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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 -
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 -
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 -
Molecules (Basel, Switzerland) Dec 2023Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro....
Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10-200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40-400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200-1000 μM significantly promoted glucose uptake (by 20-25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3-100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1-10 μM promoted glucose uptake by 20-30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.
Topics: Mice; Animals; Dictyostelium; 3T3-L1 Cells; Biological Transport; Phosphodiesterase Inhibitors; Glucose; Mammals
PubMed: 38067655
DOI: 10.3390/molecules28237926 -
Molecular Biology and Evolution Dec 2023The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine...
Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?
The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.
Topics: DCMP Deaminase; Gene Transfer, Horizontal; Escherichia coli; Amoeba; Thymidylate Synthase; Dictyostelium; Deoxycytidine Monophosphate
PubMed: 38064674
DOI: 10.1093/molbev/msad268 -
Frontiers in Cell and Developmental... 2023Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems and neutrophils over the...
Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems and neutrophils over the years have uncovered generality in their overall cell movement pattern. Under no directional cues, the centroid movement can be quantitatively characterized by their persistence to move in a straight line and the frequency of re-orientation. Mathematically, the cells essentially behave as a persistent random walker with memory of two characteristic time-scale. Such quantitative characterization is important from a cellular-level ethology point of view as it has direct connotation to their exploratory and foraging strategies. Interestingly, outside the amoebozoa and metazoa, there are largely uncharacterized species in the excavate taxon Heterolobosea including amoeboflagellate . While classical works have shown that these cells indeed show typical amoeboid locomotion on an attached surface, their quantitative features are so far unexplored. Here, we analyzed the cell movement of by employing long-time phase contrast imaging that automatically tracks individual cells. We show that the cells move as a persistent random walker with two time-scales that are close to those known in and neutrophils. Similarities were also found in the shape dynamics which are characterized by the appearance, splitting and annihilation of the curvature waves along the cell edge. Our analysis based on the Fourier descriptor and a neural network classifier point to importance of morphology features unique to including complex protrusions and the transient bipolar dumbbell morphologies.
PubMed: 38020930
DOI: 10.3389/fcell.2023.1274127 -
The EMBO Journal Dec 2023Motile cells encounter microenvironments with locally heterogeneous mechanochemical composition. Individual compositional parameters, such as chemokines and...
Motile cells encounter microenvironments with locally heterogeneous mechanochemical composition. Individual compositional parameters, such as chemokines and extracellular matrix pore sizes, are well known to provide guidance cues for pathfinding. However, motile cells face diverse cues at the same time, raising the question of how they respond to multiple and potentially competing signals on their paths. Here, we reveal that amoeboid cells require nuclear repositioning, termed nucleokinesis, for adaptive pathfinding in heterogeneous mechanochemical micro-environments. Using mammalian immune cells and the amoeba Dictyostelium discoideum, we discover that frequent, rapid and long-distance nucleokinesis is a basic component of amoeboid pathfinding, enabling cells to reorientate quickly between locally competing cues. Amoeboid nucleokinesis comprises a two-step polarity switch and is driven by myosin-II forces that readjust the nuclear to the cellular path. Impaired nucleokinesis distorts path adaptions and causes cellular arrest in the microenvironment. Our findings establish that nucleokinesis is required for amoeboid cell navigation. Given that many immune cells, amoebae, and some cancer cells utilize an amoeboid migration strategy, these results suggest that nucleokinesis underlies cellular navigation during unicellular biology, immunity, and disease.
Topics: Animals; Cell Movement; Dictyostelium; Amoeba; Extracellular Matrix; Mammals
PubMed: 37987147
DOI: 10.15252/embj.2023114557 -
BioRxiv : the Preprint Server For... Oct 2023The actin cortex is very dynamic during migration of eukaryotes. In cells that use blebs as leading-edge protrusions, the cortex reforms beneath the cell membrane (bleb...
The actin cortex is very dynamic during migration of eukaryotes. In cells that use blebs as leading-edge protrusions, the cortex reforms beneath the cell membrane (bleb cortex) and completely disassembles at the site of bleb initiation. Remnants of the actin cortex at the site of bleb nucleation are referred to as the actin scar. We refer to the combined process of cortex reformation along with the degradation of the actin scar during bleb-based cell migration as . The molecular factors that regulate the dynamic reorganization of the cortex are not fully understood. Myosin motor protein activity has been shown to be necessary for blebbing, with its major role associated with pressure generation to drive bleb expansion. Here, we examine the role of myosin in regulating cortex dynamics during bleb stabilization. Analysis of microscopy data from protein localization experiments in cells reveals a rapid formation of the bleb's cortex with a delay in myosin accumulation. In the degrading actin scar, myosin is observed to accumulate before active degradation of the cortex begins. Through a combination of mathematical modeling and data fitting, we identify that myosin helps regulate the equilibrium concentration of actin in the bleb cortex during its reformation by increasing its dissasembly rate. Our modeling and analysis also suggests that cortex degradation is driven primarily by an exponential decrease in actin assembly rate rather than increased myosin activity. We attribute the decrease in actin assembly to the separation of the cell membrane from the cortex after bleb nucleation.
PubMed: 37961169
DOI: 10.1101/2023.10.26.564082 -
Nature Methods Dec 2023Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulation of cells and organelles. However, stochastic noise inherently restricts...
Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulation of cells and organelles. However, stochastic noise inherently restricts optical interrogation quality and exacerbates observation fidelity when balancing the joint demands of high frame rate, long-term recording and low phototoxicity. Here we propose DeepSeMi, a self-supervised-learning-based denoising framework capable of increasing signal-to-noise ratio by over 12 dB across various conditions. With the introduction of newly designed eccentric blind-spot convolution filters, DeepSeMi effectively denoises images with no loss of spatiotemporal resolution. In combination with confocal microscopy, DeepSeMi allows for recording organelle interactions in four colors at high frame rates across tens of thousands of frames, monitoring migrasomes and retractosomes over a half day, and imaging ultra-phototoxicity-sensitive Dictyostelium cells over thousands of frames. Through comprehensive validations across various samples and instruments, we prove DeepSeMi to be a versatile and biocompatible tool for breaking the shot-noise limit.
Topics: Dictyostelium; Image Enhancement; Microscopy, Confocal; Signal-To-Noise Ratio; Microscopy, Fluorescence; Image Processing, Computer-Assisted
PubMed: 37957429
DOI: 10.1038/s41592-023-02058-9