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Seminars in Cell & Developmental Biology May 2023While the field of synthetic developmental biology has traditionally focused on the study of the rich developmental processes seen in metazoan systems, an attractive... (Review)
Review
While the field of synthetic developmental biology has traditionally focused on the study of the rich developmental processes seen in metazoan systems, an attractive alternate source of inspiration comes from microbial developmental models. Microbes face unique lifestyle challenges when forming emergent multicellular collectives. As a result, the solutions they employ can inspire the design of novel multicellular systems. In this review, we dissect the strategies employed in multicellular development by two model microbial systems: the cellular slime mold Dictyostelium discoideum and the biofilm-forming bacterium Bacillus subtilis. Both microbes face similar challenges but often have different solutions, both from metazoan systems and from each other, to create emergent multicellularity. These challenges include assembling and sustaining a critical mass of participating individuals to support development, regulating entry into development, and assigning cell fates. The mechanisms these microbial systems exploit to robustly coordinate development under a wide range of conditions offer inspiration for a new toolbox of solutions to the synthetic development community. Additionally, recreating these phenomena synthetically offers a pathway to understanding the key principles underlying how these behaviors are coordinated naturally.
Topics: Humans; Animals; Dictyostelium; Models, Biological
PubMed: 35537929
DOI: 10.1016/j.semcdb.2022.04.014 -
Frontiers in Physiology 2022Cullins (CULs) are a core component of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs... (Review)
Review
Cullins (CULs) are a core component of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba , has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in , which supports previous work that revealed the cullin interactome in and the roles of cullins and the CSN in regulating cellular and developmental processes during the life cycle. Here, we review the roles of cullins, neddylation, and the CSN in to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.
PubMed: 35586714
DOI: 10.3389/fphys.2022.827435 -
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 -
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 -
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 -
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 -
Biophysical Journal Dec 2022Cell shape change processes, such as proliferation, polarization, migration, and cancer metastasis, rely on a dynamic network of macromolecules. The proper function of...
Cell shape change processes, such as proliferation, polarization, migration, and cancer metastasis, rely on a dynamic network of macromolecules. The proper function of this network enables mechanosensation, the ability of cells to sense and respond to mechanical cues. Myosin II and cortexillin I, critical elements of the cellular mechanosensory machinery, preassemble in the cytoplasm of Dictyostelium cells into complexes that we have termed contractility kits (CKs). Two IQGAP proteins then differentially regulate the mechanoresponsiveness of the cortexillin I-myosin II elements within CKs. To investigate the mechanism of CK self-assembly and gain insight into possible molecular means for IQGAP regulation, we developed a coarse-grained excluded volume molecular model in which all protein polymers are represented by nm-sized spheres connected by spring-like links. The model is parameterized using experimentally measured parameters acquired through fluorescence cross-correlation spectroscopy and fluorescence correlation spectroscopy, which describe the interaction affinities and diffusion coefficients for individual molecular components, and which have also been validated via several orthogonal methods. Simulations of wild-type and null-mutant conditions implied that the temporal order of assembly of these kits is dominated by myosin II dimer formation and that IQGAP proteins mediate cluster growth. In addition, our simulations predicted the existence of "ambiguous" CKs that incorporate both classes of IQGAPs, and we confirmed this experimentally using fluorescence cross-correlation spectroscopy. The model serves to describe the formation of the CKs and how their assembly enables and regulates mechanosensation at the molecular level.
Topics: Dictyostelium
PubMed: 36273263
DOI: 10.1016/j.bpj.2022.10.031 -
Molecular Biology of the Cell Apr 2023Multinucleate cells of divide usually by unilateral cleavage furrows that ingress from the cell border. Along their path into the cell, they follow regions that are...
Multinucleate cells of divide usually by unilateral cleavage furrows that ingress from the cell border. Along their path into the cell, they follow regions that are rich in myosin II and cortexillin and leave out the areas around the spindle poles that are populated with microtubule asters. In cells of a mutant that remain spread during mitosis we observed, as a rare event, cleavage by the expansion of a hole that is initiated in the middle of the cell area and has no connection with the cell's periphery. Here we show that these ring-shaped furrows develop in two phases, the first being reversible. During the first phase, the dorsal and ventral cell cortices come in close apposition and the cell membrane detaches locally from the substrate surface. The second phase comprises formation of the hole by membrane fusion and expansion of the opening toward the border of the cell, eventually cutting the multinucleate cell into pieces. We address the three-dimensional organization of ring-shaped furrows, their interaction with lateral furrows, and their association with filamentous myosin II and cortexillin. Thus, despite their geometrical divergence, similar molecular mechanisms might link the expanding hole to the standard contractile ring.
Topics: Dictyostelium; Mitosis; Microtubules; Myosin Type II; Cell Membrane; Cytoskeletal Proteins
PubMed: 36652336
DOI: 10.1091/mbc.E22-10-0487