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Methods in Molecular Biology (Clifton,... 2024Uptaking particulate objects and bulk liquid by eucaryotic cells is critical for their growth, survival, and defense. Dictyostelium is a model organism spearheaded to...
Uptaking particulate objects and bulk liquid by eucaryotic cells is critical for their growth, survival, and defense. Dictyostelium is a model organism spearheaded to uncover mechanisms behind various types of uptaking activities. Here, we describe assays measuring phagocytosis and macropinocytosis using Dictyostelium discoideum.
Topics: Dictyostelium; Phagocytosis; Pinocytosis
PubMed: 38954198
DOI: 10.1007/978-1-0716-3894-1_5 -
Methods in Molecular Biology (Clifton,... 2024Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules...
Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules including nucleic acids, proteins, polysaccharides, and lipids. In addition to functioning within lysosomes, lysosomal enzymes are also secreted. Alterations in the levels and activities of lysosomal enzymes dysregulates lysosomes, which can lead to the intralysosomal accumulation of biological material and the development of lysosomal storage diseases (LSDs) in humans. Dictyostelium discoideum has a long history of being used to study the trafficking and functions of lysosomal enzymes. More recently, it has been used as a model system to study several LSDs. In this chapter, we outline the methods for assessing the activity of several lysosomal enzymes in D. discoideum (α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-N-acetylglucosaminidase, α-mannosidase, cathepsin B, cathepsin D, cathepsin F, palmitoyl protein thioesterase 1, and tripeptidyl peptidase 1).
Topics: Dictyostelium; Lysosomes; Tripeptidyl-Peptidase 1; Enzyme Assays; Humans; beta-Galactosidase; Lysosomal Storage Diseases; Thiolester Hydrolases
PubMed: 38954197
DOI: 10.1007/978-1-0716-3894-1_4 -
Methods in Molecular Biology (Clifton,... 2024Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or...
Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.
PubMed: 38954196
DOI: 10.1007/978-1-0716-3894-1_3 -
Methods in Molecular Biology (Clifton,... 2024Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in...
Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in resolution in ExM is not achieved by improvement of the resolution of the microscope itself but by isotropic expansion of the sample. To achieve this, the sample is cross-linked to an expandable gel matrix that swells approximately fourfold by incubation in water. We have applied the method to Dictyostelium amoebae and discuss the pros and cons of different labeling techniques in combination with pre- and post-expansion staining protocols.
Topics: Dictyostelium; Microscopy; Staining and Labeling; Microscopy, Fluorescence
PubMed: 38954195
DOI: 10.1007/978-1-0716-3894-1_2 -
Methods in Molecular Biology (Clifton,... 2024The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis,...
The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.
Topics: Dictyostelium; Cytokinesis; Gene Library; Protozoan Proteins; Signal Transduction; Fluorescence Recovery After Photobleaching
PubMed: 38954194
DOI: 10.1007/978-1-0716-3894-1_1 -
Nature Cell Biology Jul 2024Ras has been extensively studied as a promoter of cell proliferation, whereas few studies have explored its role in migration. To investigate the direct and immediate...
Ras has been extensively studied as a promoter of cell proliferation, whereas few studies have explored its role in migration. To investigate the direct and immediate effects of Ras activity on cell motility or polarity, we focused on RasGAPs, C2GAPB in Dictyostelium amoebae and RASAL3 in HL-60 neutrophils and macrophages. In both cellular systems, optically recruiting the respective RasGAP to the cell front extinguished pre-existing protrusions and changed migration direction. However, when these respective RasGAPs were recruited uniformly to the membrane, cells polarized and moved more rapidly, whereas targeting to the back exaggerated these effects. These unexpected outcomes of attenuating Ras activity naturally had strong, context-dependent consequences for chemotaxis. The RasGAP-mediated polarization depended critically on myosin II activity and commenced with contraction at the cell rear, followed by sustained mTORC2-dependent actin polymerization at the front. These experimental results were captured by computational simulations in which Ras levels control front- and back-promoting feedback loops. The discovery that inhibiting Ras activity can produce counterintuitive effects on cell migration has important implications for future drug-design strategies targeting oncogenic Ras.
PubMed: 38951708
DOI: 10.1038/s41556-024-01453-4 -
Journal of Cell Science Jun 2024Little is known about eukaryotic chemorepulsion. The enzymes Phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate...
Little is known about eukaryotic chemorepulsion. The enzymes Phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3, and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion.
PubMed: 38940195
DOI: 10.1242/jcs.262054 -
Biochemical Society Transactions Jun 2024Macropinocytosis is a broadly conserved endocytic process discovered nearly 100 years ago, yet still poorly understood. It is prominent in cancer cell feeding, immune...
Macropinocytosis is a broadly conserved endocytic process discovered nearly 100 years ago, yet still poorly understood. It is prominent in cancer cell feeding, immune surveillance, uptake of RNA vaccines and as an invasion route for pathogens. Macropinocytic cells extend large cups or flaps from their plasma membrane to engulf droplets of medium and trap them in micron-sized vesicles. Here they are digested and the products absorbed. A major problem - discussed here - is to understand how cups are shaped and closed. Recently, lattice light-sheet microscopy has given a detailed description of this process in Dictyostelium amoebae, leading to the 'stalled-wave' model for cup formation and closure. This is based on membrane domains of PIP3 and active Ras and Rac that occupy the inner face of macropinocytic cups and are readily visible with suitable reporters. These domains attract activators of dendritic actin polymerization to their periphery, creating a ring of protrusive F-actin around themselves, thus shaping the walls of the cup. As domains grow, they drive a wave of actin polymerization across the plasma membrane that expands the cup. When domains stall, continued actin polymerization under the membrane, combined with increasing membrane tension in the cup, drives closure at lip or base. Modelling supports the feasibility of this scheme. No specialist coat proteins or contractile activities are required to shape and close cups: rings of actin polymerization formed around PIP3 domains that expand and stall seem sufficient. This scheme may be widely applicable and begs many biochemical questions.
PubMed: 38934501
DOI: 10.1042/BST20231426 -
Microorganisms May 2024Protected areas are widely considered an essential strategy for biodiversity conservation. Dictyostelids are unique protists known to have important ecological functions...
Diversity of Cellular Slime Molds (Dictyostelids) in the Fanjing Mountain Nature Reserve and Geographical Distribution Comparisons with Other Representative Nature Reserves in Different Climate Zones of China.
Protected areas are widely considered an essential strategy for biodiversity conservation. Dictyostelids are unique protists known to have important ecological functions in promoting soil and plant health through their top-down regulation of ecosystem processes, such as decomposition, that involve bacterial populations. But the relationship between dictyostelid diversity within protected areas remains poorly understood, especially on a large scale. Herein, we report data on the distribution of dictyostelids, identified with ITS + SSU rRNA molecular and morphology-based taxonomy, from soil samples collected in the Fanjing Mountain protected area of Guizhou Province, Southwest China. We compared the biodiversity data of dictyostelids in Fanjing Mountain with similar data from previously sampled sites in four other protected areas, including Changbai Mountain (CB), Gushan Mountain (GS), Baiyun Mountain (BY), and Qinghai-Tibet Plateau (QT) in China. We identified four species of dictyostelids belonging to three genera (, and ) and herein provide information on the taxonomy of these species. Two species ( and ) are common and widely distributed throughout the world, but one species () was new to China. Our data indicate that there is no distinguishable significant correlation between the dictyostelid species studied and environmental factors. Overall, the similarity index between Baiyun Mountain in Henan Province and Fanjing Mountain in Guizhou Province, located at approximately the same longitude, is the highest, and the Jaccard similarity coefficients (Jaccard index) of family, genus, and species are 100%, 100%, and 12.5%, respectively. From a species perspective, species in the same climate zone are not closely related, but obvious geographical distributions are evident in different climate zones. This preliminary study provided evidence of the ecological adaptation of dictyostelids to different biological niches.
PubMed: 38930443
DOI: 10.3390/microorganisms12061061 -
International Journal of Molecular... Jun 2024Tanshinone IIA (T2A) is a bioactive compound that provides promise in the treatment of glioblastoma multiforme (GBM), with a range of molecular mechanisms including the...
Tanshinone IIA (T2A) is a bioactive compound that provides promise in the treatment of glioblastoma multiforme (GBM), with a range of molecular mechanisms including the inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy. Recently, T2A has been demonstrated to function through sestrin 2 (SESN) to inhibit mTORC1 activity, but its possible impact on autophagy through this pathway has not been investigated. Here, the model system and GBM cell lines were employed to investigate the cellular role of T2A in regulating SESN to inhibit mTORC1 and activate autophagy through a GATOR2 component MIOS. In , T2A treatment induced autophagy and inhibited mTORC1 activity, with both effects lost upon the ablation of SESN (sesn) or MIOS (mios). We further investigated the targeting of MIOS to reproduce this effect of T2A, where computational analysis identified 25 novel compounds predicted to strongly bind the human MIOS protein, with one compound (MIOS inhibitor 3; Mi3) reducing cell proliferation in two GBM cells. Furthermore, Mi3 specificity was demonstrated through the loss of potency in the mios cells regarding cell proliferation and the induction of autophagy. In GBM cells, Mi3 treatment also reduced mTORC1 activity and induced autophagy. Thus, a potential T2A mimetic showing the inhibition of mTORC1 and induction of autophagy in GBM cells was identified.
Topics: Glioblastoma; Abietanes; Humans; Mechanistic Target of Rapamycin Complex 1; Autophagy; Cell Line, Tumor; Dictyostelium; Cell Proliferation; Nuclear Proteins; Sestrins
PubMed: 38928292
DOI: 10.3390/ijms25126586