ABSTRACT
To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244-538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination.
Subject(s)
CDC2 Protein Kinase/metabolism , Calmodulin-Binding Proteins/metabolism , Cell Division , Cell Membrane/physiology , Actins/metabolism , Amino Acid Sequence , Animals , CHO Cells , Calmodulin-Binding Proteins/genetics , Cell Size , Cricetinae , Fibroblasts , Gene Expression , Humans , Mutagenesis, Site-Directed , Phosphorylation , Point Mutation , Time Factors , Tropomyosin/analysis , Tropomyosin/isolation & purificationABSTRACT
Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2, and HOS3) have been cloned from Candida albicans and characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylases HDA1 and RPD3 is down-regulated in the opaque phase of the white-opaque transition in strain WO-1. HOS3 is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase. HDA1 and RPD3 were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion of HDA1 resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion of RPD3 resulted in an increase in the frequency of switching in both directions. Deletion of HDA1 resulted in reduced white-phase-specific expression of the EFG1 3.2-kb transcript, but had no significant effect on white-phase-specific expression of WH11 or opaque-phase-specific expression of OP4, SAP1, and SAP3. Deletion of RPD3 resulted in reduced opaque-phase-specific expression of OP4, SAP1, and SAP3 and a slight reduction of white-phase-specific expression of WH11 and 3.2-kb EFG1. Deletion of neither HDA1 nor RPD3 affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein gene MCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genes HDA1 and RPD3 play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.
Subject(s)
Fungal Proteins/physiology , Histone Deacetylases/physiology , Intracellular Signaling Peptides and Proteins , Nuclear Proteins , Plant Proteins , Repressor Proteins , Saccharomyces cerevisiae Proteins , Schizosaccharomyces pombe Proteins , Amino Acid Sequence , Candida albicans/genetics , Candida albicans/physiology , Carrier Proteins/genetics , Cloning, Molecular , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Gene Expression , Histone Deacetylases/genetics , Minichromosome Maintenance 1 Protein , Molecular Sequence Data , Mutagenesis , Sequence Homology, Amino Acid , Transcription Factors/geneticsABSTRACT
Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.
Subject(s)
Candida albicans/genetics , Histone Deacetylase Inhibitors , Mutation , Antifungal Agents/pharmacology , Candida albicans/drug effects , Candida albicans/physiology , Cell Division/drug effects , Hydroxamic Acids/pharmacology , Phenotype , Up-RegulationABSTRACT
A variety of methods have emerged for genetic fingerprinting the infectious fungi. One of the most versatile is Southern blot hybridization with species-specific complex DNA probes that include sequences that identify hypervariable, moderately variable and invariant genomic sequences. These probes assess genetic relatedness at all the necessary levels including identical, highly related but non-identical, moderately related and unrelated. Methods are described for cloning complex probes, characterizing them and verifying their effectiveness at the different levels of resolution. The complex probes that have been developed for Candida albicans, C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis and Aspergillus fumigatus are described and discussed.
Subject(s)
Candida/genetics , DNA Fingerprinting/methods , DNA Probes , Aspergillus fumigatus , Blotting, Southern , Candida/classification , Candida/pathogenicity , Cloning, Molecular , DNA, Fungal/genetics , Genetic Variation , Genome, Fungal , Humans , Repetitive Sequences, Nucleic Acid , Species SpecificityABSTRACT
BACKGROUND: The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS: A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS: Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.
Subject(s)
Cell Adhesion/physiology , Myosins/physiology , Protozoan Proteins , Animals , Cell Movement/physiology , Dictyostelium/physiology , Mutagenesis , Myosins/genetics , Phagocytosis/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiologyABSTRACT
We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.
Subject(s)
Chemotaxis/physiology , Cyclic AMP/metabolism , Dictyostelium/metabolism , Mitochondria/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Cell Size , Dictyostelium/genetics , Dictyostelium/growth & development , Dictyostelium/ultrastructure , Fluorescent Dyes/metabolism , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , MAP Kinase Kinase 1 , Mitochondria/chemistry , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Molecular Sequence Data , Organic Chemicals , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , SolubilityABSTRACT
Candida parapsilosis accounts for a significant number of nosocomial fungemias, but in fact, no effective and verified genetic fingerprinting method has emerged for assessing the relatedness of independent isolates for epidemiological studies. A complex 15-kb DNA fingerprinting probe, Cp3-13, was therefore isolated from a library of C. parapsilosis genomic DNA fragments. The efficacy of Cp3-13 for DNA fingerprinting was verified by a comparison of its clustering capacity with those of randomly amplified polymorphic DNA analysis and internally transcribed spacer region sequencing, by testing species specificity, and by assessing its capacity to identify microevolutionary changes both in vitro and in vivo. Southern blot hybridization of EcoRI/SalI-digested DNA with Cp3-13 provides a fingerprinting system that (i) identifies the same strain in independent isolates, (ii) discriminates between unrelated isolates, (iii) separates independent isolates into valid groups in a dendrogram, (iv) identifies microevolution in infecting populations, and (v) is amenable to automatic computer-assisted DNA fingerprint analysis. This probe is now available for epidemiological studies.
Subject(s)
Candida/classification , Candida/genetics , DNA, Bacterial/genetics , Phylogeny , Candida/isolation & purification , Candidiasis/blood , Candidiasis/diagnosis , Candidiasis/microbiology , Cloning, Molecular/methods , DNA Fingerprinting/methods , DNA Probes , Deoxyribonuclease EcoRI , Humans , Random Amplified Polymorphic DNA Technique , Restriction MappingABSTRACT
Strains of Candida albicans obtained from human immunodeficiency virus (HIV)-positive individuals prior to their first episode of oral thrush were already in a high-frequency mode of switching and were far more resistant to a number of antifungal drugs than commensal isolates from healthy individuals. Switching in these isolates also had profound effects both on susceptibility to antifungal drugs and on the levels of secreted proteinase activity. These results suggest that commensal strains colonizing HIV-positive individuals either undergo phenotypic alterations or are replaced prior to the first episode of oral thrush. They also support the suggestion that high-frequency phenotypic switching functions as a higher-order virulence trait, spontaneously generating in colonizing populations variants with alterations in a variety of specific virulence traits.
Subject(s)
AIDS-Related Opportunistic Infections/microbiology , Antifungal Agents/pharmacology , Candida albicans/genetics , Candidiasis, Oral/microbiology , Candida albicans/drug effects , Candida albicans/isolation & purification , Candidiasis, Oral/etiology , Female , Genetic Variation , HIV Seropositivity , Humans , Male , Microbial Sensitivity Tests , PhenotypeABSTRACT
Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.
Subject(s)
Chemotaxis , Cyclic AMP-Dependent Protein Kinases/physiology , Dictyostelium/physiology , Protozoan Proteins , Pseudopodia/physiology , 3',5'-Cyclic-AMP Phosphodiesterases , Animals , Cell Aggregation , Computer Simulation , Cyclic AMP/physiology , Cyclic AMP-Dependent Protein Kinases/genetics , Dictyostelium/cytology , Dictyostelium/genetics , Genes, Protozoan , Mutation , Myosins/metabolism , Pseudopodia/metabolism , Signal Transduction , Transformation, GeneticABSTRACT
Cell behavior is three-dimensional (3-D), even when it takes place on a flat surface. Migrating cells form pseudopods on and off the substratum, and the cell body undergoes height changes associated with a 1 min behavior cycle. Inside the cell, the nucleus has a 3-D migratory cycle, and vesicles move up and down in the z-axis as a cell locomotes. For these reasons, the two-dimensional (2-D) analysis of cellular and subcellular behavior is, in many cases, inadequate. We have, therefore, developed 3-D motion analysis systems that reconstruct the cell surface, nucleus, pseudopods, and vesicles of living, crawling cells in 3-D at time intervals as short as 1 s, and compute more than 100 parameters of motility and dynamics morphology at 1-s intervals. We are now in the process of developing a multimode reconstruction system that will allow us to reconstruct and analyze fluorescently tagged molecular complexes within the differential interference contrast-imaged subcellular architecture of a crawling cell. These evolving technologies should find wide application for a host of biomedical problems.
Subject(s)
Cell Movement , Dictyostelium/physiology , Dictyostelium/ultrastructure , Image Processing, Computer-Assisted , Microscopy, Interference/instrumentation , Microscopy, Phase-Contrast/instrumentation , Animals , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cell Nucleus/physiology , Cell Nucleus/ultrastructure , Microscopy, Interference/methods , Microscopy, Phase-Contrast/methods , Organelles/physiology , Organelles/ultrastructure , Pseudopodia/physiology , Pseudopodia/ultrastructure , Software , User-Computer InterfaceABSTRACT
An in vitro model has been developed for analyzing the two developmental phases of human dendritic cell (DC) migration. Employing the age of the culture and the addition of GM-CSF, IL-4, and serum to regulate cellular phenotype, and glass coated with acid-precipitated human plasma proteins to facilitate persistent DC translocation, the model produces three sequential in vitro phenotypes with the following suggested in vivo counterparts: (1) DCs recently isolated from blood, which are highly polar and motile, and reflect the behavior of "undifferentiated" DCs that must extravasate from the blood stream and migrate into peripheral tissue; (2) large, nonmotile, stellate DCs, which reflect the highly "differentiated" signature phenotype of DCs in peripheral tissue, whose function is to capture foreign antigens; and (3) the large, motile "dedifferentiated" DCs, which reflect the behavior of "veiled cells" that have captured an antigen, retracted dendritic processes, migrated out of peripheral tissue, and are in the process of transporting a captured antigen to a proximal draining lymph node for presentation to T cells. Computer-assisted motion analysis of the three sequential phenotypes and fluorescent staining of F-actin reveal three unique behavioral states and unique cellular architecture consistent with inferred in vivo function. This in vitro model should serve as a starting point for elucidating the cues and molecular mechanisms involved in the regulation of DC differentiation and motility.
Subject(s)
Actins/metabolism , Cell Movement/immunology , Cytoskeleton/ultrastructure , Dendritic Cells/cytology , Dendritic Cells/metabolism , Cell Differentiation/immunology , Cell Membrane/physiology , Cell Size/immunology , Cytoskeleton/metabolism , Dendritic Cells/ultrastructure , Flow Cytometry , Humans , Image Cytometry , Image Processing, Computer-Assisted , In Vitro Techniques , Intracellular Membranes/physiology , Microscopy, Fluorescence , PhenotypeABSTRACT
The Candida albicans gene EFG1 encodes a putative trans-acting factor. In strain WO-1, which undergoes the white-opaque transition, EFG1 is transcribed as a 3.2-kb mRNA in white-phase cells and a less-abundant 2.2-kb mRNA in opaque-phase cells. cDNA sequencing and 5' rapid amplification of cDNA ends analysis demonstrate that the major difference in molecular mass of the two transcripts is due to different transcription start sites. EFG1 null mutants form opaque-phase colonies and express the opaque-phase cell phenotype at 25 degrees C. When shifted from 25 to 42 degrees C, mutant opaque-phase cells undergo phenotypic commitment to the white phase, which includes deactivation of the opaque-phase-specific gene OP4 and activation of the white-phase-specific gene WH11, as do wild-type opaque-phase cells. After the commitment event, EFG1 null mutant cells form daughter cells which have the smooth (pimpleless) surface of white-phase cells but the elongate morphology of opaque-phase cells. Taken together, these results demonstrate that EFG1 expression is not essential for the switch event per se, but is essential for a subset of phenotypic characteristics necessary for the full expression of the phenotype of white-phase cells. These results demonstrate that EFG1 is not the site of the switch event, but is, rather, downstream of the switch event.
Subject(s)
Candida albicans/growth & development , DNA-Binding Proteins , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Transcription Factors , 5' Untranslated Regions/genetics , Base Sequence , Blotting, Northern , Blotting, Southern , Candida albicans/genetics , Candida albicans/metabolism , Candida albicans/ultrastructure , Colony Count, Microbial , DNA, Complementary/genetics , Gene Deletion , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation , Phenotype , Promoter Regions, Genetic , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.
Subject(s)
Cell Movement/physiology , Tropomyosin/physiology , Animals , CHO Cells , Cell Adhesion , Cell Division/physiology , Cell Size , Cricetinae , Humans , Image Processing, Computer-Assisted , Recombinant Fusion Proteins/biosynthesis , Tropomyosin/biosynthesis , Tropomyosin/geneticsABSTRACT
Although Candida glabrata has emerged in recent years as a major fungal pathogen, there have been no reports demonstrating that it undergoes either the bud-hypha transition or high-frequency phenotypic switching, two developmental programs believed to contribute to the pathogenic success of other Candida species. Here it is demonstrated that C. glabrata undergoes reversible, high-frequency phenotypic switching between a white (Wh), light brown (LB), and dark brown (DB) colony phenotype discriminated on an indicator agar containing 1 mM CuSO(4). Switching regulates the transcript level of the MT-II metallothionein gene(s) and a newly discovered gene for a hemolysin-like protein, HLP. The relative MT-II transcript levels in Wh, LB, and DB cells grown in the presence of CuSO(4) are 1:27:81, and the relative transcript levels of HLP are 1:20:35. The relative MT-II and HLP transcript levels in cells grown in the absence of CuSO(4) are 1:20:30 and 1:20:25, respectively. In contrast, switching has little or no effect on the transcript levels of the genes MT-I, AMT-I, TRPI, HIS3, EPAI, and PDHI. Switching of C. glabrata is not associated with microevolutionary changes identified by the DNA fingerprinting probe Cg6 and does not involve tandem amplification of the MT-IIa gene, which has been shown to occur in response to elevated levels of copper. Finally, switching between Wh, LB, and DB occurred in all four clinical isolates examined in this study. As in Candida albicans, switching in C. glabrata may provide colonizing populations with phenotypic plasticity for rapid responses to the changing physiology of the host, antibiotic treatment, and the immune response, through the differential regulation of genes involved in pathogenesis. More importantly, because C. glabrata is haploid, a mutational analysis of switching is now feasible.
Subject(s)
Candida/genetics , Gene Expression Regulation, Fungal , Hemolysin Proteins/genetics , Metallothionein/genetics , Amino Acid Sequence , Copper Sulfate/pharmacology , Molecular Sequence Data , Phenotype , RNA, Messenger/analysisABSTRACT
DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.
Subject(s)
DNA Fingerprinting/methods , Fungi/classification , Fungi/genetics , Mycoses/microbiology , DNA Probes , DNA, Fungal/analysis , DNA, Fungal/genetics , Humans , Mycological Typing Techniques , Polymerase Chain Reaction , Random Amplified Polymorphic DNA Technique , SoftwareABSTRACT
Although the traditional role of clathrin has been in vesicle trafficking and the internalization of receptors, a novel role in cytokinesis was recently revealed in an analysis of a clathrin-minus Dictyostelium mutant (chc(-)). chc(-) cells grown in suspension were demonstrated to be defective in assembling myosin II into a normal contractile ring. To test whether this defect reflected a more general one of cytoskeletal dysfunction, chc(-) cells were analyzed for cell polarity, pseudopod formation, uropod stability, cell locomotion, chemotaxis, cytoskeletal organization and vesicle movement. chc(-) cells crawled, chemotaxed, localized F-actin in pseudopods, organized their microtubule cytoskeleton in a relatively normal fashion and exhibited normal vesicle dynamics. Although chc(-) cells extended pseudopods from the anterior half of the cell with the same frequency as normal chc(+) cells, they extended pseudopods at twice the normal frequency from the posterior half of the cell. The uropods of chc(-) cells also exhibited spatial instability. These defects resulted in an increase in roundness, a reduction in polarity, a reduction in velocity, a dramatic increase in turning, a high frequency of 180 degrees direction reversals and a decrease in the efficiency of chemotaxis. All defects were reversed in a rescued strain. These results are the first to suggest a novel role for clathrin in cell polarity, pseudopod formation, uropod stability and locomotion. It is hypothesized that clathrin functions to suppress pseudopod formation and to stabilize the uropod in the posterior half of a crawling cell, two behavioral characteristics that are essential for the maintenance of cellular polarity, efficient locomotion and efficient chemotaxis.
Subject(s)
Cell Movement , Cell Polarity , Clathrin/metabolism , Dictyostelium/cytology , Pseudopodia/metabolism , Actins/metabolism , Animals , Cell Size , Chemotaxis , Clathrin/genetics , Cyclic AMP/metabolism , Dictyostelium/genetics , Dictyostelium/physiology , Gene Deletion , Kinetics , Microtubules/metabolismABSTRACT
To have a better understanding of the role of Candida dubliniensis in clinical infections, it is essential that microbiology laboratories can identify this species rapidly and accurately in clinical specimens. C. dubliniensis has been reported to lack the ability to utilize xylose (XYL) and alpha-methyl-D-glucoside (MDG) and to grow poorly or not at all at 45 degrees C, whereas Candida albicans isolates utilize XYL and MDG and usually grow well at 45 degrees C. We tested 66 isolates of C. dubliniensis and 100 isolates of C. albicans with both the API 20C AUX and Vitek YBC systems to evaluate the ability of the XYL and MDG tests contained within each of these systems to distinguish between the two species. The ability to grow at 45 degrees C was also examined. None of the C. dubliniensis isolates grew at 45 degrees C, and 23 of 100 C. albicans isolates (23%) exhibited poor or no growth at 45 degrees C. The XYL and MDG tests contained within the API 20C AUX system were both negative for all 66 C. dubliniensis isolates and were positive for 98 (XYL) and 56 (MDG) of the 100 C. albicans isolates. With the Vitek system, 64 of 66 C. dubliniensis isolates (97.0%) were XYL negative and 63 (95.0%) were MDG negative. Conversely, 96 of 100 C. albicans isolates (96.0%) were XYL positive and 100 (100.0%) were MDG positive with the Vitek system. Clinical microbiology laboratories could use lack of growth at 45 degrees C and a negative XYL test with either the API 20C AUX or Vitek yeast identification system to provide a presumptive identification of C. dubliniensis. A negative MDG test result with either system would also be helpful but may misclassify C. albicans as C. dubliniensis, especially when the API 20C AUX system is used.
Subject(s)
Candida/classification , Candida/physiology , Candidiasis/microbiology , Methylglucosides/metabolism , Xylose/metabolism , Candida/growth & development , Candida/isolation & purification , Candida albicans/classification , Candida albicans/growth & development , Candida albicans/isolation & purification , Candida albicans/metabolism , Culture Media , Humans , Reagent Kits, Diagnostic , Reproducibility of Results , Sensitivity and Specificity , TemperatureABSTRACT
Candida albicans WO-1 switches reversibly and at high frequency between a white and an opaque colony-forming phenotype that includes dramatic changes in cell morphology and physiology. A misexpression strategy has been used to investigate the role of the opaque-phase-specific gene PEP1 (SAP1), which encodes a secreted aspartyl proteinase, in the expression of the unique opaque-phase phenotype and phase-specific virulence in two animal models. The PEP1 (SAP1) open reading frame was inserted downstream of the promoter of the white-phase-specific gene WH11 in the transforming vector pCPW7, and the resulting transformants were demonstrated to misexpress PEP1 (SAP1) in the white phase. Misexpression did not confer any of the unique morphological characteristics of the opaque phase to cells in the white phase and had no effect on the switching process. However, misexpression conferred upon white-phase cells the increased capacity of opaque-phase cells to grow in medium in which protein was the sole nitrogen source. Misexpression of PEP1 (SAP1) had no effect on the virulence of white-phase cells in a systemic mouse model, in which white-phase cells were already more virulent than opaque-phase cells. Misexpression did, however, confer upon white-phase cells the dramatic increase in colonization of skin in a cutaneous mouse model that was exhibited by opaque-phase cells. Misexpression of PEP1 (SAP1) conferred upon white-phase cells two dissociable opaque-phase characteristics: increased adhesion and the capacity to cavitate skin. The addition of pepstatin A to the cutaneous model inhibited the latter, but not the former, suggesting that the latter is effected by released enzyme, while the former is effected by cell-associated enzyme.
Subject(s)
Aspartic Acid Endopeptidases/genetics , Candida albicans/genetics , Candida albicans/pathogenicity , Candidiasis, Cutaneous/microbiology , Animals , Aspartic Acid Endopeptidases/metabolism , Blotting, Northern , Blotting, Southern , Candida albicans/enzymology , Candida albicans/growth & development , Disease Models, Animal , Gene Expression , Genes, Fungal , Mice , Plasmids , Transformation, Genetic , VirulenceABSTRACT
Using a newly developed gradient chamber to provide independent measurements of chemokinesis (stimulated motility) and chemotaxis (stimulated motility up a concentration gradient) of individual T-helper cells, it was recently demonstrated that HIV-induced T-cell syncytia release two distinct chemotactic activities that are separable by their rates of diffusion. The molecular masses of the two chemoattractant activities were estimated to be 30 and 120 kDa. The higher molecular mass activity was demonstrated to be the viral glycoprotein gp120. In an attempt to identify the lower molecular mass activity, chemotaxis and chemokinesis of T-helper cells were analyzed in individual concentration gradients of the virally encoded proteins Rev, p24, Tat and Nef. None functioned alone as a chemoattractant, but both Tat and Nef alone functioned as chemokinetic stimulants. When Tat and Nef were used together to generate parallel gradients, they stimulated chemotaxis. Antibody to either Tat or Nef neutralized the lower molecular mass chemotactic activity released by syncytia. The addition of antibody to the CD4 receptor or the addition of soluble CD4 inhibited high molecular mass chemotactic activity but not the low molecular mass chemotactic activity in HIV-induced syncytium-conditioned medium, demonstrating that the former but not the latter activity is mediated through the CD4 receptor. These results identify the combination of Nef and Tat as the lower molecular mass T cell chemoattractant released by HIV-induced syncytia, and provide the first evidence suggesting that parallel concentration gradients of two proteins are necessary for chemotaxis.