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BioRxiv : the Preprint Server For... Sep 2023Studies in the model systems, amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation...
Studies in the model systems, amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation on the membrane is spatiotemporally regulated by its activators, RasGEFs, and inhibitors, RasGAPs, which might be expected to create a stable 'front' and 'back', respectively, in migrating cells. Focusing on C2GAPB in amoebae and RASAL3 in neutrophils, we investigated how Ras activity along the cortex controls polarity. Since existing gene knockout and overexpression studies can be circumvented, we chose optogenetic approaches to assess the immediate, local effects of these Ras regulators on the cell cortex. In both cellular systems, optically targeting the respective RasGAPs to the cell front extinguished existing protrusions and changed the direction of migration, as might be expected. However, when the expression of C2GAPB was induced globally, amoebae polarized within hours. Furthermore, within minutes of globally recruiting either C2GAPB in amoebae or RASAL3 in neutrophils, each cell type polarized and moved more rapidly. Targeting the RasGAPs to the cell backs exaggerated these effects on migration and polarity. Overall, in both cell types, RasGAP-mediated polarization was brought about by increased actomyosin contractility at the back and sustained, localized F-actin polymerization at the front. These experimental results were accurately captured by computational simulations in which Ras levels control front and back feedback loops. The discovery that context-dependent Ras activity on the cell cortex has counterintuitive, unanticipated effects on cell polarity can have important implications for future drug-design strategies targeting oncogenic Ras.
PubMed: 37693515
DOI: 10.1101/2023.08.30.555648 -
BMC Ecology and Evolution Oct 2023Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid...
BACKGROUND
Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4.
RESULTS
Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells.
CONCLUSIONS
The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.
Topics: Dictyostelium; Ammonia; Phosphorus-Oxygen Lyases; Dictyosteliida; Oxygen
PubMed: 37803310
DOI: 10.1186/s12862-023-02169-z -
The Journal of Biological Chemistry Dec 2023Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires'...
Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (P or P), but not under control of its endogenous promoter (P), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.
Topics: Humans; Bacterial Proteins; Dictyostelium; Escherichia coli; Legionella; Legionella pneumophila; Legionnaires' Disease; Quorum Sensing
PubMed: 37866633
DOI: 10.1016/j.jbc.2023.105376 -
ELife Jul 2023Actin dynamics in cell motility, division, and phagocytosis is regulated by complex factors with multiple feedback loops, often leading to emergent dynamic patterns in...
Actin dynamics in cell motility, division, and phagocytosis is regulated by complex factors with multiple feedback loops, often leading to emergent dynamic patterns in the form of propagating waves of actin polymerization activity that are poorly understood. Many in the actin wave community have attempted to discern the underlying mechanisms using experiments and/or mathematical models and theory. Here, we survey methods and hypotheses for actin waves based on signaling networks, mechano-chemical effects, and transport characteristics, with examples drawn from , human neutrophils, , and oocytes. While experimentalists focus on the details of molecular components, theorists pose a central question of universality: Are there generic, model-independent, underlying principles, or just boundless cell-specific details? We argue that mathematical methods are equally important for understanding the emergence, evolution, and persistence of actin waves and conclude with a few challenges for future studies.
Topics: Humans; Actins; Dictyostelium; Cell Movement; Signal Transduction; Phagocytosis; Models, Biological; Actin Cytoskeleton
PubMed: 37428017
DOI: 10.7554/eLife.87181 -
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 -
Current Biology : CB Aug 2023Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water...
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Topics: Cytosol; Dictyostelium; Osmolar Concentration; Water-Electrolyte Balance; Vacuoles; Eukaryota; Water
PubMed: 37478864
DOI: 10.1016/j.cub.2023.06.061 -
BioRxiv : the Preprint Server For... Feb 2024The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN)...
The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P ] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ]. cells require both PTEN and the PTEN-like phosphatase CnrN to locally inhibit Ras activation to induce biased movement of cells away from the secreted chemorepellent protein AprA. Both PTEN and CnrN decrease basal levels of PI(3,4,5)P and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P levels, decrease PI(3,4,5)P levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. AprA causes PTEN, similar to CnrN, to localize to the side of the cell towards AprA in an AprA gradient. However, PTEN and CnrN also have distinct roles in some signaling pathways. PTEN, but not CnrN, decreases basal levels of PI(4,5)P , AprA requires PTEN, but not CnrN, to induce cell roundness, and CnrN and PTEN have different effects on the number of filopods and pseudopods, and the sizes of filopods. Together, our results suggest that CnrN and PTEN play unique roles in signaling pathways, and possibly dephosphorylate PI(3,4,5)P in different membrane domains, to mediate chemorepulsion away from AprA.
PubMed: 38464111
DOI: 10.1101/2024.02.23.581751 -
Frontiers in Cell and Developmental... 2024Eukaryotic cells exhibit random migration in the absence of extracellular directional cues. This random migration acts as basal motility for various migratory responses... (Review)
Review
Eukaryotic cells exhibit random migration in the absence of extracellular directional cues. This random migration acts as basal motility for various migratory responses such as chemotaxis. The self-organization of random motility requires the internal signals that determine the anterior side of the migrating cell be generated spontaneously from the intrinsic activities of intracellular signaling networks. Recent studies have identified an excitable system as the mechanism of the spontaneous signal generation. Here, we discuss how the excitable system of Ras, a small G protein, regulates signaling networks in as a model organism. The excitability produces a domain where an active form of Ras is enriched on the cell membrane without extracellular directional cues, such that Ras serves as the anterior signal. The typical spatiotemporal characteristics are mathematically explained by reaction-diffusion models. These models further enable a quantitative analysis of the dynamics that depends on the internal cellular states and surrounding environments. Downstream of the Ras excitable system, a phosphoinositide metabolic network composed of PI3K, PTEN, PI(3,4,5)P and PI(4,5)P exhibits bistability to discretize the anterior and posterior regions of the cell membrane. Upstream, a local excitation and global inhibition local excitation global inhibition network, which works for gradient sensing in the presence of chemoattractant gradients, spatiotemporally biases the excitability of Ras for chemotaxis. In parallel with the Ras excitable system, the cGMP signaling pathway constitutes another excitable system of its own periodicity to ensure flexible migratory dynamics. In addition to these intracellular signaling networks, an intercellular signaling network activated by secreted cAMP is coupled with the Ras excitable system for collective cell migration. Finally, we discuss how the excitable system of Ras operates as a platform of information integration by receiving multiple intrinsic and extrinsic signals to ensure spontaneous cellular activity and robust responses in eukaryotic cell migration under natural complex environments.
PubMed: 38481533
DOI: 10.3389/fcell.2024.1373609 -
BioRxiv : the Preprint Server For... Feb 2024To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit...
To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit chemical cues to the cytoskeleton, but the cytoskeleton must also relay mechanical information back to those signaling systems. Using novel synthetic tools to acutely control specific elements of the cytoskeleton in and neutrophils, we delineate feedback mechanisms that alter the signaling network and promote front- or back-states of the cell membrane and cortex. First, increasing branched actin assembly increases Ras/PI3K activation while reducing polymeric actin levels overall decreases activation. Second, reducing myosin II assembly immediately increases Ras/PI3K activation and sensitivity to chemotactic stimuli. Third, inhibiting branched actin alone increases cortical actin assembly and strongly blocks Ras/PI3K activation. This effect is mitigated by reducing filamentous actin levels and in cells lacking myosin II. Finally, increasing actin crosslinking with a controllable activator of cytoskeletal regulator RacE leads to a large decrease in Ras activation both globally and locally. Curiously, RacE activation can trigger cell spreading and protrusion with no detectable activation of branched actin nucleators. Taken together with legacy data that Ras/PI3K promotes branched actin assembly and myosin II disassembly, our results define front- and back-promoting positive feedback loops. We propose that these loops play a crucial role in establishing cell polarity and mediating signal integration by controlling the excitable state of the signal transduction networks in respective regions of the membrane and cortex. This interplay enables cells to navigate intricate topologies like tissues containing other cells, the extracellular matrix, and fluids.
PubMed: 38405988
DOI: 10.1101/2024.02.13.580131 -
Cells Feb 2024The cell membrane is frequently subjected to damage, either through physical or chemical means. The swift restoration of the cell membrane's integrity is crucial to... (Review)
Review
The cell membrane is frequently subjected to damage, either through physical or chemical means. The swift restoration of the cell membrane's integrity is crucial to prevent the leakage of intracellular materials and the uncontrolled influx of extracellular ions. Consequently, wound repair plays a vital role in cell survival, akin to the importance of DNA repair. The mechanisms involved in wound repair encompass a series of events, including ion influx, membrane patch formation, endocytosis, exocytosis, recruitment of the actin cytoskeleton, and the elimination of damaged membrane sections. Despite the absence of a universally accepted general model, diverse molecular models have been proposed for wound repair in different organisms. Traditional wound methods not only damage the cell membrane but also impact intracellular structures, including the underlying cortical actin networks, microtubules, and organelles. In contrast, the more recent improved laserporation selectively targets the cell membrane. Studies on cells utilizing this method have introduced a novel perspective on the wound repair mechanism. This review commences by detailing methods for inducing wounds and subsequently reviews recent developments in the field.
Topics: Dictyostelium; Cell Membrane; Actins; Microtubules; Actin Cytoskeleton
PubMed: 38391954
DOI: 10.3390/cells13040341