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Alan R Kimmel - One of the best experts on this subject based on the ideXlab platform.
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integrated actions of mtor complexes 1 and 2 for growth and development of Dictyostelium
The International Journal of Developmental Biology, 2019Co-Authors: Pundrik Jaiswal, Daniel Rosel, Xinhua Liao, Amit R Majithia, Taruna Khurana, Alan R KimmelAbstract:Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation, whereas, mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We also review functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments have suggested novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions.
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biochemical responses to chemically distinct chemoattractants during the growth and development of Dictyostelium
Methods of Molecular Biology, 2016Co-Authors: Netra Pal Meena, Alan R KimmelAbstract:Dictyostelium discoideum has proven an excellent model for the study of eukaryotic chemotaxis. During growth in its native environment, Dictyostelium phagocytose bacteria and fungi for primary nutrient capture. Growing Dictyostelium can detect these nutrient sources through chemotaxis toward the metabolic by-product folate. Although Dictyostelium grow as individual cells, nutrient depletion induces a multicellular development program and a separate chemotactic response pathway. During development, Dictyostelium synthesize and secrete cAMP, which serves as a chemoattractant to mobilize and coordinate cells for multicellular formation and development. Separate classes of GPCRs and Gα proteins mediate chemotactic signaling to the chemically distinct ligands. We discuss common and separate component responses of Dictyostelium to folate and cAMP during growth and development, and the advantages and disadvantages for each. As examples, we present biochemical assays to characterize the chemoattractant-induced kinase activations of mTORC2 and the ERKs.
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an ancestral non proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum
Journal of Cell Science, 2014Co-Authors: Marthe H R Ludtmann, Alan R Kimmel, Grant Otto, Christina Schilde, Zhi Hui Chen, Claire Y Allan, Selina Brace, Philip W Beesley, Paul R Fisher, Richard KillickAbstract:Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.
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biochemical responses to chemoattractants in Dictyostelium ligand receptor interactions and downstream kinase activation
Methods of Molecular Biology, 2009Co-Authors: Xinhua Liao, Alan R KimmelAbstract:Dictyostelium discoideum is one of the most facile eukaryotic systems for the study of chemotactic response to secreted chemical ligands. Dictyostelium grow as individual cells, using bacteria and fungi as primary nutrient sources; during growth, Dictyostelium moves directionally toward folate, a bacterial byproduct. Upon nutrient depletion Dictyostelium initiates a multicellular development program characterized by the production and secretion of cAMP. Cell surface receptors specifically recognize extracellular cAMP, which serves as both a morphogen to promote development and a chemoattractant to organize multicellularity. We discuss several approaches for the study of ligand-receptor interaction, with focus on affinity class determination and quantification of ligand binding sites (i.e., receptors) per cell. We further present examples for the application of biochemical assays to characterize the ligand-induced kinase activation of PI3K, GSK3, and ERK2.
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The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum.
European journal of cell biology, 2006Co-Authors: Daniel Rosel, Alan R KimmelAbstract:Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.
Rex L Chisholm - One of the best experts on this subject based on the ideXlab platform.
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Making Permanent Stocks of Dictyostelium
CSH protocols, 2008Co-Authors: Pascale Gaudet, Petra Fey, Rex L ChisholmAbstract:INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. Dictyostelium can be grown axenically or in the presence of bacteria, either on agar plates or in suspension. Because Dictyostelium growth rates are relatively slow compared to those of bacteria or yeast, laboratories commonly maintain stocks of growing cultures in order to start experiments rapidly. However, it is important to remember that the genome of Dictyostelium, like that of any living organism, is subject to genetic modification. It is well documented that cell lines that are kept in culture for an extended period of time exhibit undesirable changes that yield unreliable experimental results (Hughes et al. 2007). Dictyostelium strains from different laboratories are known to contain various large genome duplications, presumably due to clone selection. Thus, good handling of the cells is essential. To obtain consistent results, new cultures must be started every 2-4 wk, and cultures should never be allowed to grow beyond 4 × 10(6) cells/mL. If overgrowth occurs, a new culture should be started. This protocol describes two methods for preparing long-term stocks of Dictyostelium, either as frozen cells or as spores.
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An anatomy ontology to represent biological knowledge in Dictyostelium discoideum
BMC Genomics, 2008Co-Authors: Pascale Gaudet, Jeffery G Williams, Rex L ChisholmAbstract:Background Dictyostelium discoideum is a model system for studying many important physiological processes including chemotaxis, phagocytosis, and signal transduction. The recent sequencing of the genome has revealed the presence of over 12,500 protein-coding genes. The model organism database dictyBase hosts the genome sequence as well as a large amount of manually curated information. Results We present here an anatomy ontology for Dictyostelium based upon the life cycle of the organism. Conclusion Anatomy ontologies are necessary to annotate species-specific events such as phenotypes, and the Dictyostelium anatomy ontology provides an essential tool for curation of the Dictyostelium genome.
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an anatomy ontology to represent biological knowledge in Dictyostelium discoideum
BMC Genomics, 2008Co-Authors: Pascale Gaudet, Petra Fey, Jeffery G Williams, Rex L ChisholmAbstract:Dictyostelium discoideum is a model system for studying many important physiological processes including chemotaxis, phagocytosis, and signal transduction. The recent sequencing of the genome has revealed the presence of over 12,500 protein-coding genes. The model organism database dictyBase hosts the genome sequence as well as a large amount of manually curated information. We present here an anatomy ontology for Dictyostelium based upon the life cycle of the organism. Anatomy ontologies are necessary to annotate species-specific events such as phenotypes, and the Dictyostelium anatomy ontology provides an essential tool for curation of the Dictyostelium genome.
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Protocols for growth and development of Dictyostelium discoideum
Nature Protocols, 2007Co-Authors: Petra Fey, Anthony S Kowal, Pascale Gaudet, Karen E Pilcher, Rex L ChisholmAbstract:Dictyostelium discoideum , a unicellular organism capable of developing into a multicellular structure, is a powerful model system to study a variety of biological processes. Because it is inexpensive and relatively easy to grow, Dictyostelium is also frequently used in teaching laboratories. Here we describe conditions for successfully growing and developing Dictyostelium cells and methods for long-term storage of Dictyostelium amoebae and spores.
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dictybase the model organism database for Dictyostelium discoideum
Nucleic Acids Research, 2006Co-Authors: Rex L Chisholm, Petra Fey, Pascale Gaudet, Karen E Pilcher, Eric M Just, Sohel N Merchant, Warren A KibbeAbstract:dictyBase (http://dictybase.org) is the model organism database (MOD) for the social amoeba Dictyostelium discoideum. The unique biology and phylogenetic position of Dictyostelium offer a great opportunity to gain knowledge of processes not characterized in other organisms. The recent completion of the 34 MB genome sequence, together with the sizable scientific literature using Dictyostelium as a research organism, provided the necessary tools to create a well-annotated genome. dictyBase has leveraged software developed by the Saccharomyces Genome Database and the Generic Model Organism Database project. This has reduced the time required to develop a full-featured MOD and greatly facilitated our ability to focus on annotation and providing new functionality. We hope that manual curation of the Dictyostelium genome will facilitate the annotation of other genomes.
Robert J. Huber - One of the best experts on this subject based on the ideXlab platform.
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Cln5 is secreted and functions as a glycoside hydrolase in Dictyostelium.
Cellular signalling, 2017Co-Authors: Robert J. Huber, Sabateeshan MathavarajahAbstract:Abstract Ceroid lipofuscinosis neuronal 5 (CLN5) is a member of a family of proteins that are linked to neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, known commonly as Batten disease, affects all ages and ethnicities and is currently incurable. The precise function of CLN5, like many of the NCL proteins, remains to be elucidated. In this study, we report the localization, molecular function, and interactome of Cln5, the CLN5 homolog in the social amoeba Dictyostelium discoideum. Residues that are glycosylated in human CLN5 are conserved in the Dictyostelium homolog as are residues that are mutated in patients with CLN5 disease. Dictyostelium Cln5 contains a putative signal peptide for secretion and we show that the protein is secreted during growth and starvation. We also reveal that both Dictyostelium Cln5 and human CLN5 are glycoside hydrolases, providing the first evidence in any system linking a molecular function to CLN5. Finally, immunoprecipitation coupled with mass spectrometry identified 61 proteins that interact with Cln5 in Dictyostelium. Of the 61 proteins, 67% localize to the extracellular space, 28% to intracellular vesicles, and 20% to lysosomes. A GO term enrichment analysis revealed that a majority of the interacting proteins are involved in metabolism, catabolism, proteolysis, and hydrolysis, and include other NCL-like proteins (e.g., Tpp1/Cln2, cathepsin D/Cln10, cathepsin F/Cln13) as well as proteins linked to Cln3 function in Dictyostelium (e.g., AprA, CfaD, CadA). In total, this work reveals a CLN5 homolog in Dictyostelium and further establishes this organism as a complementary model system for studying the functions of proteins linked to NCL in humans.
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loss of cln3 function in the social amoeba Dictyostelium discoideum causes pleiotropic effects that are rescued by human cln3
PLOS ONE, 2014Co-Authors: Robert J. Huber, Michael A Myre, Susan L CotmanAbstract:The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3− cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3− cells was precocious and cln3− slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3− cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3− cells, strongly supports the use of this new model for JNCL research.
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The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum
Cellular and Molecular Life Sciences, 2014Co-Authors: Robert J. HuberAbstract:Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum , several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium . Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.
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The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum
Cellular and Molecular Life Sciences, 2014Co-Authors: Robert J. HuberAbstract:Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum , several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium . Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.
Pascale Gaudet - One of the best experts on this subject based on the ideXlab platform.
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Making Permanent Stocks of Dictyostelium
CSH protocols, 2008Co-Authors: Pascale Gaudet, Petra Fey, Rex L ChisholmAbstract:INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. Dictyostelium can be grown axenically or in the presence of bacteria, either on agar plates or in suspension. Because Dictyostelium growth rates are relatively slow compared to those of bacteria or yeast, laboratories commonly maintain stocks of growing cultures in order to start experiments rapidly. However, it is important to remember that the genome of Dictyostelium, like that of any living organism, is subject to genetic modification. It is well documented that cell lines that are kept in culture for an extended period of time exhibit undesirable changes that yield unreliable experimental results (Hughes et al. 2007). Dictyostelium strains from different laboratories are known to contain various large genome duplications, presumably due to clone selection. Thus, good handling of the cells is essential. To obtain consistent results, new cultures must be started every 2-4 wk, and cultures should never be allowed to grow beyond 4 × 10(6) cells/mL. If overgrowth occurs, a new culture should be started. This protocol describes two methods for preparing long-term stocks of Dictyostelium, either as frozen cells or as spores.
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An anatomy ontology to represent biological knowledge in Dictyostelium discoideum
BMC Genomics, 2008Co-Authors: Pascale Gaudet, Jeffery G Williams, Rex L ChisholmAbstract:Background Dictyostelium discoideum is a model system for studying many important physiological processes including chemotaxis, phagocytosis, and signal transduction. The recent sequencing of the genome has revealed the presence of over 12,500 protein-coding genes. The model organism database dictyBase hosts the genome sequence as well as a large amount of manually curated information. Results We present here an anatomy ontology for Dictyostelium based upon the life cycle of the organism. Conclusion Anatomy ontologies are necessary to annotate species-specific events such as phenotypes, and the Dictyostelium anatomy ontology provides an essential tool for curation of the Dictyostelium genome.
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an anatomy ontology to represent biological knowledge in Dictyostelium discoideum
BMC Genomics, 2008Co-Authors: Pascale Gaudet, Petra Fey, Jeffery G Williams, Rex L ChisholmAbstract:Dictyostelium discoideum is a model system for studying many important physiological processes including chemotaxis, phagocytosis, and signal transduction. The recent sequencing of the genome has revealed the presence of over 12,500 protein-coding genes. The model organism database dictyBase hosts the genome sequence as well as a large amount of manually curated information. We present here an anatomy ontology for Dictyostelium based upon the life cycle of the organism. Anatomy ontologies are necessary to annotate species-specific events such as phenotypes, and the Dictyostelium anatomy ontology provides an essential tool for curation of the Dictyostelium genome.
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Protocols for growth and development of Dictyostelium discoideum
Nature Protocols, 2007Co-Authors: Petra Fey, Anthony S Kowal, Pascale Gaudet, Karen E Pilcher, Rex L ChisholmAbstract:Dictyostelium discoideum , a unicellular organism capable of developing into a multicellular structure, is a powerful model system to study a variety of biological processes. Because it is inexpensive and relatively easy to grow, Dictyostelium is also frequently used in teaching laboratories. Here we describe conditions for successfully growing and developing Dictyostelium cells and methods for long-term storage of Dictyostelium amoebae and spores.
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dictybase the model organism database for Dictyostelium discoideum
Nucleic Acids Research, 2006Co-Authors: Rex L Chisholm, Petra Fey, Pascale Gaudet, Karen E Pilcher, Eric M Just, Sohel N Merchant, Warren A KibbeAbstract:dictyBase (http://dictybase.org) is the model organism database (MOD) for the social amoeba Dictyostelium discoideum. The unique biology and phylogenetic position of Dictyostelium offer a great opportunity to gain knowledge of processes not characterized in other organisms. The recent completion of the 34 MB genome sequence, together with the sizable scientific literature using Dictyostelium as a research organism, provided the necessary tools to create a well-annotated genome. dictyBase has leveraged software developed by the Saccharomyces Genome Database and the Generic Model Organism Database project. This has reduced the time required to develop a full-featured MOD and greatly facilitated our ability to focus on annotation and providing new functionality. We hope that manual curation of the Dictyostelium genome will facilitate the annotation of other genomes.
Sarah E. Rice - One of the best experts on this subject based on the ideXlab platform.
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Temperature dependence of myosin-II tail fragment assembly.
Journal of muscle research and cell motility, 2008Co-Authors: Peggy M. Mcmahon, Daniel R. Hostetter, Sarah E. RiceAbstract:Dictyostelium myosin-II bipolar thick filament (BTF) assembly is heavily dependent on ionic strength and temperature and is reversible by the phosphorylation of just three threonines. Truncated tail fragments of Dictyostelium myosin-II are commonly used as models for BTF assembly, as they self-assemble into regular paracrystals that recapitulate the ionic strength and phosphorylation dependence of full-length Dictyostelium myosin-II BTF assembly. Here we show that Dictyostelium myosin-II tail fragment assembly is highly temperature dependent, similar to full-length Dictyostelium myosin-II. Assembly of paracrystals was far more robust at 4 degrees C than at higher temperatures. Pre-assembled paracrystals disassembled completely when shifted to 37 degrees C, indicating that assembly does not greatly improve the thermostability of these tail fragments. The melting temperatures of individual Dictyostelium myosin-II tail coiled-coils under both low and high ionic strength conditions that prohibit paracrystal assembly are extremely low, 21 degrees C and 28 degrees C, respectively. These data are consistent with reversible thermal denaturation of the coiled-coil as the most likely explanation for assembly incompetence under either very low ionic strength or high temperature conditions. Assembled paracrystals of a structurally similar fragment of nonmuscle myosin-IIA were far more thermodynamically stable than their Dictyostelium counterparts at the temperatures examined here.
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Temperature dependence of myosin-II tail fragment assembly
Journal of Muscle Research and Cell Motility, 2008Co-Authors: Peggy M. Mcmahon, Daniel R. Hostetter, Sarah E. RiceAbstract:Dictyostelium myosin-II bipolar thick filament (BTF) assembly is heavily dependent on ionic strength and temperature and is reversible by the phosphorylation of just three threonines. Truncated tail fragments of Dictyostelium myosin-II are commonly used as models for BTF assembly, as they self-assemble into regular paracrystals that recapitulate the ionic strength and phosphorylation dependence of full-length Dictyostelium myosin-II BTF assembly. Here we show that Dictyostelium myosin-II tail fragment assembly is highly temperature dependent, similar to full-length Dictyostelium myosin-II. Assembly of paracrystals was far more robust at 4°C than at higher temperatures. Pre-assembled paracrystals disassembled completely when shifted to 37°C, indicating that assembly does not greatly improve the thermostability of these tail fragments. The melting temperatures of individual Dictyostelium myosin-II tail coiled-coils under both low and high ionic strength conditions that prohibit paracrystal assembly are extremely low, 21°C and 28°C, respectively. These data are consistent with reversible thermal denaturation of the coiled-coil as the most likely explanation for assembly incompetence under either very low ionic strength or high temperature conditions. Assembled paracrystals of a structurally similar fragment of nonmuscle myosin-IIA were far more thermodynamically stable than their Dictyostelium counterparts at the temperatures examined here.