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ELife May 2023The amoeba-resistant bacterium causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated -containing vacuole...
The amoeba-resistant bacterium causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated -containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba . Dually fluorescence-labeled producing LCV and LD markers revealed that Sey1 as well as the T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δ mutant indicated that Sey1 and GTP promote this process. Sey1 and the fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular .
Topics: Humans; Legionella pneumophila; GTP Phosphohydrolases; Macrophages; Dictyostelium; Lipid Droplets; Vacuoles; Legionella; Legionnaires' Disease; Bacterial Proteins
PubMed: 37158597
DOI: 10.7554/eLife.85142 -
The Journal of Biological Chemistry May 2024Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested...
Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.
PubMed: 38815864
DOI: 10.1016/j.jbc.2024.107423 -
BMC Research Notes Apr 2020The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion,...
OBJECTIVE
The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion, macropinocytosis and phagocytosis, host-pathogen interactions and multicellular development. However, the relative small size of the Dictyostelium community hampers the production and distribution of reagents and tools, such as antibodies, by commercial vendors.
RESULTS
For the past 5 years, our laboratory has worked to promote an increased use of recombinant antibodies (rAbs) by academic laboratories. Here we report our efforts to ensure that Dictyostelium researchers have access to rAbs. Using hybridoma sequencing and phage display techniques, we generated a panel of recombinant antibodies against D. discoideum antigens, providing a useful and reliable set of reagents for labelling and characterization of proteins and subcellular compartments in D. discoideum, accessible to the entire Dictyostelium community.
Topics: Antibodies; Dictyostelium; Hybridomas; Models, Biological; Recombinant Proteins
PubMed: 32276653
DOI: 10.1186/s13104-020-05048-8 -
Current Biology : CB Aug 2023Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an...
Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.
Topics: Actins; Dictyostelium; Cell Membrane Structures; Actin Cytoskeleton; Endocytosis
PubMed: 37379843
DOI: 10.1016/j.cub.2023.06.017 -
Biophysical Journal Jul 2019A living cell's interior is one of the most complex and intrinsically dynamic systems, providing an elaborate interplay between cytosolic crowding and ATP-driven motion...
A living cell's interior is one of the most complex and intrinsically dynamic systems, providing an elaborate interplay between cytosolic crowding and ATP-driven motion that controls cellular functionality. Here, we investigated two distinct fundamental features of the merely passive, non-biomotor-shuttled material transport within the cytoplasm of Dictyostelium discoideum cells: the anomalous non-linear scaling of the mean-squared displacement of a 150-nm-diameter particle and non-Gaussian distribution of increments. Relying on single-particle tracking data of 320,000 data points, we performed a systematic analysis of four possible origins for non-Gaussian transport: 1) sample-based variability, 2) rarely occurring strong motion events, 3) ergodicity breaking/aging, and 4) spatiotemporal heterogeneities of the intracellular medium. After excluding the first three reasons, we investigated the remaining hypothesis of a heterogeneous cytoplasm as cause for non-Gaussian transport. A, to our knowledge, novel fit model with randomly distributed diffusivities implementing medium heterogeneities suits the experimental data. Strikingly, the non-Gaussian feature is independent of the cytoskeleton condition and lag time. This reveals that efficiency and consistency of passive intracellular transport and the related anomalous scaling of the mean-squared displacement are regulated by cytoskeleton components, whereas cytoplasmic heterogeneities are responsible for the generic, non-Gaussian distribution of increments.
Topics: Actins; Biological Transport; Dictyostelium; Intracellular Space; Microtubules; Models, Biological; Motion; Nanoparticles; Probability
PubMed: 31278001
DOI: 10.1016/j.bpj.2019.06.009 -
BioEssays : News and Reviews in... Apr 2021Ancient pathways promoting unicellularity and multicellularity are associated with cancer, the former being pro-oncogenic and the latter acting to suppress oncogenesis.... (Review)
Review
Ancient pathways promoting unicellularity and multicellularity are associated with cancer, the former being pro-oncogenic and the latter acting to suppress oncogenesis. However, there are only a limited number of non-vertebrate models for studying these pathways. Here, we review Dictyostelium discoideum and describe how it can be used to understand these gene networks. D. discoideum has a unicellular and multicellular life cycle, making it possible to study orthologs of cancer-associated genes in both phases. During development, differentiated amoebae form a fruiting body composed of a mass of spores that are supported atop a stalk. A portion of the cells sacrifice themselves to become non-reproductive stalk cells. Cheating disrupts the principles of multicellularity, as cheater cells alter their cell fate to preferentially become spores. Importantly, D. discoideum has gene networks and several strategies for maintaining multicellularity. Therefore, D. discoideum can help us better understand how conserved genes and pathways involved in multicellularity also influence cancer development, potentially identifying new therapeutic avenues.
Topics: Amoeba; Cell Differentiation; Dictyostelium; Humans; Neoplasms
PubMed: 33448043
DOI: 10.1002/bies.202000156 -
Frontiers in Cellular and Infection... 2022A variety of bacteria have evolved the ability to interact with environmental phagocytic predators such as amoebae, which may have facilitated their subsequent...
A variety of bacteria have evolved the ability to interact with environmental phagocytic predators such as amoebae, which may have facilitated their subsequent interactions with phagocytes in animal hosts. Our recent study found that the animal pathogen can evade predation by the common soil amoeba , survive within, and hijack its complex life cycle as a propagation and dissemination vector. However, it is uncertain whether the mechanisms allowing interactions with predatory amoebae are conserved among species, because divergence, evolution, and adaptation to different hosts and ecological niches was accompanied by acquisition and loss of many genes. Here we tested 9 diverse species in three assays representing distinct aspects of their interactions with . Several human and animal pathogens retained the abilities to survive within single-celled amoeba, to inhibit amoebic plaque expansion, and to translocate with amoebae to the fruiting body and disseminate along with the fruiting body. In contrast, these abilities were partly degraded for the bird pathogen , and for the human-restricted species and . Interestingly, a different lineage of only known to infect sheep retained the ability to interact with , demonstrating that these abilities were lost in multiple lineages independently, correlating with niche specialization and recent rapid genome decay apparently mediated by insertion sequences. has been isolated sporadically from diverse human and environmental sources, has acquired insertion sequences, undergone genome decay and has also lost the ability to interact with amoebae, suggesting some specialization to some unknown niche. A genome-wide association study (GWAS) identified a set of genes that are potentially associated with the ability to interact with . These results suggest that massive gene loss associated with specialization of some species to a closed life cycle in a particular host was repeatedly and independently accompanied by loss of the ability to interact with amoebae in an environmental niche.
Topics: Amoeba; Animals; Bordetella; Bordetella bronchiseptica; Dictyostelium; Genome-Wide Association Study; Sheep
PubMed: 35223538
DOI: 10.3389/fcimb.2022.798317 -
Applied Microbiology and Biotechnology May 2020Regulation of gene expression is fundamental for cellular function. Upon manipulation of the mechanism of gene expression in Escherichia coli, various bioproducts have... (Review)
Review
Regulation of gene expression is fundamental for cellular function. Upon manipulation of the mechanism of gene expression in Escherichia coli, various bioproducts have been developed that are valuable industrially and medically in the last four decades. To efficiently produce bioproducts, numerous molecular tools are used for enhancing expression at the transcriptional and translational levels. Our recent discovery identified a new approach that enhances the gene expression in E. coli using the gene sequence of the eukaryote, Dictyostelium discoideum. In this review, we highlight the current molecular strategies used for high-level gene expression techniques commonly utilized in basic and applied microbiology.
Topics: Biological Products; Cloning, Molecular; Dictyostelium; Escherichia coli; Gene Expression Regulation, Bacterial; Protein Biosynthesis; Transcription Factors
PubMed: 32125482
DOI: 10.1007/s00253-020-10430-4 -
Animal Cells and Systems 2021There are three Rap proteins in . RapA is a key regulator of cell adhesion and cytoskeletal rearrangement. Recently, RapC has been reported to be involved in...
There are three Rap proteins in . RapA is a key regulator of cell adhesion and cytoskeletal rearrangement. Recently, RapC has been reported to be involved in cytokinesis, cell migration, and multicellular development. Here, we compare the functions of RapA and RapC using cells expressing or lacking Rap proteins, and confirm that RapA and RapC have opposite functions in cell spreading, adhesion, and migration. On the other hand, RapC has a unique function in cytokinesis and multicellular development. Activated RapA appears to stimulate spreading and adhesion of the cells to the substrate, possibly resulting in a decrease in the migration speed of the cells during chemotaxis without affecting the directionality, whereas RapC suppresses cell spreading and adhesion, thereby increasing the migration speed. Cells lacking RapC were defective in cytokinesis and multicellular development and showed multinucleation and formation of multiple tips from a mound during development. At the C-terminus, RapC has an additional stretch of amino acids, which is not found in RapA. The mechanism through which RapA and RapC perform their opposite functions in diverse cellular processes should be characterized further to understand the Rap signaling pathways in detail. GAP; GTPase-activating proteins; GEF; guanine nucleotide exchanging factor; WT; wild type; CA; constitutively active; DN; dominantly negative.
PubMed: 34413965
DOI: 10.1080/19768354.2021.1947372 -
Biochemical Society Transactions Apr 2020Directed cell migration poses a rich set of theoretical challenges. Broadly, these are concerned with (1) how cells sense external signal gradients and adapt; (2) how... (Review)
Review
Directed cell migration poses a rich set of theoretical challenges. Broadly, these are concerned with (1) how cells sense external signal gradients and adapt; (2) how actin polymerisation is localised to drive the leading cell edge and Myosin-II molecular motors retract the cell rear; and (3) how the combined action of cellular forces and cell adhesion results in cell shape changes and net migration. Reaction-diffusion models for biological pattern formation going back to Turing have long been used to explain generic principles of gradient sensing and cell polarisation in simple, static geometries like a circle. In this minireview, we focus on recent research which aims at coupling the biochemistry with cellular mechanics and modelling cell shape changes. In particular, we want to contrast two principal modelling approaches: (1) interface tracking where the cell membrane, interfacing cell interior and exterior, is explicitly represented by a set of moving points in 2D or 3D space and (2) interface capturing. In interface capturing, the membrane is implicitly modelled analogously to a level line in a hilly landscape whose topology changes according to forces acting on the membrane. With the increased availability of high-quality 3D microscopy data of complex cell shapes, such methods will become increasingly important in data-driven, image-based modelling to better understand the mechanochemistry underpinning cell motion.
Topics: Actins; Biochemistry; Cell Membrane; Cell Movement; Computer Simulation; Dictyostelium; Diffusion; Hydrodynamics; Models, Theoretical; Molecular Motor Proteins; Myosin Type II; Polymerization
PubMed: 32239187
DOI: 10.1042/BST20190311