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Molecular Biology of the Cell Dec 2023Although the oncogene has been extensively studied, new aspects concerning its role and regulation in normal biology and cancer continue to be discovered. Recently,...
CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells.
Although the oncogene has been extensively studied, new aspects concerning its role and regulation in normal biology and cancer continue to be discovered. Recently, others and we have shown that the mechanistic Target of Rapamycin Complex 2 (mTORC2) is a Ras effector in and mammalian cells. mTORC2 plays evolutionarily conserved roles in cell survival and migration and has been linked to tumorigenesis. Because is often mutated in lung cancer, we investigated whether a Ras-mTORC2 pathway contributes to enhancing the migration of lung cancer cells expressing oncogenic Ras. We used A549 cells and CRISPR/Cas9 to revert the cells' G12S mutation to wild-type and establish A549 revertant (REV) cell lines, which we then used to evaluate the Ras-mediated regulation of mTORC2 and cell migration. Interestingly, our results suggest that K-Ras and mTORC2 promote A549 cell migration but as part of different pathways and independently of Ras's mutational status. Moreover, further characterization of the A549 cells revealed that loss of mutant K-Ras expression for the wild-type protein leads to an increase in cell growth and proliferation, suggesting that the A549 cells have low mutant dependency and that recovering expression of wild-type K-Ras protein increases these cells tumorigenic potential.
Topics: Animals; Humans; Lung Neoplasms; Genes, ras; A549 Cells; Proto-Oncogene Proteins p21(ras); Mechanistic Target of Rapamycin Complex 2; Dictyostelium; Cell Proliferation; Mutation; Cell Line, Tumor; Mammals
PubMed: 37729017
DOI: 10.1091/mbc.E23-05-0152 -
Scientific Reports May 2024The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which...
The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell.
Topics: Microtubules; Dictyostelium; Mitosis; Microfilament Proteins; Actin-Related Protein 2-3 Complex; Protozoan Proteins; Protein Transport; Cytokinesis; Actins
PubMed: 38755233
DOI: 10.1038/s41598-024-61967-7 -
BioRxiv : the Preprint Server For... Jul 2023In facultative symbioses, only a fraction of hosts are associated with a symbiont. Understanding why specific host and symbiont strains are associated can inform us of...
In facultative symbioses, only a fraction of hosts are associated with a symbiont. Understanding why specific host and symbiont strains are associated can inform us of the evolutionary forces affecting facultative symbioses. Possibilities include ongoing host-symbiont coevolution driven by reciprocal selection, or priority effects that are neutral in respect to the host-symbiont interaction. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness estimates for naturally co-occurring (native) host and symbiont combinations compared to nonnative combinations. We used the - system to test this hypothesis. features a reduced genome size relative to another symbiont of , indicating a significant history of coevolution with its host. Facultative symbionts may experience continued genome reduction if coevolution is ongoing, or their genome size may have reached a stable state if the symbiosis has also stabilized. Our work demonstrates that ongoing coevolution is unlikely for and The system instead represents a stable facultative symbiosis. Specifically associated host and symbiont strains in this system are the result of priority effects, and presently unassociated hosts are simply uncolonized. We find evidence for a virulence-transmission trade-off without host strain specificity, and identify candidate virulence factors in the genomes of strains that may contribute to variation in benevolence.
PubMed: 36824889
DOI: 10.1101/2023.02.16.528903 -
Royal Society Open Science Aug 2023Some endosymbionts living within a host must modulate their hosts' immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a...
Some endosymbionts living within a host must modulate their hosts' immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a facultatively multicellular social amoeba host. Aggregates of the amoeba contain a subpopulation of sentinel cells that function akin to the immune systems of more conventional multicellular organisms. Sentinel cells sequester and discard toxins from aggregates and may play a central role in defence against pathogens. We measured the number and functionality of sentinel cells in aggregates of infected by bacterial endosymbionts in the genus . Infected produced fewer and less functional sentinel cells, suggesting that may interfere with its host's immune system. Despite impaired sentinel cells, however, infected were less sensitive to ethidium bromide toxicity, suggesting that may also have a protective effect on its host. By contrast, infected by did not show differences in their sensitivity to two non-symbiotic pathogens. Our results expand previous work on yet another aspect of the complicated relationship between and , which has considerable potential as a model for the study of symbiosis.
PubMed: 37593719
DOI: 10.1098/rsos.230727 -
Nature Communications May 2024Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards...
Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards have been described in various organisms but direct evidence for all the properties in one system is scarce. The tgrB1-tgrC1 allorecognition system of Dictyostelium discoideum encodes two polymorphic membrane proteins which protect cells from chimerism-associated perils. During development, TgrC1 functions as a ligand-signal and TgrB1 as its receptor, but evidence for altruism has been indirect. Here, we show that mixing wild-type and activated tgrB1 cells increases wild-type spore production and relegates the mutants to the altruistic stalk, whereas mixing wild-type and tgrB1-null cells increases mutant spore production and wild-type stalk production. The tgrB1-null cells cheat only on partners that carry the same tgrC1-allotype. Therefore, TgrB1 activation confers altruism whereas TgrB1 inactivation causes allotype-specific cheating, supporting the greenbeard concept and providing insight into the relationship between allorecognition, altruism, and exploitation.
Topics: Dictyostelium; Protozoan Proteins; Spores, Protozoan; Signal Transduction; Mutation; Altruism; Membrane Proteins; Chemotaxis
PubMed: 38734736
DOI: 10.1038/s41467-024-48380-4 -
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 -
PeerJ 2024The evolution of symbiotic interactions may be affected by unpredictable conditions. However, a link between prevalence of these conditions and symbiosis has not been...
The evolution of symbiotic interactions may be affected by unpredictable conditions. However, a link between prevalence of these conditions and symbiosis has not been widely demonstrated. We test for these associations using social amoebae and their bacterial endosymbionts. commonly hosts endosymbiotic bacteria from three taxa: and Chlamydiae. Three species of facultative endosymbionts are the best studied and give hosts the ability to carry prey bacteria through the dispersal stage to new environments. and Chlamydiae are obligate endosymbiont lineages with no measurable impact on host fitness. We tested whether the frequency of both single infections and coinfections of these symbionts were associated with the unpredictability of their soil environments by using symbiont presence-absence data from isolates from 21 locations across the eastern United States. We found that symbiosis across all infection types, symbiosis with and Chlamydiae obligate endosymbionts, and symbiosis involving coinfections were not associated with any of our measures. However, unpredictable precipitation was associated with symbiosis in two species of , suggesting a link between unpredictable conditions and symbiosis.
Topics: Symbiosis; Soil Microbiology; Dictyostelium; Burkholderiaceae; Soil; United States; Chlamydia
PubMed: 38784393
DOI: 10.7717/peerj.17445 -
The ISME Journal Dec 2023The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from...
The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from transiently infected wild D. discoideum for ability to escape predation and infect D. discoideum fruiting bodies. Three predation-resistant strains frequently caused extracellular infections of fruiting bodies but were not found within spores. Furthermore, infection by one of these species induces secondary infections and suppresses predation of otherwise edible bacteria. Another strain can persist inside of amoebae after being phagocytosed but is rarely taken up. We sequenced isolate genomes and discovered that predation-resistant isolates are not monophyletic. Many Pseudomonas isolates encode secretion systems and toxins known to improve resistance to phagocytosis in other species, as well as diverse secondary metabolite biosynthetic gene clusters that may contribute to predation resistance. However, the distribution of these genes alone cannot explain why some strains are edible and others are not. Each lineage may employ a unique mechanism for resistance.
Topics: Animals; Predatory Behavior; Dictyostelium; Pseudomonas; Amoeba; Bacteria
PubMed: 37884792
DOI: 10.1038/s41396-023-01535-5 -
Biophysical Journal May 2024Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on...
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on cell physiology and diseases. It is responsible for the protruding edge formation, cell polarization, macropinocytosis, and other membrane remodeling dynamics in cells. It has been shown that the membrane confinement and curvature affects the wave formation of PIP3 and F-actin. But, even in the absence of F-actin, a complex self-organization of the spatiotemporal PIP3 waves is observed. In recent findings, we have shown that these waves can be guided and pinned on strongly bended Dictyostelium membranes caused by molecular crowding and curvature-limited diffusion. Based on these experimental findings, we investigate the spatiotemporal PIP3 wave dynamics on realistic three-dimensional cell-like membranes to explore the effect of curvature-limited diffusion, as observed experimentally. We use an established stochastic reaction-diffusion model with enzymatic Michaelis-Menten-type reactions that mimics the dynamics of Dictyostelium cells. As these cells mimic the three-dimensional shape and size observed experimentally, we found that the PIP3 wave directionality can be explained by a Hopf-like and a reverse periodic-doubling bifurcation for uniform diffusion and curvature-limited diffusion properties. Finally, we compare the results with recent experimental findings and discuss the discrepancy between the biological and numerical results.
Topics: Cell Membrane; Dictyostelium; Phosphatidylinositol Phosphates; Models, Biological; Diffusion
PubMed: 38515298
DOI: 10.1016/j.bpj.2024.03.022 -
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