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Cells Feb 2020Individual gene analyses of microtubule-based motor proteins in have provided a rough draft of its machinery for cytoplasmic organization and division. This review... (Review)
Review
Individual gene analyses of microtubule-based motor proteins in have provided a rough draft of its machinery for cytoplasmic organization and division. This review collates their activities and looks forward to what is next. A comprehensive approach that considers the collective actions of motors, how they balance rates and directions, and how they integrate with the actin cytoskeleton will be necessary for a complete understanding of cellular dynamics.
Topics: Cell Movement; Dictyostelium; Microtubules; Protozoan Infections; Time Factors
PubMed: 32106406
DOI: 10.3390/cells9030528 -
ELife Aug 2021Using a self-generated hypoxic assay, we show that the amoeba displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume...
Using a self-generated hypoxic assay, we show that the amoeba displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume the available oxygen (O) and form a dense ring moving outwards at constant speed and density. To decipher this collective process, we combined two technological developments: porphyrin-based O -sensing films and microfluidic O gradient generators. We showed that cells exhibit aerotactic and aerokinetic response in a low range of O concentration indicative of a very efficient detection mechanism. Cell behaviors under self-generated or imposed O gradients were modeled using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis.
Topics: Anaerobiosis; Chemotaxis; Dictyostelium; Oxygen
PubMed: 34415238
DOI: 10.7554/eLife.64731 -
BMC Biology Jul 2019Kinases mTORC1 and AMPK act as energy sensors, controlling nutrient responses and cellular growth. Changes in nutrient levels affect diverse transcriptional networks,...
BACKGROUND
Kinases mTORC1 and AMPK act as energy sensors, controlling nutrient responses and cellular growth. Changes in nutrient levels affect diverse transcriptional networks, making it challenging to identify downstream paths that regulate cellular growth or a switch to development via nutrient variation. The life cycle of Dictyostelium presents an excellent model to study the mTORC1 signaling function for growth and development. Dictyostelium grow as single cells in nutrient-rich media, but, upon nutrient withdrawal, growth ceases and cells enter a program for multi-cell development. While nearly half the genome shows gene expression changes upon nutrient removal, we hypothesized that not all of these genes are required for the switch to program development. Through manipulation of mTORC1 activity alone, without nutrient removal, we focused on a core network of genes that are required for switching between growth and development for regulation of cell fate decisions.
RESULTS
To identify developmentally essential genes, we sought ways to promote development in the absence of nutrient loss. We first examined the activities of mTORC1 and AMPK in Dictyostelium during phases of rapid growth and starvation-induced development and showed they exhibited reciprocal patterns of regulation under various conditions. Using these as initial readouts, we identified rich media conditions that promoted rapid cell growth but, upon mTORC1 inactivation by rapamycin, led to a growth/development switch. Examination of gene expression during cell fate switching showed that changes in expression of most starvation-regulated genes were not required for developmental induction. Approximately 1000 genes which become downregulated upon rapamycin treatment comprise a cellular growth network involving ribosome biogenesis, protein synthesis, and cell cycle processes. Conversely, the upregulation of ~ 500 genes by rapamycin treatment defines essential signaling pathways for developmental induction, and ~ 135 of their protein products intersect through the well-defined cAMP/PKA network. Many of the rapamycin-induced genes we found are currently unclassified, and mutation analyses of 5 such genes suggest a novel gene class essential for developmental regulation.
CONCLUSIONS
We show that manipulating activities of mTORC1/AMPK in the absence of nutrient withdrawal is sufficient for a growth-to-developmental fate switch in Dictyostelium, providing a means to identify transcriptional networks and signaling pathways essential for early development.
Topics: Adenylate Kinase; Cell Cycle; Dictyostelium; Mechanistic Target of Rapamycin Complex 1; Protozoan Proteins; Signal Transduction
PubMed: 31319820
DOI: 10.1186/s12915-019-0673-1 -
Cellular Microbiology Nov 2019Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a brief and personal view of the emergence of cellular... (Review)
Review
Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a brief and personal view of the emergence of cellular microbiology research. From the very first observations of phagocytosis by Goeze in 1777 to the exhaustive analysis of the cellular defence mechanisms performed in modern laboratories, the studies by cell biologists and microbiologists have converged into an integrative research field distinct from, but fully coupled to immunity: cellular microbiology. In addition, this brief article is thought as a humble patchwork of the motivations that have guided the research in my group over a quarter century.
Topics: Animals; Dictyostelium; History, 18th Century; History, 19th Century; History, 21st Century; Host-Pathogen Interactions; Humans; Immunity, Innate; Microbiology; Mycobacterium marinum; Phagocytosis; Phagosomes
PubMed: 31290267
DOI: 10.1111/cmi.13083 -
Methods in Molecular Biology (Clifton,... 2018The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation,...
The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum cells make use of Rho-regulated signaling pathways to reorganize the actin cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation-competent cells with the chemoattractant cAMP, and for monitoring the localization and dynamics of Rac activity in live cells.
Topics: Chemotactic Factors; Chemotaxis; Cyclic AMP; Dictyostelium; Endocytosis; Protozoan Proteins; Signal Transduction; cdc42 GTP-Binding Protein; rac GTP-Binding Proteins
PubMed: 30062425
DOI: 10.1007/978-1-4939-8612-5_25 -
Biomolecules Nov 2019Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these -substituted adenine...
Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these -substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, . Within , CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that produces 6 CK forms (-Zeatin (Z), discadenine (DA), -isopentenyladenine (iP), -isopentenyladenine-9-riboside (iPR), -isopentenyladenine-9-riboside-5' phosphate (iPRP), and 2-methylthio--isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in , this research offers new insight into the nature of CK biosynthesis, secretion, and function during growth, development, and spore germination.
Topics: Cell Line; Cell Proliferation; Chromatography, High Pressure Liquid; Cytokinins; Dictyostelium; Life Cycle Stages; Metabolomics; Spectrometry, Mass, Electrospray Ionization; Spores, Protozoan; Up-Regulation
PubMed: 31694277
DOI: 10.3390/biom9110702 -
Biochemical and Biophysical Research... Jan 2015The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to...
The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during nucleolar disruption as a result of either AM-D treatment or mitosis support these subcompartments. A model for the AM-D-induced redistribution patterns is proposed.
Topics: Biomarkers; Cell Compartmentation; Cell Line; Cell Nucleolus; Dictyostelium; Green Fluorescent Proteins; Humans; Models, Biological; Protein Transport; Protozoan Proteins; Recombinant Fusion Proteins
PubMed: 25522879
DOI: 10.1016/j.bbrc.2014.12.050 -
ELife Mar 2022Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower...
Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis shares both similarities and differences with chemical-gradient-sensing pathways. We examine a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 min after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display 'U-turn' behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.
Topics: Cell Movement; Dictyostelium; Electricity; Signal Transduction; Wound Healing
PubMed: 35318938
DOI: 10.7554/eLife.73198 -
Scientific Reports Sep 2020The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene...
The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations in the targeted region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n + 1 and 3n + 2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.
Topics: Amino Acid Sequence; Base Sequence; CRISPR-Cas Systems; Dictyostelium; Gene Editing; Genes, Essential; Protozoan Proteins; Sequence Homology
PubMed: 32901070
DOI: 10.1038/s41598-020-71690-8 -
BMC Ecology and Evolution Oct 2023Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid...
BACKGROUND
Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4.
RESULTS
Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells.
CONCLUSIONS
The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.
Topics: Dictyostelium; Ammonia; Phosphorus-Oxygen Lyases; Dictyosteliida; Oxygen
PubMed: 37803310
DOI: 10.1186/s12862-023-02169-z