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Cell Adhesion & Migration Dec 2024Copines are a family of calcium-dependent membrane-binding proteins. To study these proteins, anull mutant for was created in , which has six copines genes (). During...
Copines are a family of calcium-dependent membrane-binding proteins. To study these proteins, anull mutant for was created in , which has six copines genes (). During development, cells were able to aggregate, but did not form streams. Once aggregated into mounds, they formed large ring structures. cells were less adherent to plastic substrates, but more adherent to other cells. These phenotypes correlated with changes in adhesion protein expression with decreased expression of SibA and increased expression of CsaA in developing cells. We also measured the expression of RegA, a cAMP phosphodiesterase, and found that cells have reduced RegA expression. The reduced RegA expression in cells is most likely responsible for the observed phenotypes.
Topics: Dictyostelium; Carrier Proteins
PubMed: 38378453
DOI: 10.1080/19336918.2024.2315629 -
Methods in Molecular Biology (Clifton,... 2024Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has...
Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.
Topics: Dictyostelium; Autophagy; Microscopy, Confocal; Green Fluorescent Proteins; Lysosomes; Protozoan Proteins
PubMed: 38954200
DOI: 10.1007/978-1-0716-3894-1_7 -
Cells Mar 2024Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, ) and Polycystin-2 (PC2, ) contain mutations. PC1 is a large membrane...
Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, ) and Polycystin-2 (PC2, ) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca signalling also impacted other downstream Ca-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that is a useful model to study Polycystin-2 mediated calcium signalling.
Topics: Humans; Polycystic Kidney, Autosomal Dominant; Dictyostelium; TRPP Cation Channels; Calcium; Calcium Signaling; Calcium Channels
PubMed: 38607049
DOI: 10.3390/cells13070610 -
ACS Infectious Diseases Feb 2024The emergence of hypervirulent (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired...
The emergence of hypervirulent (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired infections in healthy individuals. The ability to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Polyphosphate kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using amoeba as a model host. We found that the PPK1 null mutant was impaired in biofilm and capsule formation and showed attenuated virulence in compared to the wild-type strain. We performed a proteomic analysis to gain further insights into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins involved in capsule synthesis (Wzi-Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB-LpxC-PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hvKP. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in and provides insights into potential therapeutic targets for treating infections.
Topics: Humans; Virulence; Klebsiella pneumoniae; Polyphosphates; Dictyostelium; Proteomics; Biofilms
PubMed: 38205780
DOI: 10.1021/acsinfecdis.3c00509 -
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 -
Life Sciences Dec 2023Differentiation-inducing factor-1 (DIF-1), a compound in Dictyostelium discoideum, exhibits anti-cancer effects by inhibiting cell proliferation and motility of various...
AIMS
Differentiation-inducing factor-1 (DIF-1), a compound in Dictyostelium discoideum, exhibits anti-cancer effects by inhibiting cell proliferation and motility of various mammalian cancer cells in vitro and in vivo. In addition, DIF-1 suppresses lung colony formation in a mouse model, thus impeding cancer metastasis. However, the precise mechanism underlying its anti-metastatic effect remains unclear. In the present study, we aim to elucidate this mechanism by investigating the adhesion of circulating tumor cells to blood vessels using in vitro and in vivo systems.
MAIN METHODS
Melanoma cells (1.0 × 10 cells) were injected into the tail vein of 8-week-old male C57BL/6 mice after administration of DIF-1 (300 mg/kg per day) and/or lipopolysaccharide (LPS: 2.5 mg/kg per day). To investigate cell adhesion and molecular mechanisms, cell adhesion assay, western blotting, immunofluorescence staining, and flow cytometry were performed.
KEY FINDINGS
Intragastric administration of DIF-1 suppressed lung colony formation. DIF-1 also substantially inhibited the adhesion of cancer cells to human umbilical vein endothelial cells. Notably, DIF-1 did not affect the expression level of adhesion-related proteins in cancer cells, but it did decrease the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells by suppressing its mRNA-to-protein translation through inhibition of mTORC1-p70 S6 kinase signaling.
SIGNIFICANCE
DIF-1 reduced tumor cell adhesion to blood vessels by inhibiting mTORC1-S6K signaling and decreasing the expression of adhesion molecule VCAM-1 on vascular endothelial cells. These findings highlight the potential of DIF-1 as a promising compound for the development of anti-cancer drugs with anti-metastatic properties.
Topics: Mice; Animals; Male; Humans; Vascular Cell Adhesion Molecule-1; Lipopolysaccharides; Dictyostelium; Mice, Inbred C57BL; Proteins; Human Umbilical Vein Endothelial Cells; Mechanistic Target of Rapamycin Complex 1; Cell Differentiation; Cell Adhesion; Mammals
PubMed: 37981227
DOI: 10.1016/j.lfs.2023.122278 -
Scientific Data Jun 2024Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition...
Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition from uni- to multicellular life. The D. firmibasis genome assembly that is currently available is of limited use due to its low contiguity, large number of undetermined bases, and lack of annotations. Here we used Nanopore long read sequencing, complemented with Illumina sequencing, and developmental transcriptomics as well as small RNA-sequencing, to present a new, fully annotated, chromosome-level D. firmibasis genome assembly. The new assembly contains no undetermined bases, and consists mainly of six large contigs representing the chromosomes, as well as a complete mitochondrial genome. This new genome assembly will be a valuable tool, allowing comprehensive comparison to Dictyostelium discoideum, the dictyostelid genetically tractable model. Further, the new genome will be important for studies of evolutionary processes governing the transition from unicellular to multicellular organisms and will aid in the sequencing and annotation of other dictyostelids genomes, many of which are currently of poor quality.
Topics: Dictyostelium; Genome, Protozoan; Chromosomes; Molecular Sequence Annotation
PubMed: 38909042
DOI: 10.1038/s41597-024-03513-8 -
Proceedings. Biological Sciences Dec 2023Many microbes interact with one another, but the difficulty of directly observing these interactions in nature makes interpreting their adaptive value complicated. The...
Many microbes interact with one another, but the difficulty of directly observing these interactions in nature makes interpreting their adaptive value complicated. The social amoeba forms aggregates wherein some cells are sacrificed for the benefit of others. Within chimaeric aggregates containing multiple unrelated lineages, cheaters can gain an advantage by undercontributing, but the extent to which wild has adapted to cheat is not fully clear. In this study, we experimentally evolved in an environment where there were no selective pressures to cheat or resist cheating in chimaeras. lines grown in this environment evolved reduced competitiveness within chimaeric aggregates and reduced ability to migrate during the slug stage. By contrast, we did not observe a reduction in cell number, a trait for which selection was not relaxed. The observed loss of traits that our laboratory conditions had made irrelevant suggests that these traits were adaptations driven and maintained by selective pressures faces in its natural environment. Our results suggest that faces social conflict in nature, and illustrate a general approach that could be applied to searching for social or non-social adaptations in other microbes.
Topics: Dictyostelium; Social Evolution
PubMed: 38113942
DOI: 10.1098/rspb.2023.1722 -
Frontiers in Cell and Developmental... 2023Like most eukaryotes, the pre-metazoan social amoeba depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In ,...
Like most eukaryotes, the pre-metazoan social amoeba depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In , starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas -disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.
PubMed: 37779900
DOI: 10.3389/fcell.2023.1259844 -
Methods in Molecular Biology (Clifton,... 2024Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium...
Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.
Topics: Dictyostelium; ras Proteins; rap GTP-Binding Proteins; Protozoan Proteins; Signal Transduction; Protein Binding
PubMed: 38954205
DOI: 10.1007/978-1-0716-3894-1_12