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Open Biology Jul 2017Starved cells aggregate into groups of nearly 10 cells. AMPK is a highly conserved serine/threonine protein kinase consisting of a catalytic and two regulatory...
Starved cells aggregate into groups of nearly 10 cells. AMPK is a highly conserved serine/threonine protein kinase consisting of a catalytic and two regulatory subunits. As multi-cellular development in is initiated upon starvation, we explored the role of the energy sensor, AMPK, which shows significant similarity to human AMPK and is expressed throughout development. Deletion of the gene results in the formation of numerous small-sized aggregates that develop asynchronously to form few fruiting bodies with small sori and long stalks. On the other hand, cells form fruiting bodies with small stalks and large sori when compared with wild-type, Ax2. A minimum of 5% cells in a chimaera with Ax2 cells was sufficient to reduce the aggregate size. Also, the conditioned media collected from cells triggered Ax2 cells to form smaller aggregates. The starved cells showed low glucose levels and formed large aggregates when glucose was supplied exogenously. Interestingly, cells exhibit abnormal cell-type patterning with increased prestalk region and a concomitant reduction of prespore region. In addition, there was a loss of distinct prestalk/prespore boundary in the slugs.
Topics: AMP-Activated Protein Kinases; Body Patterning; Cell Proliferation; Dictyostelium; Gene Expression; Gene Knockout Techniques; Glucose; Models, Biological; Mutation; Organ Specificity; RNA, Messenger; Transcription, Genetic
PubMed: 28701378
DOI: 10.1098/rsob.170055 -
Journal of Cell Science Mar 2018Macropinocytosis is a conserved endocytic process used by amoebae for feeding on liquid medium. To further as a model for macropinocytosis, we developed a...
Macropinocytosis is a conserved endocytic process used by amoebae for feeding on liquid medium. To further as a model for macropinocytosis, we developed a high-throughput flow cytometry assay to measure macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis. has two feeding states: phagocytic and macropinocytic. When cells are switched from phagocytic growth on bacteria to liquid media, the rate of macropinocytosis slowly increases, due to increased size and frequency of macropinosomes. Upregulation is triggered by a minimal medium containing three amino acids plus glucose and likely depends on macropinocytosis itself. The presence of bacteria suppresses macropinocytosis while their product, folate, partially suppresses upregulation of macropinocytosis. Starvation, which initiates development, does not of itself suppress macropinocytosis: this can continue in isolated cells, but is shut down by a conditioned-medium factor or activation of PKA signalling. Thus macropinocytosis is a facultative ability of cells, regulated by environmental conditions that are identified here.This article has an associated First Person interview with the first author of the paper.
Topics: Amino Acids; Dictyostelium; Glucose; Phagocytosis; Pinocytosis; Protozoan Proteins
PubMed: 29440238
DOI: 10.1242/jcs.213736 -
Cells Jan 2022cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To...
cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in , we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Topics: Centrosome; Dictyostelium; Mitosis; Nuclear Pore; Nuclear Pore Complex Proteins
PubMed: 35159217
DOI: 10.3390/cells11030407 -
BMC Genomics Jun 2021Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG)...
BACKGROUND
Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. However, the functions of these two core ATG proteins still need further elucidation. Here, we applied RNA and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in Dictyostelium discoideum ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells.
RESULT
In total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNA and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS).
CONCLUSIONS
Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells. Our results suggest, that most changes in protein abundance were not caused by transcriptional changes, but were rather due to changes in protein homeostasis. In particular, knock-out of atg9 and/or atg16 appears to cause dysregulation of lipid metabolism and oxidative phosphorylation.
Topics: Autophagy; Dictyostelium; Proteomics; Protozoan Proteins; RNA
PubMed: 34126926
DOI: 10.1186/s12864-021-07756-2 -
ELife Apr 2020Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing cells undergo highly efficient...
Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing cells undergo highly efficient dedifferentiation, completed by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration.
Topics: Cell Dedifferentiation; Dictyostelium; Gene Expression; Gene Expression Profiling; Mutation; Single-Cell Analysis; Transcription Factors
PubMed: 32255425
DOI: 10.7554/eLife.55435 -
MSystems Oct 2022The social amoeba Dictyostelium discoideum is a predatory soil protist frequently used for studying host-pathogen interactions. A subset of D. discoideum strains...
The social amoeba Dictyostelium discoideum is a predatory soil protist frequently used for studying host-pathogen interactions. A subset of D. discoideum strains isolated from soil persistently carry symbiotic , recently formally described as , , and . The three facultative symbiont species of D. discoideum present a unique opportunity to study a naturally occurring symbiosis in a laboratory model protist. There is a large difference in genome size between (8.7 million base pairs [Mbp]) versus and (4.1 Mbp). We took a comparative genomics approach and compared the three genomes of D. discoideum symbionts to 12 additional genomes to test for genome evolution patterns that frequently accompany host adaptation. Overall, is difficult to distinguish from other based on its genome size and content, but the reduced genomes of and display characteristics indicative of genome streamlining rather than deterioration during adaptation to their protist hosts. In addition, D. discoideum-symbiont genomes have increased secretion system and motility genes that may mediate interactions with their host. Specifically, adjacent BurBor-like type 3 and T6SS-5-like type 6 secretion system operons shared among all three D. discoideum-symbiont genomes may be important for host interaction. Horizontal transfer of these secretion system operons within the amoeba host environment may have contributed to the unique ability of these symbionts to establish and maintain a symbiotic relationship with D. discoideum. Protists are a diverse group of typically single cell eukaryotes. Bacteria and archaea that form long-term symbiotic relationships with protists may evolve in additional ways than those in relationships with multicellular eukaryotes such as plants, animals, or fungi. Social amoebas are a predatory soil protist sometimes found with symbiotic bacteria living inside their cells. They present a unique opportunity to explore a naturally occurring symbiosis in a protist frequently used for studying host-pathogen interactions. We show that one amoeba-symbiont species is similar to other related bacteria in genome size and content, while the two reduced-genome-symbiont species show characteristics of genome streamlining rather than deterioration during adaptation to their host. We also identify sets of genes present in all three amoeba-symbiont genomes that are potentially used for host-symbiont interactions. Because the amoeba symbionts are distantly related, the amoeba host environment may be where these genes were shared among symbionts.
Topics: Animals; Amoeba; Dictyostelium; Eukaryota; Burkholderiaceae; Bacteria; Soil
PubMed: 36098425
DOI: 10.1128/msystems.00562-22 -
ACS Chemical Biology Mar 2023Protein aggregation is a hallmark of the polyglutamine diseases. One potential treatment for these diseases is suppression of polyglutamine aggregation. Previous work...
Protein aggregation is a hallmark of the polyglutamine diseases. One potential treatment for these diseases is suppression of polyglutamine aggregation. Previous work identified the cellular slime mold as being naturally resistant to polyglutamine aggregation. Further work identified serine-rich chaperone protein 1 (SRCP1) as a protein that is both necessary in and sufficient in human cells to suppress polyglutamine aggregation. Therefore, understanding how SRCP1 suppresses aggregation may be useful for developing therapeutics for the polyglutamine diseases. Here we utilized a protein modeling approach to generate predictions of SRCP1's structure. Using our best-fit model, we generated mutants that were predicted to alter the stability of SRCP1 and tested these mutants' stability in cells. Using these data, we identified top models of SRCP1's structure that are consistent with the C-terminal region of SRCP1 forming a β-hairpin with a highly dynamic N-terminal region. We next generated a series of peptides that mimic the predicted β-hairpin and validated that they inhibit aggregation of a polyglutamine-expanded mutant huntingtin exon 1 fragment . To further assess mechanistic details of how SRCP1 inhibits polyglutamine aggregation, we utilized biochemical assays to determine that SRCP1 inhibits secondary nucleation in a manner dependent upon the regions flanking the polyglutamine tract. Finally, to determine if SRCP1 more could generally suppress protein aggregation, we confirmed that it was sufficient to inhibit aggregation of polyglutamine-expanded ataxin-3. Together these studies provide details into the structural and mechanistic basis of the inhibition of protein aggregation by SRCP1.
Topics: Humans; Protein Aggregates; Dictyostelium; Serine; Molecular Chaperones; Peptides; Huntingtin Protein
PubMed: 36791332
DOI: 10.1021/acschembio.2c00893 -
MBio Feb 2021Ingestion and killing of bacteria by phagocytic cells protect the human body against infections. While many mechanisms have been proposed to account for bacterial...
Ingestion and killing of bacteria by phagocytic cells protect the human body against infections. While many mechanisms have been proposed to account for bacterial killing in phagosomes, their relative importance, redundancy, and specificity remain unclear. In this study, we used the amoeba as a model phagocyte and quantified the requirement of 11 individual gene products, including nine putative effectors, for the killing of bacteria. This analysis revealed that radically different mechanisms are required to kill , , , , and AlyL, a lysozyme-like protein equipped with a distinct bacteriolytic region, plays a specific role in the intracellular killing of , with assistance from BpiC and Aoah, two lipopolysaccharide (LPS)-binding proteins. Rapid killing of and requires the presence of BpiC and of the NoxA NADPH oxidase. No single effector tested is essential for rapid killing of or Overall, our observations reveal an unsuspected degree of specificity in the elimination of bacteria in phagosomes. Phagocytic cells ingest and kill bacteria, a process essential for the defense of the human body against infections. Many potential killing mechanisms have been identified in phagocytic cells, including free radicals, toxic ions, enzymes, and permeabilizing peptides. Yet fundamental questions remain unanswered: what is the relative importance of these mechanisms, how redundant are they, and are different mechanisms used to kill different species of bacteria? We addressed these questions using , a model phagocytic cell amenable to genetic manipulations and quantitative analysis. Our results reveal that vastly different mechanisms are required to kill different species of bacteria. This very high degree of specificity was unexpected and indicates that a lot remains to be discovered about how phagocytic cells eliminate bacteria.
Topics: Bacteria; Dictyostelium; Klebsiella pneumoniae; Phagocytes; Phagocytosis; Phagosomes; Pseudomonas aeruginosa; Staphylococcus aureus
PubMed: 33593980
DOI: 10.1128/mBio.03169-20 -
Frontiers in Cellular and Infection... 2019
Topics: Acanthamoeba; Bacteria; Dictyostelium; Fungi; Host-Pathogen Interactions; Models, Theoretical
PubMed: 30941316
DOI: 10.3389/fcimb.2019.00047 -
Disease Models & Mechanisms Jan 2018Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical...
Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.
Topics: Antioxidants; Curcumin; Dictyostelium; Ligands; Molecular Docking Simulation; Presenilin-1; Protein Phosphatase 2; Sequence Homology, Amino Acid
PubMed: 29361519
DOI: 10.1242/dmm.032375