scholarly journals In vivo reconstitution of autophagy in Saccharomyces cerevisiae

2008 ◽  
Vol 182 (4) ◽  
pp. 703-713 ◽  
Author(s):  
Yang Cao ◽  
Heesun Cheong ◽  
Hui Song ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Networks ◽  
In Vitro System ◽  
Atg Genes ◽  
Minimum Requirements ◽  
Autophagy Pathway ◽  
Cellular Dysfunction

Autophagy is a major intracellular degradative pathway that is involved in various human diseases. The role of autophagy, however, is complex; although the process is generally considered to be cytoprotective, it can also contribute to cellular dysfunction and disease progression. Much progress has been made in our understanding of autophagy, aided in large part by the identification of the autophagy-related (ATG) genes. Nonetheless, our understanding of the molecular mechanism remains limited. In this study, we generated a Saccharomyces cerevisiae multiple-knockout strain with 24 ATG genes deleted, and we used it to carry out an in vivo reconstitution of the autophagy pathway. We determined minimum requirements for different aspects of autophagy and studied the initial protein assembly steps at the phagophore assembly site. In vivo reconstitution enables the study of autophagy within the context of the complex regulatory networks that control this process, an analysis that is not possible with an in vitro system.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

1997 ◽  
Vol 328 (2) ◽  
pp. 669-675 ◽  
Author(s):  
L. Tamara DOERING ◽  
Randy SCHEKMAN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Proteins ◽  
Attachment Site ◽  
Gpi Anchor ◽  
Mating Pheromone ◽  
Peptide Substrates ◽  
In Vitro System ◽  
Glycosyl Phosphatidylinositol

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

scholarly journals Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus

2006 ◽  
Vol 80 (12) ◽  
pp. 6182-6187 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Recombination Frequency ◽  
In Vivo Studies ◽  
Rna Recombination ◽  
In Vitro System ◽  
Brome Mosaic Bromovirus ◽  
Subgenomic Promoter

ABSTRACT Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (−)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.

scholarly journals Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. VII. Idiotype-specific suppression of plaque-forming cell responses.

1981 ◽  
Vol 153 (3) ◽  
pp. 640-652 ◽  
Author(s):  
D H Sherr ◽  
S T Ju ◽  
J Z Weinberger ◽  
B Benacerraf ◽  
M E Dorf
Keyword(s):  
B Cells ◽  
Suppressor Cells ◽  
Spleen Cells ◽  
In Vitro System ◽  
Specific Suppression ◽  
Cell Responses ◽  
T Suppressor Cells

The ability of suppressor cells induced by the intravenous administration of 4-hydro-3-nitrophenyl acetyl (NP)-modified syngeneic cells to reduce an idiotypic B cell response was studied in both an in vivo and an in vitro system. Idiotype-positive B cells were assayed by the ability of guinea pig anti-idiotypic antiserum to specifically inhibit idiotype-positive plaque formation. It was found that up to 57% of the PFC response in vivo and 100% of the PFC response in vitro was inhibitable with antiidiotypic antiserum. The expression of these idiotype-positive B cells could be suppressed by the transfer of spleen cells form mice treated 7 d previously with NP coupled syngeneic cels. T cells are both required and sufficient for the transfer of idiotype specific suppression. The induction of these idiotype-specific T suppressor cells directly with antigen suggests that recognition of unique determinants on cell surfaces is important for regulation of lymphoid cell interactions. The role of idiotype-specific suppressor cells in the network of lymphoid interactions is discussed.

scholarly journals Genome-wide analysis of cis-regulatory changes in the metabolic adaptation of cavefish

2020 ◽  
Author(s):  
Jaya Krishnan ◽  
Chris W. Seidel ◽  
Ning Zhang ◽  
Jake VanCampen ◽  
Robert Peuß ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Metabolic Adaptation ◽  
Enhancer Activity ◽  
Genome Wide ◽  
Cave Environment

AbstractChanges in cis-regulatory elements play important roles in adaptation and phenotypic evolution. However, their contribution to metabolic adaptation of organisms is less understood. Here we have utilized a unique vertebrate model, Astyanax mexicanus, different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave water to uncover gene regulatory networks in metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissue of one surface and two independently derived cave populations. We find that many cis-regulatory elements differ in their epigenetic status/chromatin accessibility between surface fish and cavefish, while the two independently derived cave populations have evolved remarkably similar regulatory signatures. These differentially accessible regions are associated with genes of key pathways related to lipid metabolism, circadian rhythm and immune system that are known to be altered in cavefish. Using in vitro and in vivo functional testing of the candidate cis-regulatory elements, we find that genetic changes within them cause quantitative expression differences. We characterized one cis-regulatory element in the hpdb gene and found a genomic deletion in cavefish that abolishes binding of the transcriptional repressor IRF2 in vitro and derepresses enhancer activity in reporter assays. Genetic experiments further validated a cis-mediated role of the enhancer and suggest a role of this deletion in the upregulation of hpdb in wild cavefish populations. Selection of this mutation in multiple independent cave populations supports its importance in the adaptation to the cave environment, providing novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to a food-deprived cave environment.

scholarly journals The Ubiquitin-Dependent Targeting Pathway in Saccharomyces cerevisiae Plays a Critical Role in Multiple Chromatin Assembly Regulatory Steps

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 615-632 ◽  
Author(s):  
Troy A A Harkness ◽  
Gerald F Davies ◽  
Vijay Ramaswamy ◽  
Terra G Arnason
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translational Control ◽  
Regulatory Networks ◽  
Critical Role ◽  
Chromatin Assembly ◽  
Temperature Sensitive ◽  
Anaphase Promoting Complex ◽  
Free Media

Abstract In a screen designed to isolate Saccharomyces cerevisiae strains defective for in vitro chromatin assembly, two temperature-sensitive (ts) mutants were obtained: rmc1 and rmc3 (remodeling of chromatin). Cloning of RMC1 and RMC3 revealed a broad role for the ubiquitin-dependent targeting cascade as the ubiquitin-protein ligases (E3s), the anaphase promoting complex (APC; RMC1 encodes APC5) and Rsp5p, respectively, were identified. Genetic studies linked the rmc1/apc5 chromatin assembly defect to APC function: rmc1/apc5 genetically interacted with apc9Δ, apc10Δ, and cdc26Δ mutants. Furthermore, phenotypes associated with the rmc1/apc5 allele were consistent with defects in chromatin metabolism and in APC function: (i) UV sensitivity, (ii) plasmid loss, (iii) accumulation of G2/M cells, and (iv) suppression of the ts defect by growth on glucose-free media and by expression of ubiquitin. On the other hand, the multifunctional E3, Rsp5p, was shown to be required for both in vitro and in vivo chromatin assembly, as well as for the proper transcriptional and translational control of at least histone H3. The finding that the distinctly different E3 enzymes, APC and Rsp5p, both play roles in regulating chromatin assembly highlight the depth of the regulatory networks at play. The significance of these findings will be discussed.

scholarly journals Progress and Challenges in the Use of MAP1LC3 as a Legitimate Marker for Measuring Dynamic Autophagy In Vivo

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1321 ◽  
Author(s):  
Srinivasa Reddy Bonam ◽  
Jagadeesh Bayry ◽  
Mario P. Tschan ◽  
Sylviane Muller
Keyword(s):  
Ex Vivo ◽  
Biological Processes ◽  
Tissue Samples ◽  
Pros And Cons ◽  
Autophagy Pathway ◽  
The Many ◽  
Autophagy Activity

Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy in tissue samples and in vivo via a protein, which centrally intervenes in the autophagy pathway, the microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3), which is the most widely used marker and the first identified to associate with autophagosomal structures. These approaches are presented and discussed in terms of pros and cons. Some recommendations are provided to improve the reliability of the interpretation of results.

scholarly journals Role of Nucleotide Binding in Septin-Septin Interactions and Septin Localization in Saccharomyces cerevisiae

2008 ◽  
Vol 28 (16) ◽  
pp. 5120-5137 ◽  
Author(s):  
Satish Nagaraj ◽  
Ashok Rajendran ◽  
Charles E. Jackson ◽  
Mark S. Longtine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Binding ◽  
Temperature Sensitive ◽  
Associated Proteins ◽  
And Function ◽  
Bud Site ◽  
Two Hybrid

ABSTRACT Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins.

scholarly journals Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro.

1997 ◽  
Vol 17 (5) ◽  
pp. 2764-2773 ◽  
Author(s):  
P Fiorentini ◽  
K N Huang ◽  
D X Tishkoff ◽  
R D Kolodner ◽  
L S Symington
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Biochemical Properties ◽  
Recombination Activity ◽  
Exonuclease I ◽  
In Vitro Recombination ◽  
Double Stranded Dna

We previously described a 5'-3' exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5'-3' exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.

scholarly journals Neutrophils use selective autophagy receptor p62/SQSTM1 to target Staphylococcus aureus for degradation in vivo in zebrafish

2019 ◽  
Author(s):  
Josie F Gibson ◽  
Tomasz K Prajsnar ◽  
Christopher J Hill ◽  
Amy K Tooke ◽  
Justyna J Serba ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Degradation ◽  
Intracellular Pathogens ◽  
Life Threatening ◽  
Autophagy Pathway ◽  
Cellular Components ◽  
First Time

AbstractAutophagy leads to degradation of cellular components and has an important role in restricting intracellular pathogens. Autophagy receptors, including p62, target invading intracellular pathogens to the autophagy pathway for degradation. Staphylococcus aureus is a significant pathogen of humans and often life-threatening in the immunocompromised. Increasing evidence demonstrates that S. aureus is an intracellular pathogen of immune cells and may use neutrophils as proliferative niche but the intracellular fate of S. aureus following phagocytosis by neutrophils has not previously been analysed in vivo. In vitro, p62 is able to co-localise with intracellular Staphylococcus aureus, but whether p62 is beneficial or detrimental in host defence against S. aureus in vivo had not been determined.Here we use zebrafish to determine the fate and location of S. aureus within neutrophils throughout infection. We show that Lc3 and p62 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil. We also show rapid Lc3 marking of bacterial phagosomes within neutrophils which may be associated with subsequent bacterial degradation. Finally, we find that p62 is important for controlling cytosolic bacteria demonstrating for the first time a key role of p62 in autophagic control of S. aureus in neutrophils.

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