scholarly journals Role of ATG8 and Autophagy in Programmed Nuclear Degradation in Tetrahymena thermophila

2012 ◽  
Vol 11 (4) ◽  
pp. 494-506 ◽  
Author(s):  
Ming-Liang Liu ◽  
Meng-Chao Yao
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Critical Role ◽  
Nuclear Periphery ◽  
Unicellular Eukaryote ◽  
Knockout Mutant ◽  
Content Type ◽  
Nuclear Degradation

ABSTRACT Autophagy is an evolutionarily conserved mechanism for the degradation of cellular components, but its role in enucleation during differentiation has not been established. Tetrahymena thermophila is a unicellular eukaryote with two functionally distinct nuclei, the somatic (macro-) and the germ line (micro-) nuclei. These nuclei are produced during sexual reproduction (conjugation), which involves differentiation and selective degradation of several specific nuclei. To examine the role of autophagy in nuclear degradation, we studied the function of two ATG8 genes in Tetrahymena . Through fluorescent protein tagging, we found that both proteins are targeted to degrading nuclei at specific stages, with some enrichment on the nuclear periphery, suggesting the formation of autophagosomes surrounding these nuclei. In addition, ATG8 knockout mutant cells showed a pronounced delay in nuclear degradation without apparently preventing the completion of other developmental events. This evidence provided direct support for a critical role for autophagy in programmed nuclear degradation. The results also showed differential roles for two ATG8 genes, with ATG8-65 playing a more significant role in starvation than ATG8-2 , although both are important in nuclear degradation.

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.

scholarly journals SUMOylation Is Developmentally Regulated and Required for Cell Pairing during Conjugation in Tetrahymena thermophila

2014 ◽  
Vol 14 (2) ◽  
pp. 170-181 ◽  
Author(s):  
Amjad M. Nasir ◽  
Qianyi Yang ◽  
Douglas L. Chalker ◽  
James D. Forney
Keyword(s):  
Sexual Reproduction ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Covalent Attachment ◽  
Sexual Life ◽  
Dynamic Regulation ◽  
Content Type ◽  
Sumo Pathway

ABSTRACT The covalent attachment of s mall u biquitin-like mo difier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena . The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena , including a dynamic regulation associated with the sexual life cycle.

scholarly journals The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa.

2022 ◽  
Author(s):  
Alfredo Figueroa-Melendez ◽  
Leonora Martinez-Nunez ◽  
Adriana Maria Rico-Ramirez ◽  
Juan Manuel Martinez-Andrade ◽  
Mary Munson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Complex Function ◽  
Mass Spectrometry Analysis ◽  
Knockout Mutant ◽  
Transmission Electron Microscopy Analysis ◽  
C Terminus

The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the apices of the cells, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

scholarly journals Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila

2015 ◽  
Vol 14 (12) ◽  
pp. 1240-1252 ◽  
Author(s):  
Qianyi Yang ◽  
Amjad M. Nasir ◽  
Robert S. Coyne ◽  
James D. Forney
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Fusion Gene ◽  
Life Cycles ◽  
Sexual Life ◽  
Proper Function ◽  
Content Type ◽  
Macronuclear Development ◽  
Green Fluorescent

ABSTRACT Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila , the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both MIC and MAC genomes is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl 2 -dependent expression of green fluorescent protein (GFP)-Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs, but MACs continued to divide. In contrast, expression of catalytically inactive Ubc9p resulted in the accumulation of multiple MICs. Critical roles for Ubc9p were also identified during the sexual life cycle of Tetrahymena . Cell lines that were depleted for Ubc9p did not form mating pairs and therefore could not complete any of the subsequent stages of conjugation, including meiosis and macronuclear development. Mating between cells expressing catalytically inactive Ubc9p resulted in arrest during macronuclear development, consistent with our observation that Ubc9p accumulates in the developing macronucleus.

scholarly journals Mutations in Pdd1 Reveal Distinct Requirements for Its Chromodomain and Chromoshadow Domain in Directing Histone Methylation and Heterochromatin Elimination

2013 ◽  
Vol 13 (2) ◽  
pp. 190-201 ◽  
Author(s):  
Rachel M. Schwope ◽  
Douglas L. Chalker
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Histone H3 ◽  
Amino Acid Residues ◽  
Focus Formation ◽  
Content Type ◽  
Genome Wide ◽  
Somatic Genome ◽  
Low Levels

ABSTRACTPdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation ofTetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.

scholarly journals A Developmentally Regulated Gene, ASI2, Is Required for Endocycling in the Macronuclear Anlagen of Tetrahymena

2010 ◽  
Vol 9 (9) ◽  
pp. 1343-1353 ◽  
Author(s):  
Lihui Yin ◽  
Susan T. Gater ◽  
Kathleen M. Karrer
Keyword(s):  
Signal Transduction ◽  
Mitotic Cycle ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Ciliated Protozoa ◽  
Dna Rearrangement ◽  
Developmentally Regulated ◽  
Content Type ◽  
Molecular Events ◽  
Multicellular Organisms

ABSTRACT Ciliated protozoa contain two types of nuclei, germ line micronuclei (Mic) and transcriptionally active macronuclei (Mac). During sexual reproduction, the parental Mac degenerates and a new Mac develops from a mitotic product of the zygotic Mic. Macronuclear development involves extensive endoreplication of the genome. The present study shows that endoreplication of macronuclear DNA in Tetrahymena is an example of endocyling, a variant of the mitotic cycle with alternating S and G phases in the absence of cell division. Thus, endocycling is conserved from ciliates to multicellular organisms. The gene ASI2 in Tetrahymena thermophila encodes a putative signal transduction receptor. ASI2 is nonessential for vegetative growth, but it is upregulated during development of the new Mac. Cells that lack ASI2 in the developing Mac anlagen are arrested in endoreplication of the DNA and die. This study shows that ASI2 is also transcribed in the parental Mac early in conjugation and that transcription of ASI2 in the parental Mac supports endoreplication of the DNA during early stages of development of the Mac anlagen. Other molecular events in Mac anlage development, including developmentally regulated DNA rearrangement, occur normally in matings between ASI2 knockouts, suggesting that ASI2 specifically regulates endocycling in Tetrahymena.

AMPK blocks chromatin activation and consequent R-loop formation to protect genome stability following acute starvation

2021 ◽  
Author(s):  
Bing Sun ◽  
McLean Sherrin ◽  
Richard Roy
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Germ Line ◽  
Critical Role ◽  
Significant Proportion ◽  
Loop Formation ◽  
C Elegans ◽  
Chromatin Activation ◽  
Acute Starvation

Abstract During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result from abnormally elevated H3K4me3 levels in the germ line following recovery from acute starvation1. Here we show that H3K4me3 is dramatically increased at promoters, driving aberrant transcription elongation that results in the accumulation of R-loops in the starved AMPK mutants. DRIP-seq analysis demonstrated that a significant proportion of the genome was affected by R-loop formation with a dramatic expansion in the number of R-loops at numerous loci, most pronounced at the promoter-TSS regions of genes in the starved AMPK mutants. The R-loops are transmissible into subsequent generations, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK null germ lines show considerably more RAD-51 foci at sites of R-loop formation, potentially sequestering it from its critical role at meiotic breaks and/or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation, where in its absence R-loops accumulate, resulting in reproductive compromise and DNA damage hypersensitivity.

Understanding destination evangelism: a social media viewpoint

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Purvendu Sharma
Keyword(s):  
Social Media ◽  
Information Seeking ◽  
Structural Equation ◽  
Structural Equation Models ◽  
Critical Role ◽  
Content Type ◽  
Mediating Role ◽  
Information Seeking Behaviors ◽  
Role Of Information

PurposeThe present research aims to introduce and understand the promising nature of destination evangelism in the context of social media-based tourism communities (SMTCs). Further, factors that influence evangelism and information-seeking behaviors on SMTCs are examined.Design/methodology/approachA conceptual model is developed that features an interplay of destination distinctiveness, destination evangelism, travel commitment and information-seeking engagement. Data were collected from 215 active users of SMTCs and analyzed using structural equation models.FindingsThe research findings indicate that destination distinctiveness and information-seeking positively lead to destination evangelism. Information-seeking is found to mediate the relationship between (1) destination evangelism and travel commitment and (2) destination evangelism and distinctiveness.Research limitations/implicationsThe research offers meaningful insights into exploring constituents of destination evangelism. The research also understands and highlights the critical role of information-seeking engagement about distinct destinations.Practical implicationsThis research highlights key areas to build, improve and inspire destination evangelism on SMTCs.Originality/valueThis study offers a fresh contribution to tourism literature by investigating destination evangelism and its drivers. This is explained by closely uniting vital research streams of evangelism, tourism and engagement. It further highlights the dual mediating role of information seeking, suggesting that these engagements are critical to evangelizing destinations.

Institutional effect on born global firms in China: the role of Sun Tzu’s The Art of War strategies

2016 ◽  
Vol 10 (1) ◽  
pp. 1-19 ◽  
Author(s):  
Man Zhang ◽  
Qian Gao ◽  
Jane V. Wheeler ◽  
Jungsook Kwon
Keyword(s):  
Emerging Markets ◽  
Institutional Environment ◽  
Critical Role ◽  
Born Global ◽  
Internationalization Process ◽  
Content Type ◽  
International Performance ◽  
The Relationship ◽  
Born Global Firms

Purpose – This paper aims to investigate the role of Sun Tzu’s significant strategies on the relationship between the institutional environment and international performance of Chinese born global firms, a type of small- and medium-sized enterprise (SME) characterized by the company’s limited resources and its early efforts to internationalize. Design/methodology/approach – The methodology is based on a multi-case analysis of interviews conducted with four chosen born global firms, coupled with public database and Web site searches. Through the use of qualitative methods, propositions were developed. Findings – This paper provides insights regarding how the institutional environment, both formal and informal, has a strong positive relationship with born global firm’s international performance. Moreover, Sun Tzu’s significant strategies play a critical role in the internationalization process of born global firms in emerging markets. Originality/value – Although existing studies discuss the application of Eastern philosophical strategies adopted by firms in emerging markets, to the best of our knowledge, this is one of the earliest studies which evaluates the moderation effect of Sun Tzu’s significant strategies on the relationship between institutional environment and business performance. The paper contributes to scholarly discourse on the influencing factors of born global firm’s internationalization process. It also has practical relevance to international entrepreneurs and SMEs from emerging markets.

scholarly journals Abnormal Micronuclear Telomeres Lead to an Unusual Cell Cycle Checkpoint and Defects in Tetrahymena Oral Morphogenesis

2008 ◽  
Vol 7 (10) ◽  
pp. 1712-1723 ◽  
Author(s):  
Karen E. Kirk ◽  
Christina Christ ◽  
Jennifer M. McGuire ◽  
Arun G. Paul ◽  
Mithaq Vahedi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Telomeric Dna ◽  
Spindle Apparatus ◽  
Cycle Checkpoint

ABSTRACT Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.

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