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Journal of Immunology (Baltimore, Md. :... Jul 2022A considerable amount is known about how eukaryotic cells move toward an attractant, and the mechanisms are conserved from to human neutrophils. Relatively little is...
A considerable amount is known about how eukaryotic cells move toward an attractant, and the mechanisms are conserved from to human neutrophils. Relatively little is known about chemorepulsion, where cells move away from a repellent signal. We previously identified pathways mediating chemorepulsion in , and here we show that these pathways, including Ras, Rac, protein kinase C, PTEN, and ERK1 and 2, are required for human neutrophil chemorepulsion, and, as with chemorepulsion, PI3K and phospholipase C are not necessary, suggesting that eukaryotic chemorepulsion mechanisms are conserved. Surprisingly, there were differences between male and female neutrophils. Inhibition of Rho-associated kinases or Cdc42 caused male neutrophils to be more repelled by a chemorepellent and female neutrophils to be attracted to the chemorepellent. In the presence of a chemorepellent, compared with male neutrophils, female neutrophils showed a reduced percentage of repelled neutrophils, greater persistence of movement, more adhesion, less accumulation of PI(3,4,5)P, and less polymerization of actin. Five proteins associated with chemorepulsion pathways are differentially abundant, with three of the five showing sex dimorphism in protein localization in unstimulated male and female neutrophils. Together, this indicates a fundamental difference in a motility mechanism in the innate immune system in men and women.
Topics: Actins; Chemotaxis; Dictyostelium; Female; Humans; Male; Neutrophils; Sex Characteristics
PubMed: 35793910
DOI: 10.4049/jimmunol.2101103 -
Scientific Reports Dec 2021It has been experimentally reported that chemotactic cells exhibit cellular memory, that is, a tendency to maintain the migration direction despite changes in the...
It has been experimentally reported that chemotactic cells exhibit cellular memory, that is, a tendency to maintain the migration direction despite changes in the chemoattractant gradient. In this study, we analyzed a phenomenological model assuming the presence of cellular inertia, as well as a response time in motility, resulting in the reproduction of the cellular memory observed in the previous experiments. According to the analysis, the cellular motion is described by the superposition of multiple oscillative functions induced by the multiplication of the oscillative polarity and motility. The cellular intertia generates cellular memory by regulating phase differences between those oscillative functions. By applying the theory to the experimental data, the cellular inertia was estimated at [Formula: see text] min. In addition, physiological parameters, such as response time in motility and intracellular processing speed, were also evaluated. The agreement between the experiemental data and theory suggests the possibility of the presence of the response time in motility, which has never been biologically verified and should be explored in the future.
Topics: Algorithms; Animals; Cell Physiological Phenomena; Chemotaxis; Dictyostelium; Humans; Models, Biological
PubMed: 34893617
DOI: 10.1038/s41598-021-02384-y -
Methods in Molecular Biology (Clifton,... 2022The unicellular eukaryotic amoeba, Dictyostelium discoideum, represents a superb model for examining the molecular mechanism of chemotaxis. Under vegetative conditions,...
The unicellular eukaryotic amoeba, Dictyostelium discoideum, represents a superb model for examining the molecular mechanism of chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folic acid. Under starved conditions, they lose their sensitivity to pterins and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including ease of growth, genetic tractability, and the conservation of mammalian signaling pathways. In this chapter, we describe the use of the under-agarose chemotaxis assay to understand the signaling pathways controlling directional sensing and motility in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect conserved pathways involved in eukaryotic chemotaxis.
Topics: Amoeba; Animals; Chemotaxis; Cyclic AMP; Dictyostelium; Pterins; Sepharose
PubMed: 34542861
DOI: 10.1007/978-1-0716-1661-1_16 -
Structure (London, England : 1993) Aug 2019In this issue of Structure, Jasnin et al. (2019) shows how actin waves on the ventral membrane of Dictyostelium cells propagate by de novo nucleation of oblique...
In this issue of Structure, Jasnin et al. (2019) shows how actin waves on the ventral membrane of Dictyostelium cells propagate by de novo nucleation of oblique filaments that are polarized toward the ventral membrane without a preference to the wave direction.
Topics: Actins; Cytoskeleton; Dictyostelium; Electron Microscope Tomography
PubMed: 31390543
DOI: 10.1016/j.str.2019.07.010 -
MBio Oct 2023Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate...
Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the / system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as .
Topics: Humans; Vacuoles; Dictyostelium; Endoplasmic Reticulum; Mycobacterium marinum; Mycobacterium tuberculosis; Tuberculosis
PubMed: 37676004
DOI: 10.1128/mbio.00943-23 -
Scientific Reports Jul 2022Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a...
Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a unique life cycle and is often used as a crucial research model for a wide range of biological processes and rare metabolites. To address the inadequacies of conventional genetic screening approaches, we developed a highly efficient CRISPR/Cas9-based genome-wide screening system for Dictyostelium. A genome-wide library of 27,405 gRNAs and a kinase library of 4,582 gRNAs were compiled and mutant pools were generated. The resulting mutants were screened for defects in cell growth and more than 10 candidate genes were identified. Six of these were validated and five recreated mutants presented with growth abnormalities. Finally, the genes implicated in developmental defects were screened to identify the unknown genes associated with a phenotype of interest. These findings demonstrate the potential of the CRISPR/Cas9 system as an efficient genome-wide screening method.
Topics: CRISPR-Cas Systems; Dictyostelium; Gene Library; Genome; RNA, Guide, CRISPR-Cas Systems
PubMed: 35780186
DOI: 10.1038/s41598-022-15500-3 -
BioEssays : News and Reviews in... Apr 2020
Topics: Circadian Clocks; Dictyostelium; Gene Expression; Glycolysis; Humans; Neoplasms; Oxidative Phosphorylation; Saccharomyces cerevisiae; Tumor Microenvironment
PubMed: 32189387
DOI: 10.1002/bies.202000048 -
Cells May 2022Directional cell migration and the establishment of polarity play an important role in development, wound healing, and host cell defense. While actin polymerization...
Directional cell migration and the establishment of polarity play an important role in development, wound healing, and host cell defense. While actin polymerization provides the driving force at the cell front, the microtubule network assumes a regulatory function, in coordinating front protrusion and rear retraction. By using cells as a model for amoeboid movement in different 2D and 3D environments, the position of the centrosome relative to the nucleus was analyzed using live-cell microscopy. Our results showed that the centrosome was preferentially located rearward of the nucleus under all conditions tested for directed migration, while the nucleus was oriented toward the expanding front. When cells are hindered from straight movement by obstacles, the centrosome is displaced temporarily from its rearward location to the side of the nucleus, but is reoriented within seconds. This relocalization is supported by the presence of intact microtubules and their contact with the cortex. The data suggest that the centrosome is responsible for coordinating microtubules with respect to the nucleus. In summary, we have analyzed the orientation of the centrosome during different modes of migration in an amoeboid model and present evidence that the basic principles of centrosome positioning and movement are conserved between and human leukocytes.
Topics: Cell Movement; Cell Nucleus; Centrosome; Dictyostelium; Humans; Microtubules
PubMed: 35681473
DOI: 10.3390/cells11111776 -
Sub-cellular Biochemistry 2022Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups...
Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.
Topics: Actin Cytoskeleton; Amoeba; Dictyostelium; Phosphatidylinositol 3-Kinases; Pinocytosis
PubMed: 35378702
DOI: 10.1007/978-3-030-94004-1_3 -
Methods in Molecular Biology (Clifton,... 2022Chemotaxis-directional cell movement steered by chemical gradients-involved in many biological processes including embryonic morphogenesis and immune cell function....
Chemotaxis-directional cell movement steered by chemical gradients-involved in many biological processes including embryonic morphogenesis and immune cell function. Eukaryotic cells, in response to external gradients of attractants, use conserved mechanisms to achieve chemotaxis by regulating the actin cytoskeleton at their fronts and myosin II at their rears. Dictyostelium discoideum, an amoeba that is widely used to study chemotaxis, uses chemotaxis to move up gradients of folate to identify and locate its bacterial prey. Similarly, when starved, Dictyostelium cells synthesize and secrete cyclic AMP (cAMP) while simultaneously expressing cAMP receptors. This allows them to chemotax toward their neighbors and aggregate together. The chemotactic behavior of cells can be studied using several techniques. One such, under-agarose chemotaxis, is a robust, easy, and inexpensive assay that allows direct quantification of chemotactic parameters such as speed and directionality. With the use of high-resolution imaging, for example confocal microscopy, detailed examination of the distribution of actin and membrane proteins in migrating wild type and mutant cells can be performed. In this chapter, we describe simple and optimized methods for studying folate and cAMP chemotaxis in Dictyostelium cells under agarose.
Topics: Cell Migration Assays; Chemotaxis; Cyclic AMP; Dictyostelium; Sepharose
PubMed: 35147958
DOI: 10.1007/978-1-0716-2035-9_27