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Developmental Cell Mar 2024Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic...
Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions.
Topics: Animals; Dictyostelium; Pinocytosis; Cytoplasm; Cell Nucleus; Transcription Factors; Mammals
PubMed: 38325371
DOI: 10.1016/j.devcel.2024.01.012 -
PloS One 2024Amoeboid cell motility is relevant in a wide variety of biomedical processes such as wound healing, cancer metastasis, and embryonic morphogenesis. It is characterized...
Amoeboid cell motility is relevant in a wide variety of biomedical processes such as wound healing, cancer metastasis, and embryonic morphogenesis. It is characterized by pronounced changes of the cell shape associated with expansions and retractions of the cell membrane, which result in a crawling kind of locomotion. Despite existing computational models of amoeboid motion, the inference of expansion and retraction components of individual cells, the corresponding classification of cells, and the a priori specification of the parameter regime to achieve a specific motility behavior remain challenging open problems. We propose a novel model of the spatio-temporal evolution of two-dimensional cell contours comprising three biophysiologically motivated components: a stochastic term accounting for membrane protrusions and two deterministic terms accounting for membrane retractions by regularizing the shape and area of the contour. Mathematically, these correspond to the intensity of a self-exciting Poisson point process, the area-preserving curve-shortening flow, and an area adjustment flow. The model is used to generate contour data for a variety of qualitatively different, e.g., polarized and non-polarized, cell tracks that visually resemble experimental data very closely. In application to experimental cell tracks, we inferred the protrusion component and examined its correlation to common biomarkers: the F-actin density close to the membrane and its local motion. Due to the low model complexity, parameter estimation is fast, straightforward, and offers a simple way to classify contour dynamics based on two locomotion types: the amoeboid and a so-called fan-shaped type. For both types, we use cell tracks segmented from fluorescence imaging data of the model organism Dictyostelium discoideum. An implementation of the model is provided within the open-source software package AmoePy, a Python-based toolbox for analyzing and simulating amoeboid cell motility.
Topics: Amoeba; Dictyostelium; Cell Movement; Actins; Locomotion
PubMed: 38277351
DOI: 10.1371/journal.pone.0297511 -
Traffic (Copenhagen, Denmark) Jan 2024Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5...
Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.
Topics: Humans; Neuronal Ceroid-Lipofuscinoses; Cathepsin D; Dictyostelium; Protein Sorting Signals; Lysosomal Membrane Proteins
PubMed: 38272448
DOI: 10.1111/tra.12925 -
MSystems Feb 2024Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A...
Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A key feature of pathogenesis, conserved between the human pathogen and the model pathogen is the metabolic switch to lipid catabolism and altered expression of virulence genes at different stages of infection. This study aims to identify genes involved in sustaining viable intracellular infection. We applied transposon sequencing (Tn-Seq) to , an unbiased genome-wide strategy combining saturation insertional mutagenesis and high-throughput sequencing. This approach allowed us to identify the localization and relative abundance of insertions in pools of transposon mutants. Gene essentiality and fitness cost of mutations were quantitatively compared between growth and different stages of infection in two evolutionary distinct phagocytes, the amoeba and the murine BV2 microglial cells. In the genome, 57% of TA sites were disrupted and 568 genes (10.2%) were essential, which is comparable to previous Tn-Seq studies on and . Major pathways involved in the survival of during infection of are related to DNA damage repair, lipid and vitamin metabolism, the type VII secretion system (T7SS) ESX-1, and the Mce1 lipid transport system. These pathways, except Mce1 and some glycolytic enzymes, were similarly affected in BV2 cells. These differences suggest subtly distinct nutrient availability or requirement in different host cells despite the known predominant use of lipids in both amoeba and microglial cells.IMPORTANCEThe emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system / to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for to identify conservation of virulence pathways between hosts.
Topics: Animals; Mice; Humans; Virulence; Microglia; Mycobacterium marinum; Amoeba; Dictyostelium; Tuberculosis; Mycobacterium tuberculosis; Lipids
PubMed: 38270456
DOI: 10.1128/msystems.01326-23 -
Open Biology Jan 2024RasG is a major regulator of macropinocytosis in . Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase...
RasG is a major regulator of macropinocytosis in . Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC and correlates with IqgC dissociation from endosomes , the physiological significance of the Rab5A-IqgC interaction remains elusive.
Topics: Animals; Dictyostelium; Endosomes; Biological Transport; Mammals
PubMed: 38263885
DOI: 10.1098/rsob.230372 -
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 -
International Journal of Molecular... Dec 2023In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps....
In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps. Our approach was designed for highly complex intracellular architectures in which a long-range cytoskeleton network extends throughout the cell bodies and protrusions. The irregular organization of the actin network, as well as cryo-electron-tomography-specific noise, missing wedge artifacts, and map dimensions call for a specialized implementation that is both robust and efficient. Our proposed solution, , accumulates densities along paths of a specific length in various directions, starting from locally determined seed points. The highest-density paths originating from the seed points form short linear candidate filament segments, which are further scrutinized and classified by users via inspection of a novel , which visualizes the likelihood of being a part of longer filaments. The pruned linear candidate filament segments are then iteratively fused into continuous, longer, and curved filaments based on their relative orientations, gap spacings, and extendibility. When applied to the simulated phantom tomograms of a filopodium under experimental conditions, demonstrated high efficacy, with F1-scores ranging from 0.85 to 0.90, depending on the noise level. Furthermore, when applied to a previously untraced experimental tomogram of mouse fibroblast lamellipodia, the filaments predicted by exhibited a good visual agreement with the experimental map. The framework is highly time efficient and can complete the tracing process in just a few minutes. The source code is publicly available with version 3.2 of the free and open-source software package.
Topics: Mice; Animals; Dictyostelium; Actin Cytoskeleton; Cytoskeleton; Actins; Electron Microscope Tomography
PubMed: 38139012
DOI: 10.3390/ijms242417183 -
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 -
Trends in Microbiology May 2024Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability... (Review)
Review
Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth's history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth's history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.
Topics: Metals; Phagocytosis; Dictyostelium; Biological Evolution; Acanthamoeba; Animals; Phagosomes; Zinc; Metalloids; Copper; Biological Availability; Mitochondria
PubMed: 38103995
DOI: 10.1016/j.tim.2023.11.008 -
FASEB Journal : Official Publication of... Jan 2024Cytokinins (CKs) are a class of growth-promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in...
From biosynthesis and beyond-Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum.
Cytokinins (CKs) are a class of growth-promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA-IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate-IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single- and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA-deficiency results in cytokinesis defects, and both iptA-deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.
Topics: Dictyostelium; Cytokinesis; Cytokinins; RNA, Transfer; Amino Acids
PubMed: 38102957
DOI: 10.1096/fj.202301936RR