scholarly journals Ras GTPase-Activating Protein Regulation of Actin Cytoskeleton and Hyphal Polarity in Aspergillus nidulans

2007 ◽  
Vol 7 (1) ◽  
pp. 141-153 ◽  
Author(s):  
Laura Harispe ◽  
Cecilia Portela ◽  
Claudio Scazzocchio ◽  
Miguel A. Peñalva ◽  
Lisette Gorfinkiel
Keyword(s):  
Carbon Source ◽  
Actin Cytoskeleton ◽  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Protein Domain ◽  
Ras Signaling ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Polarity Establishment

ABSTRACT Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more “true” Ras proteins. A gapAΔ strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapAΔ strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapAΔ disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapAΔ conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapAΔ cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapAΔ cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

scholarly journals MoTea4-Mediated Polarized Growth Is Essential for Proper Asexual Development and Pathogenesis in Magnaporthe oryzae

2010 ◽  
Vol 9 (7) ◽  
pp. 1029-1038 ◽  
Author(s):  
Rajesh N. Patkar ◽  
Angayarkanni Suresh ◽  
Naweed I. Naqvi
Keyword(s):  
Actin Cytoskeleton ◽  
Magnaporthe Oryzae ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Coiled Coil ◽  
Polarized Growth ◽  
Wild Type ◽  
Cortical Actin ◽  
Content Type ◽  
Aerial Hyphae

ABSTRACT Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in the rice blast fungus Magnaporthe oryzae (MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispensable for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the tea4Δ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4Δ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The tea4Δ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the tea4Δ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in Magnaporthe.

scholarly journals Tumor Suppression of Ras GTPase-Activating Protein RASA5 through Antagonizing Ras Signaling Perturbation in Carcinomas

iScience ◽  
2019 ◽  
Vol 21 ◽  
pp. 1-18 ◽  
Author(s):  
Lili Li ◽  
Yichao Fan ◽  
Xin Huang ◽  
Jie Luo ◽  
Lan Zhong ◽  
...  
Keyword(s):  
Tumor Suppression ◽  
Ras Signaling ◽  
Gtpase Activating Protein ◽  
Ras Gtpase

Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium

2000 ◽  
Vol 113 (12) ◽  
pp. 2253-2265 ◽  
Author(s):  
M. Dumontier ◽  
P. Hocht ◽  
U. Mintert ◽  
J. Faix
Keyword(s):  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Fluorescent Protein ◽  
Colony Growth ◽  
Dominant Negative ◽  
Related Protein ◽  
Wild Type ◽  
Equal Capacity ◽  
Green Fluorescent

The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.

scholarly journals Exploring the Function of Alcohol Dehydrogenases During the Endophytic Life of Azoarcus Sp. Strain BH72

2011 ◽  
Vol 24 (11) ◽  
pp. 1325-1332 ◽  
Author(s):  
Andrea Krause ◽  
Birte Bischoff ◽  
Lucie Miché ◽  
Federico Battistoni ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Carbon Source ◽  
Fluorescent Protein ◽  
Endophytic Bacterium ◽  
Microtiter Plate ◽  
Wild Type ◽  
Protein Fusion ◽  
High Concentration ◽  
Rice Roots ◽  
Genes Encoding ◽  
Green Fluorescent

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

Phosphatidylinositol phospholipase C mediates carbon sensing and vegetative nuclear duplication rates in Aspergillus nidulans

2011 ◽  
Vol 57 (7) ◽  
pp. 611-616 ◽  
Author(s):  
Ana Paula de Figueiredo Conte Vanzela ◽  
Suraia Said ◽  
Rolf Alexander Prade
Keyword(s):  
Molecular Weight ◽  
Carbon Source ◽  
Phospholipase C ◽  
Aspergillus Nidulans ◽  
Nuclear Division ◽  
Deficient Mutant ◽  
Wild Type ◽  
Pectinolytic Enzymes ◽  
In The Wild ◽  
Phosphatidylinositol Phospholipase C

In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6–7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.

Aspergillus galactose metabolism is more complex than that of Saccharomyces: the story of GalDGAL7 and GalEGAL1

Botany ◽  
2013 ◽  
Vol 91 (7) ◽  
pp. 467-477 ◽  
Author(s):  
Md. Kausar Alam ◽  
Susan G.W. Kaminskyj
Keyword(s):  
Aspergillus Nidulans ◽  
Minimal Medium ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Galactose Metabolism ◽  
Sequence Identity ◽  
Functional Homology ◽  
Wild Type Phenotype ◽  
Polymerase Chain ◽  
Green Fluorescent

Saccharomyces cerevisiae Hansen GAL1 (galactokinase) generates galactose-1-phosphate; GAL7 (galactose-1-phosphate uridylyltransferase) transfers UDP between galactose or glucose and their respective sugar-1-phosphate conjugates, and both are essential on galactose. Aspergillus nidulans ANID_04957 has 41% amino acid sequence identity with GAL1; ANID_06182 has 50% sequence identity with GAL7. The names Aspergillus nidulans GalE (galactokinase) and GalD (galactose-1-phosphate uridylyltransferase) are consistent with prior studies. Complemented galDΔ:ScGAL7 and galEΔ:ScGAL1 strains had wild-type phenotype, demonstrating functional homology. The galD5 and galE9 alleles were truncated. Strains galDΔ and galD5 were impaired on minimal medium containing 1% galactose (MM-Gal) at pH 7.5 and did not grow on MM-Gal pH 4.5. Strains galEΔ and galE9 grew on MM-Gal at both pH levels. Strains galDΔ and galEΔ produced wild-type conidiophores on minimal medium containing 1% glucose (MM-Glu) but few spores; for both, sporulation was lower on MM-Gal pH 7.5. GalD-GFP (green fluorescent protein) and GalE-GFP were cytosolic and upregulated on MM-Gal, consistent with quantitative real-time polymerase chain reaction. Galactofuranose immunolocalization in galDΔ resembled wild type on MM-Glu but was reduced on MM-Gal. The galEΔ strains had immunolocalizable Galf on all these media. Strains galDΔ and galEΔ were more sensitive to calcofluor, caspofungin, and itraconazole on MM-Gal. Neither galD nor galE is essential on galactose at high pH, implying additional routes for galactose metabolism in Aspergillus. Aspergillus galactose metabolism is more complex than that of S. cerevisiae.

Conidial Germination in Aspergillus nidulans Requires RAS Signaling and Protein Synthesis

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 647-656 ◽  
Author(s):  
Nir Osherov ◽  
Gregory May
Keyword(s):  
Protein Synthesis ◽  
Carbon Source ◽  
Aspergillus Nidulans ◽  
Nuclear Division ◽  
Conidial Germination ◽  
Ras Signaling ◽  
Restrictive Temperature ◽  
Malonyl Coa ◽  
Initiation Of Translation ◽  
High Degree

Abstract The dormant spores of Aspergillus nidulans become competent for growth and nuclear division in a process called conidial germination. To analyze the molecular details of conidial germination, we developed a genetic screen in which we identified spore germination-deficient mutants that are blocked in this process at the restrictive temperature. These mutants defined eight genes, of which we identified five. Four of the five were directly involved in translation and protein folding, and the fifth showed a high degree of homology to a malonyl CoA synthetase. These results suggest that out of a wide array of processes occurring during conidial germination, translation is essential if germination is to proceed. We also show that conidia containing a mutant-activated form of rasA, the ras homologue in A. nidulans, germinate in the absence of an inducing carbon source, suggesting an important role for rasA signaling in conidial germination. Together these data suggest a model by which a carbon source activates a ras-dependent sensory mechanism, inducing translation and leading to conidial germination. This study shows that conidial germination in A. nidulans requires protein synthesis and that the initiation of translation is linked, through an as yet to be determined signaling cascade that includes rasA, to a carbon-source-sensing apparatus.

Nf1 deficiency cooperates with oncogenic K-RAS to induce acute myeloid leukemia in mice

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3629-3632 ◽  
Author(s):  
Briony A. Cutts ◽  
Anna-Karin M. Sjogren ◽  
Karin M. E. Andersson ◽  
Annika M. Wahlstrom ◽  
Christin Karlsson ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Myeloproliferative Disorders ◽  
Ras Signaling ◽  
Gtpase Activating Protein ◽  
Oncogenic Ras ◽  
Ras Genes ◽  
Ras Gtpase ◽  
Acute Myeloid

Abstract Hyperactive RAS signaling is caused by mutations in RAS genes or a deficiency of the neurofibromatosis gene (NF1) and is common in myeloid malignancies. In mice, expression of oncogenic K-RAS or inactivation of Nf1 in hematopoietic cells results in myeloproliferative disorders (MPDs) that do not progress to acute myeloid leukemia (AML). Because NF1 is a RAS-GTPase–activating protein it has been proposed that NF1 deficiency is functionally equivalent to an oncogenic RAS. It is not clear, however, whether Nf1 deficiency would be redundant in K-RAS–induced MPD development or whether the 2 mutations would cooperate in leukemogenesis. Here, we show that the simultaneous inactivation of Nf1 and expression of K-RASG12D in mouse hematopoietic cells results in AML that was fatal in primary mice within 4 weeks and transplantable to sublethally irradiated secondary recipients. The data point to a strong cooperation between Nf1 deficiency and oncogenic K-RAS.

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