scholarly journals Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Paul Roberts ◽  
Sharon Moshitch-Moshkovitz ◽  
Erik Kvam ◽  
Eileen O'Toole ◽  
Mark Winey ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Starvation ◽  
Nutrient Sensing ◽  
Dependent Manner ◽  
Vacuole Membrane ◽  
Carbon And Nitrogen ◽  
Transmission Electron ◽  
Biochemical Assays ◽  
Dividing Cells ◽  
Tor Kinase

Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promotepiecemeal microautophagy of thenucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-Δcells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole.

scholarly journals Protein Kinase A and Sch9 Cooperatively Regulate Induction of Autophagy in Saccharomyces cerevisiae

2007 ◽  
Vol 18 (10) ◽  
pp. 4180-4189 ◽  
Author(s):  
Tomohiro Yorimitsu ◽  
Shadia Zaman ◽  
James R. Broach ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Nutrient Sensing ◽  
Regulatory Mechanisms ◽  
Eukaryotic Cells ◽  
Tor Kinase ◽  
Kinase A

Autophagy is a highly conserved, degradative process in eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that the protein kinase A (PKA) and Sch9 signaling pathways regulate autophagy cooperatively in yeast. Autophagy is induced in cells when PKA and Sch9 are simultaneously inactivated. Mutant alleles of these kinases bearing a mutation that confers sensitivity to the ATP-analogue inhibitor C3-1′-naphthyl-methyl PP1 revealed that autophagy was induced independently of effects on Tor kinase. The PKA–Sch9-mediated autophagy depends on the autophagy-related 1 kinase complex, which is also essential for TORC1-regulated autophagy, the transcription factors Msn2/4, and the Rim15 kinase. The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1.

scholarly journals Piecemeal Microautophagy of the Nucleus Requires the Core Macroautophagy Genes

2008 ◽  
Vol 19 (10) ◽  
pp. 4492-4505 ◽  
Author(s):  
R. Krick ◽  
Y. Muehe ◽  
T. Prick ◽  
S. Bremer ◽  
P. Schlotterhose ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Fusion Genes ◽  
Vacuole Membrane ◽  
The Core ◽  
Atg Proteins ◽  
Biochemical Assays ◽  
Atg Genes ◽  
Mutant Cells ◽  
Specific Membrane

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.

scholarly journals Multilayered regulation of TORC1-body formation in budding yeast

2019 ◽  
Vol 30 (3) ◽  
pp. 400-410 ◽  
Author(s):  
Arron Sullivan ◽  
Ryan L. Wallace ◽  
Rachel Wellington ◽  
Xiangxia Luo ◽  
Andrew P. Capaldi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Nitrogen Starvation ◽  
Potent Inhibitor ◽  
Budding Yeast ◽  
Binding Protein ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
Body Formation ◽  
Level Model

The target of rapamycin kinase complex 1 (TORC1) regulates cell growth and metabolism in eukaryotes. In Saccharomyces cerevisiae, TORC1 activity is known to be controlled by the conserved GTPases, Gtr1/2, and movement into and out of an inactive agglomerate/body. However, it is unclear whether/how these regulatory steps are coupled. Here we show that active Gtr1/2 is a potent inhibitor of TORC1-body formation, but cells missing Gtr1/2 still form TORC1-bodies in a glucose/nitrogen starvation-dependent manner. We also identify 13 new activators of TORC1-body formation and show that seven of these proteins regulate the Gtr1/2-dependent repression of TORC1-body formation, while the remaining proteins drive the subsequent steps in TORC1 agglomeration. Finally, we show that the conserved phosphatidylinositol-3-phosphate (PI(3)P) binding protein, Pib2, forms a complex with TORC1 and overrides the Gtr1/2-dependent repression of TORC1-body formation during starvation. These data provide a unified, systems-level model of TORC1 regulation in yeast.

scholarly journals Antitumor Potential of Green Synthesized ZnONPs Using Root Extract of Withania somnifera against Human Breast Cancer Cell Line

Separations ◽  
2021 ◽  
Vol 8 (1) ◽  
pp. 8
Author(s):  
Kollur Shiva Prasad ◽  
Shashanka K Prasad ◽  
Ravindra Veerapur ◽  
Ghada Lamraoui ◽  
Ashwini Prasad ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Cell Line ◽  
Withania Somnifera ◽  
Root Extract ◽  
Dependent Manner ◽  
Sem Images ◽  
Xrd Pattern ◽  
Transmission Electron

Herein we report the synthesis of zinc oxide nanoparticles (ZnONPs) using Withania somnifera root extract (WSE) as an effective chelating agent. The microscopic techniques viz., X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and selected area electron diffraction (SAED) were employed to analyze the as-obtained ZnONPs. The crystalline planes observed from the XRD pattern agrees with the hexagonal wurtzite structure of the as-prepared ZnONPs. The aggregations and agglomerations observed in the SEM images indicated that the size of the as-prepared ZnONPs was between 30 and 43 nm. The interplanar distance between the lattice fringes observed in the HRTEM image was found to be 0.253 nm, which is in good agreement with the (100) plane obtained in the XRD pattern. Furthermore, the anti-breast cancer cytotoxic evaluation was carried out using the MCF-7 cell line, and the results showed significant cytotoxic effects in a dose-dependent manner.

scholarly journals Mot3, a Zn Finger Transcription Factor That Modulates Gene Expression and Attenuates Mating Pheromone Signaling in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 879-892 ◽  
Author(s):  
Anatoly V Grishin ◽  
Michael Rothenberg ◽  
Maureen A Downs ◽  
Kendall J Blumer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Signaling Pathway ◽  
G Protein ◽  
Binding Sites ◽  
Dependent Manner ◽  
Pheromone Response ◽  
Mating Pheromone ◽  
Pheromone Signaling ◽  
Yeast Genes

Abstract In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein β subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5′ flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

scholarly journals Subcellular Investigation of Photosynthesis-Driven Carbon Assimilation in the Symbiotic Reef Coral Pocillopora damicornis

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Christophe Kopp ◽  
Isabelle Domart-Coulon ◽  
Stephane Escrig ◽  
Bruno M. Humbel ◽  
Michel Hignette ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Secondary Ion Mass Spectrometry ◽  
Reef Coral ◽  
Carbon Assimilation ◽  
Isotopic Labeling ◽  
Cellular Level ◽  
Coral Tissue ◽  
Pocillopora Damicornis ◽  
Carbon And Nitrogen ◽  
Transmission Electron

ABSTRACT  Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [13C]bicarbonate and [15N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. IMPORTANCE  Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association.

scholarly journals Comparison of the effects of difenacoum and brodifacoum on the ultrastructure of rat liver cells

2016 ◽  
Vol 67 (3) ◽  
pp. 204-209 ◽  
Author(s):  
Nursel Gül ◽  
Nuri Yiğit ◽  
Fulya Saygılı ◽  
Ebru Demirel ◽  
Ceren Geniş
Keyword(s):  
Rat Liver ◽  
Lipid Droplets ◽  
Liver Cells ◽  
Mitochondrial Morphology ◽  
Cytotoxic Effects ◽  
Cytoplasmic Material ◽  
Transmission Electron ◽  
Biochemical Assays ◽  
Rat Liver Cells ◽  
Contrast Exposure

Abstract We used transmission electron microscopy to examine the cytotoxic effects of the second-generation anticoagulant rodenticides difenacoum and brodifacoum on rat liver. A single dose of difenacoum or brodifacoum was administered to rats by gastric gavage and liver samples were taken after 24 h, four days or seven days. In the livers of rats treated with difenacoum for 24 h, hepatocytes typically showed increased numbers of lysosomes, as well as enlargement of both the perinuclear space and the cisternae of the rough endoplasmic reticulum (RER), while sinusoids were irregularly shaped and contained Kupffer cells. Similar irregularities occurred in brodifacoum-treated rats at the same time point, but additionally increased numbers of vacuoles, damaged mitochondrial cristae, and clumping of chromatin were observed in hepatocytes, and hemolysed erythrocytes were noted in the sinusoids. Comparable findings were made in each group of rats after four days. After seven days of difenacoum treatment, hepatocytes suffered loss of cytoplasmic material and mitochondrial shrinkage, while RER cisternae became discontinuous. In contrast, exposure to brodifacoum for seven days caused the formation of numerous vacuoles and lipid droplets, disordered mitochondrial morphology, chromatin clumping and invagination of the nuclear envelope in hepatocytes. Sinusoids in the livers of rodenticide-treated rats contained an accumulation of dense material, lipid droplets, cells with pycnotic nuclei and hemolysed erythrocytes. Overall, our results show that brodifacoum causes more severe effects in liver cells than difenacoum. Thus our microscopic data along with additional biochemical assays point to a severe effect of rodenticide on vertebrates.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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