transitions that were accompanied by upshift or downshift in growth rate. These differences can be explained on the basis that growth rate affected the rate of synthesis of cylindrical but not septal wall.     

Characterization of a major cell wall antigen and potential adhesin in three strains of Candida albicans

Selected strains of Candida albicans were examined to reveal the surface antigenicity and biochemical nature of major cell wall proteins that also were shown to serve as cellular adhesins on human buccal epithelial cells. Confirmation of the adhesive properties of these cells was made by scanning electron microscopy and immunofluorescence microscopy. Particular attention was directed at the clinical isolate KM-302. By means of indirect immunofluorescence staining, the KM-302 blastoconidia absorbed rabbit anti-C. albicans ATCC-32354 serum, revealing specific localization of surface antigens on germ tubes and pseudohyphae. Extracellular polymeric material and the cell wall extract of C. albicans KM-302 blastoconidia were found to contain a major surface antigen of 49 kDa that exhibited 42% adhesion inhibition in vitro. Of considerable significance is that immunogold localization by electron microscopy showed the antigen to be almost exclusively cell wall bound. This major antigen, identified in affinity and gel filtration chromatography fractions, was composed of 4% carbohydrate and 95.7% protein and had an isoelectric point of 6.1. The major antigen also showed a high level of similarity with that of C. albicans strain SC-5314 inasmuch as the major antigen of that strain had carbohydrate and protein compositions of 4 and 95.5%, respectively. Both of these strains also possessed the same percent of adhesion inhibition of human buccal epithelial cells.Abbreviations BECs buccal epithelial cells - CWE cell wall extract - EPP extracellular polymers and proteins - FITC fluorescein isothiocyanate - mAg major antigen - OD ₆₀₀ optical density at 600 nm - PBS phosphate buffered saline - TEM transmission electron microscopy - YNB yeast nitrogen base     

Chemical components and their locations in the Verticillium fungicola cell wall

The chemical structure of cell walls and fractions of Verticillium fungicola, a pathogen of Agaricus bisporus, as well as their corresponding ultrastructures were studied. There are at least three chemically distinct types of carbohydrate polymers: one yielding mannose with lower amounts of galactose and glucose (glucogalactomannan), another one composed mainly of glucose (glucan), and a third one containing only N-acetylglucosamine (chitin). Attempts were made to locate these materials in situ by comparing electron micrographs of shadowed and sectioned cell walls, and also by indirect immunofluorescence. It was shown that none of these polymers constituted a completely physically distinct layer, but there seem to be different solubility properties in the outer, inner, and intermediate layers. It was also shown that fibrillar material (chitin) embedded in cementing glucan constituted the residual inner fraction of the original wall material. Indirect immunofluorescence showed the location of a significant amount of glucogalactomannan on the surface of the walls in which rodlet structures were visualized by electron microscopy.     

Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobium-legume symbiosis

A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.     

Regeneration and maturation of daughter cell walls in the autospore-forming green alga<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis> (Chlorophyta,Trebouxiophyceae)

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17–21 nm) emerged as a small, but complete, autospore.     

Localization of α-galactomannan and of wheat germ agglutinin receptors inSchizosaccharomyces pombe

The location of galactomannan on the surface ofSchizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized onS. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections ofS. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed thatS. pombe cell wall is devoid of chitin.Non-Standard Abbreviations Au gold colloid - RCA_I Ricinus communis lectin - SEM scanning electron microscopy - TEM transmission electron microscopy - WGA wheat germ agglutinin     

Adhesion,Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.     

Formation and ultrastructure of Mucor rouxii arthrospores

The formation of arthrospores in Mucor rouxii was studied by transmission and scanning electron microscopy and light microscopy. The arthrospores formed in a random manner in terminal and internal regions of the hyphae. The earliest appearance of the arthrospores was seen by scanning electron microscopy as compartments delineated by double ridges. These ridges probably corresponded to the site of septal wall formation. The elongated compartments varied considerably in size. As the arthrospores matured, they tended to separate as a result of a gradual change in the shape of the arthrospores to a nearly spherical form and also as the result of simultaneous degradation of the outermost cell wall layer. The mature arthrospores were surrounded by a complex cell wall consisting of at least three distinct layers in addition to the original hyphal cell wall. Crystal-like structures were seen in the cytoplasm of some of the arthrospores in addition to the usual organelles such as mitochondria, nuclei, and ribosomes. Septum formation by centripetal cell wall growth from the lateral hyphal wall was documented by transmission electron microscopy. However, evidence was also found which suggested that not all internal cell wall development in the fungal hyphae during arthrosporogenesis necessarily led to the formation of mature arthrospores.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

Localization of bacteriophage receptor, clumping factor, and protein A on the cell surface of Staphylococcus aureus.

The surface of several laboratory strains of Staphylococcus aureus were observed with a scanning electron microscope, and the presence of two morphologically characteristic structures--a ridge separating cell surface into old and new surfaces and a concentric circular structure--are described. These two structures seemed to be present universally on the surfaces of cells of the genus Staphylococcus. The removal of the circular structures by a mild treatment of the cell with trichloroacetic acid suggested that this structure seemed to represent circularly arranged teichoic acid. With experiments using morphologically recognizable markers among three of the cell wall components, clumping factor, phage receptor, and protein A, the clumping factor was proven to be specifically localized on the old surface; and more phage receptors were detected on the old surface than on the new surface, but protein A was present all over the cell surface. This indicated that the clumping factor and most of the phage receptors appeared on the cell wall surface in a late stage of the cell growth cycle, but protein A was present in an early stage of the growth. The idea of aging of the cell wall is discussed.     

Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.     

Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study.

A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells. The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells. When increasing dilutions of mAb KT4 were employed, a 'punctuated' labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin. Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension. These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides. Indirect immunodetection was performed in electron microscopy on ultra-thin sections of cells embedded in Araldite resin. The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation. Indirect immunodetection, performed on ultra-thin frozen sections, showed a cytoplasmic and cell wall labelling. However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence. In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4.     

碳/碳材料含硅羟基磷灰石涂层体外成骨细胞相容性研究

目的:成骨细胞在碳/碳复合材料表面羟基磷灰石涂层上有良好的长入,该实验在羟基磷灰石(hydroxyapatite HA)涂层中掺入硅后,研究成骨细胞对该涂层的生物活性,为临床骨科应用提供实验基础。方法:本研究采用化学液相气化沉积/水热法在碳/碳材料表面制备了含硅羟基磷灰石(silicon-hydroxyapatite Si-HA)涂层。在体外成骨细胞相容性的研究中,以HA涂层为对照,通过甲基噻唑基四唑(methylthiazolyl tetrazolium MTT)法测定细胞增殖反应和对细胞毒性反应,碱性磷酸酶(alkaline phosphatase ALP)测定细胞的分化,扫描电镜观察细胞生长形态,免疫荧光显影技术测定细胞的长入。结果:在HA涂层中引入硅后,Si-HA涂层和HA涂层在第2天、第4天的成骨细胞增殖实验表明前者的细胞长入数较后者多,两者的差异有统计学意义,而Si-HA涂层浸泡液中的ALP活性下降较HA涂层而言更明显,两者的差异有统计学意义,电镜扫描及荧光染色均提示在Si-HA涂层中成骨细胞的增值数更多。结论:在HA涂层中引入硅后,改变了涂层自身的晶体结构及表面电荷,同时诱导成骨细胞分泌胶原,使得成骨细胞更好的贴壁生长和增殖,成骨能力增加。在临床骨科植入物表面涂层改性上有很好的应用前景。     

Role of cell shape in determination of the division plane in Schizosaccharomyces pombe: random orientation of septa in spherical cells

The establishment of growth polarity in Schizosaccharomyces pombe cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.     

Structure of the Staphylococcus aureus cell wall determined by the freeze-substitution method.

The fine structure of the Staphylococcus aureus cell wall was determined by electron microscopy with the new technique of rapid freezing and substitution fixation. The surface of the cell wall was covered with a fuzzy coat which consisted of fine fibers or an electron-dense mass. Morphological examination of the cell wall, which was treated sequentially with sodium dodecyl sulfate, trypsin, and trichloroacetic acid, revealed that this coat was partially removed by trypsin digestion and was completely removed by trichloroacetic acid extraction but was not affected by sodium dodecyl sulfate treatment, suggesting that the fuzzy coat consists mostly of a complex of teichoic acids and proteins. This was confirmed by the application of the concanavalin A-ferritin technique for teichoic acid and antiferritin immunoglobulin G technique for protein A.     

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Involvement of [beta]-glucans in the wide-spectrum antimicrobial activity of Williopsis saturnus var. mrakii MUCL 41968 killer toxin

BACKGROUND: Williopsis saturnus var. mrakii MUCL 41968 secretes a 85-kDa glycoprotein killer toxin (WmKT) that displays a cytocidal activity against a wide range of microorganisms, making WmKT a promising candidate for the development of new antimicrobial molecules. Although the killing mechanism of WmKT is still unknown, the toxin was recently proposed to bind to the surface of sensitive microorganisms through the recognition of beta-glucans. Indeed, Saccharomyces cerevisiae strains sensitive to the toxin become resistant when mutated in their beta-glucan synthesis pathway. MATERIALS AND METHODS: To investigate the interaction of WmKT with beta-glucans, we examined in agar diffusion assays the WmKT activity in the presence of enzymes displaying beta-glucanase activity. The toxin activity was also investigated using spheroplasts derived from sensitive yeast cells. The hydrolytic activity of WmKT was studied using specific glucosidase inhibitors as well as various sugar molecules covalently linked to p-nitrophenyl as potential substrates. Finally, the ultrastructural modifications induced by WmKT activity on sensitive yeasts were assessed by scanning electron microscopy. RESULTS: The data reported here support the hypothesis that WmKT binds to sensitive cells using surface-exposed beta-glucans. Indeed beta-glucanase exerts an antagonistic effect on WmKT activity and spheroplasts derived from WmKT-sensitive yeast cells are shown to be resistant to WmKT, suggesting that cell wall beta-glucans are required for WmKT lethal effect. Because WmKT exhibits amino acid sequence similarities with proteins suspected to be glucanase, we also investigated the effect of castanospermine, a potent glucosidase inhibitor, on WmKT activity. Castanospermine completely abolished WmKT killer activity as well as its hydrolytic enzymatic activity against p-nitrophenyl beta-D-glucopyranoside. The scanning electron microscopy analysis of sensitive yeast cells treated with the toxin reveals that WmKT causes cell wall modifications similar to those observed with zymolyase. CONCLUSION: The results reported in this study show that WmKT activity requires an interaction between the mycocin and the cell wall beta-glucans. Moreover, they indicate that WmKT acts on sensitive yeast cells through a hydrolytic activity directed against cell wall beta-glucans that disrupts the yeast cell wall integrity leading to death.     

Mucor rouxii ultrastructure: cyclic AMP and actin cytoskeleton

Summary. A comparative analysis of the effect of two compounds, dibutyryl–cyclic-AMP (dbcAMP) and latrunculin B, on the morphology and ultrastructure of the dimorphic fungus Mucor rouxii under aerobic growth conditions is presented. dbcAMP acts through the sustained activation of protein kinase A, and latrunculin B through the disruption of the actin cytoskeleton. Upon addition of these compounds to the growth medium at any stage of the germination process, cells lost polarised growth and switched to isodiametric growth. The effect was reversible. The morphologies, visualised by light microscopy or scanning electron microscopy (SEM), were alike. A switch from a rough to a smooth surface was observed by SEM when cells were repolarised by removal of the added compound. Ultrastructural changes under both conditions, as observed by transmission electron microscopy, were similar, the main feature being the enlargement of the cell wall, with irregular depositions, and detachment from the cell membrane. dbcAMP-treated cells showed a decrease in the number of glycogen granules compared with control and latrunculin B-treated cells. F-actin staining with fluorescein isothiocyanate–phalloidin showed that both dbcAMP- and latrunculin B-treated cells displayed a much lower fluorescence than control cells, with only a few pale plaques. The results suggest that the sustained activation of protein kinase A, which impairs polarised growth, might exert its effect through a modification of actin cytoskeleton organisation, very probably also involving an integrinlike pathway, as judged by the cell wall detachment and loss of cell adhesiveness of the dbcAMP-treated isodiametric cells. Correspondence and reprints: Departamento de Química Biológica, Pabellón 2, Piso 4, Ciudad Universitaria, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.