scholarly journals The Mitochondrial Dnm1-Like Fission Component Is Required for lga2 -Induced Mitophagy but Dispensable for Starvation-Induced Mitophagy in Ustilago maydis

2012 ◽  
Vol 11 (9) ◽  
pp. 1154-1166 ◽  
Author(s):  
Fernanda Nieto-Jacobo ◽  
Denise Pasch ◽  
Christoph W. Basse
Keyword(s):  
Sexual Development ◽  
Fluorescent Protein ◽  
Developmental Process ◽  
Mitochondrial Fission ◽  
Mitochondrial Protein ◽  
Ustilago Maydis ◽  
Mitochondrial Rna ◽  
Type Locus ◽  
Content Type

ABSTRACT Selective elimination of mitochondria by autophagy (mitophagy) is a crucial developmental process to dispose of disintegrated or superflous organelles. However, little is known about underlying regulatory mechanisms. We have investigated mitophagy in response to conditional overexpression of the a2 mating-type locus gene lga2 , which encodes a small mitochondrial protein critically involved in uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis . In this study, we show that conditional overexpression of lga2 efficiently triggers mitophagy that is dependent on atg8 and atg11 , consistent with selective autophagy. lga2 -triggered mitophagy is preceded by mitochondrial dysfunction, including depletion of mitochondrial RNA transcripts, and is mechanistically distinct from starvation-induced mitophagy despite a common requirement for atg11 . In particular, lga2 -triggered mitophagy strongly depends on the mitochondrial fission factor Dnm1, but it is only slightly affected by N -acetylcysteine, which is an inhibitor of starvation-induced mitophagy. To further delineate the role of mitochondrial fission, we analyzed lga2 effects in Δfis1 mutants. This revealed that mitochondrial fragmentation was only attenuated and mitophagy was largely unaffected. In further support of a Fis1-independent role for Dnm1, mitochondrial association of green fluorescent protein-tagged Dnm1 as well as Dnm1-opposed mitochondrial fusion during sexual development were fis1 independent. In conclusion, our results specify the role of the mitochondrial fission factor Dnm1 in mitophagy and uncover differences between mitophagy pathways in the same cellular system.

scholarly journals Deletion of ptn1, a PTEN/TEP1 Orthologue, in Ustilago maydis Reduces Pathogenicity and Teliospore Development

2018 ◽  
Vol 5 (1) ◽  
pp. 1 ◽  
Author(s):  
Lalu Vijayakrishnapillai ◽  
John Desmarais ◽  
Michael Groeschen ◽  
Michael Perlin
Keyword(s):  
Defense Mechanisms ◽  
Developmental Process ◽  
Signal Transduction Pathway ◽  
Ustilago Maydis ◽  
Suppressor Gene ◽  
Tumor Formation ◽  
Proliferation And Apoptosis ◽  
Mating Efficiency ◽  
Percent Germination

The PTEN/PI3K/mTOR signal transduction pathway is involved in the regulation of biological processes such as metabolism, cell growth, cell proliferation, and apoptosis. This pathway has been extensively studied in mammals, leading to the conclusion that PTEN is a major tumor suppressor gene. PTEN orthologues have been characterized in a variety of organisms, both vertebrates and non-vertebrates, and studies of the associated PTEN/PI3K/mTOR pathway indicate that it is widely conserved. Studies in fungal systems indicated a role of PTEN in fungal defense mechanisms in Candida albicans, and in the developmental process of sporulation in Saccharomyces cerevisiae. The present study was aimed at investigating the role of the PTEN ortholog, ptn1, in Ustilago maydis, the pathogen of maize. U. maydis ptn1 mutant strains where ptn1 gene is deleted or overexpressed were examined for phenotypes associate with mating, virulence and spore formation. While the overexpression of ptn1 had no substantial effects on virulence, ptn1 deletion strains showed slight reductions in mating efficiency and significant reductions in virulence; tumor formation on stem and/or leaves were severely reduced. Moreover, tumors, when present, had significantly lower levels of mature teliospores, and the percent germination of such spores was similarly reduced. Thus, ptn1 is required for these important aspects of virulence in this fungus.

scholarly journals Regulation of Pathogenic Spore Germination by CgRac1 in the Fungal Plant Pathogen Colletotrichum gloeosporioides

2011 ◽  
Vol 10 (8) ◽  
pp. 1122-1130 ◽  
Author(s):  
Iris Nesher ◽  
Anna Minz ◽  
Leonie Kokkelink ◽  
Paul Tudzynski ◽  
Amir Sharon
Keyword(s):  
Cell Division ◽  
Plant Pathogen ◽  
Germ Tube ◽  
Colletotrichum Gloeosporioides ◽  
Fluorescent Protein ◽  
Nuclear Division ◽  
Simultaneous Formation ◽  
Content Type ◽  
Germ Tubes

ABSTRACT Colletotrichum gloeosporioides is a facultative plant pathogen: it can live as a saprophyte on dead organic matter or as a pathogen on a host plant. Different patterns of conidial germination have been recognized under saprophytic and pathogenic conditions, which also determine later development. Here we describe the role of CgRac1 in regulating pathogenic germination. The hallmark of pathogenic germination is unilateral formation of a single germ tube following the first cell division. However, transgenic strains expressing a constitutively active CgRac1 (CA-CgRac1) displayed simultaneous formation of two germ tubes, with nuclei continuing to divide in both cells after the first cell division. CA-CgRac1 also caused various other abnormalities, including difficulties in establishing and maintaining cell polarity, reduced conidial and hyphal adhesion, and formation of immature appressoria. Consequently, CA-CgRac1 isolates were completely nonpathogenic. Localization studies with cyan fluorescent protein (CFP)-CgRac1 fusion protein showed that the CgRac1 protein is abundant in conidia and in hyphal tips. Although the CFP signal was equally distributed in both cells of a germinating conidium, reactive oxygen species accumulated only in the cell that produced a germ tube, indicating that CgRac1 was active only in the germinating cell. Collectively, our results show that CgRac1 is a major regulator of asymmetric development and that it is involved in the regulation of both morphogenesis and nuclear division. Modification of CgRac1 activity disrupts the morphogenetic program and prevents fungal infection.

scholarly journals Neurospora crassa NKIN2, a Kinesin-3 Motor, Transports Early Endosomes and Is Required for Polarized Growth

2013 ◽  
Vol 12 (7) ◽  
pp. 1020-1032 ◽  
Author(s):  
Constanze Seidel ◽  
Sergio David Moreno-Velásquez ◽  
Meritxell Riquelme ◽  
Reinhard Fischer
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Early Endosome ◽  
Mechanical Forces ◽  
Structural Components ◽  
Content Type ◽  
Early Endosomes ◽  
Polarized Cells ◽  
Green Fluorescent

ABSTRACT Biological motors are molecular nanomachines, which convert chemical energy into mechanical forces. The combination of mechanoenzymes with structural components, such as the cytoskeleton, enables eukaryotic cells to overcome entropy, generate molecular gradients, and establish polarity. Hyphae of filamentous fungi are among the most polarized cells, and polarity defects are most obvious. Here, we studied the role of the kinesin-3 motor, NKIN2, in Neurospora crassa . We found that NKIN2 localizes as fast-moving spots in the cytoplasm of mature hyphae. To test whether the spots represented early endosomes, the Rab5 GTPase YPT52 was used as an endosomal marker. NKIN2 colocalized with YPT52. Deletion of nkin2 caused strongly reduced endosomal movement. Combined, these results confirm the involvement of NKIN2 in early endosome transport. Introduction of a rigor mutation into NKIN2 labeled with green fluorescent protein (GFP) resulted in decoration of microtubules. Interestingly, NKIN2 rigor was associated with a subpopulation of microtubules, as had been shown earlier for the Aspergillus nidulans orthologue UncA. Other kinesins did not show this specificity.

scholarly journals Functional Characterization of a New Member of the Cdk9 Family in Aspergillus nidulans

2010 ◽  
Vol 9 (12) ◽  
pp. 1901-1912 ◽  
Author(s):  
Friederike Bathe ◽  
Claudia Kempf ◽  
Stephen A. Osmani ◽  
Aysha H. Osmani ◽  
Sabrina Hettinger ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Transcription Elongation ◽  
Functional Characterization ◽  
Asexual Development ◽  
Full Spectrum ◽  
Content Type ◽  
Essential Components

ABSTRACT Cdk9-like kinases in complex with T-type cyclins are essential components of the eukaryotic transcription elongation machinery. The full spectrum of Cdk9/cyclin T targets, as well as the specific consequences of phosphorylations, is still largely undefined. We identify and characterize here a Cdk9 kinase (PtkA) in the filamentous ascomycete Aspergillus nidulans. Deletion of ptkA had a lethal effect in later stages of vegetative growth and completely impeded asexual development. Overexpression of ptkA affected directionality of polarized growth and the initiation of new branching sites. A green fluorescent protein-tagged PtkA version localized inside the nucleus during interphase, supporting a role of PtkA in transcription elongation, as observed in other organisms. We also identified a putative cyclin T homolog, PchA, in the A. nidulans genome and confirmed its interaction with PtkA in vivo. Surprisingly, the Pcl-like cyclin PclA, previously described to be involved in asexual development, was also found to interact with PtkA, indicating a possible role of PtkA in linking transcriptional activity with development and/or morphogenesis in A. nidulans. This is the first report of a Cdk9 kinase interacting with a Pcl-like cyclin, revealing interesting new aspects about the involvement of this Cdk-subfamily in differential gene expression.

scholarly journals A Mutant Defective in Sexual Development Produces Aseptate Ascogonia

2010 ◽  
Vol 9 (12) ◽  
pp. 1856-1866 ◽  
Author(s):  
Sandra Bloemendal ◽  
Kathryn M. Lord ◽  
Christine Rech ◽  
Birgit Hoff ◽  
Ines Engh ◽  
...  
Keyword(s):  
Sexual Development ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Sexual Cycle ◽  
Sequence Analyses ◽  
Gene Encoding ◽  
Content Type ◽  
Domain Of Unknown Function ◽  
Sterile Mutant

ABSTRACT The transition from the vegetative to the sexual cycle in filamentous ascomycetes is initiated with the formation of ascogonia. Here, we describe a novel type of sterile mutant from Sordaria macrospora with a defect in ascogonial septum formation. This mutant, named pro22, produces only small, defective protoperithecia and carries a point mutation in a gene encoding a protein that is highly conserved throughout eukaryotes. Sequence analyses revealed three putative transmembrane domains and a C-terminal domain of unknown function. Live-cell imaging showed that PRO22 is predominantly localized in the dynamic tubular and vesicular vacuolar network of the peripheral colony region close to growing hyphal tips and in ascogonia; it is absent from the large spherical vacuoles in the vegetative hyphae of the subperipheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar network, and the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

Mitochondrial protein 18 is a positive apoptotic regulator in cardiomyocytes under oxidative stress

2019 ◽  
Vol 133 (9) ◽  
pp. 1067-1084 ◽  
Author(s):  
Lynn H.H. Aung ◽  
Yu-Zhen Li ◽  
Hua Yu ◽  
Xiatian Chen ◽  
Zhongjie Yu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Mitochondrial Protein ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cardiac Apoptosis ◽  
Inner Membrane Protein ◽  
Artery Disease ◽  
Subsequent Activation

AbstractAccumulation of reactive oxygen species is a common phenomenon in cardiac stress conditions, for instance, coronary artery disease, aging-related cardiovascular abnormalities, and exposure to cardiac stressors such as hydrogen peroxide (H2O2). Mitochondrial protein 18 (Mtp18) is a novel mitochondrial inner membrane protein, shown to involve in the regulation of mitochondrial dynamics. Although Mtp18 is abundant in cardiac muscles, its role in cardiac apoptosis remains elusive. The present study aimed to detect the role of Mtp18 in H2O2-induced mitochondrial fission and apoptosis in cardiomyocytes. We studied the effect of Mtp18 in cardiomyocytes by modulating its expression with lentiviral construct of Mtp18-shRNA and Mtp18 c-DNA, respectively. We then analyzed mitochondrial morphological dynamics with MitoTracker Red staining; apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) and cell death detection assays; and protein expression with immunoblotting. Here, we observed that Mtp18 could regulate oxidative stress- mediated mitochondrial fission and apoptosis in cardiac myocytes. Mechanistically, we found that Mtp8 induced mitochondrial fission and apoptosis by enhancing dynamin-related protein 1 (Drp1) accumulation. Conversely, knockdown of Mtp18 interfered with Drp1-associated mitochondrial fission and subsequent activation of apoptosis in both HL-1 cells and primary cardiomyocytes. However, overexpression of Mtp18 alone was not sufficient to execute apoptosis when Drp1 was minimally expressed, suggesting that Mtp18 and Drp1 are interdependent in apoptotic cascade. Together, these data highlight the role of Mtp18 in cardiac apoptosis and provide a novel therapeutic insight to minimize cardiomyocyte loss via targetting mitochondrial dynamics.

scholarly journals FigA, a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae, Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

2013 ◽  
Vol 13 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Shizhu Zhang ◽  
Hailin Zheng ◽  
Nanbiao Long ◽  
Natalia Carbó ◽  
Peiying Chen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aspergillus Nidulans ◽  
Sexual Development ◽  
Calcium Uptake ◽  
Fluorescent Protein ◽  
Calcium Influx ◽  
Hyphal Growth ◽  
Uptake System ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTCalcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. InSaccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 fromS. cerevisiae, was functionally characterized in the filamentous fungusAspergillus nidulans. Loss offigAresulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACSmidA. Interestingly, in afigAdeletion mutant, adding extracellular Ca2+rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed thatfigAdeletion sharply decreased the expression ofbrlAandnsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system inA. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

scholarly journals Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus

2015 ◽  
Vol 60 (2) ◽  
pp. 806-817 ◽  
Author(s):  
Joy Y. Feng ◽  
Yili Xu ◽  
Ona Barauskas ◽  
Jason K. Perry ◽  
Shekeba Ahmadyar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adverse Events ◽  
Mitochondrial Protein ◽  
Mitochondrial Rna ◽  
Mitochondrial Toxicity ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Rna Polymerase

ABSTRACTToxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4′-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2′-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2′-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.

scholarly journals Potassium Uptake Modulates Staphylococcus aureus Metabolism

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Casey M. Gries ◽  
Marat R. Sadykov ◽  
Logan L. Bulock ◽  
Sujata S. Chaudhari ◽  
Vinai C. Thomas ◽  
...  
Keyword(s):  
Carbon Flux ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Normal Growth ◽  
Cytoplasmic Ph ◽  
Carbon Utilization ◽  
Content Type ◽  
Alkaline Conditions ◽  
Green Fluorescent

ABSTRACT Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization.

Sign in / Sign up

Export Citation Format

Share Document