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1.
Methods Mol Biol ; 1407: 123-30, 2016.
Article in English | MEDLINE | ID: mdl-27271898

ABSTRACT

Increasing concern regarding the use of animals in research has triggered a growing need for non-animal research models in a range of fields. The development of 3Rs (replacement, refinement, and reduction) approaches in research, to reduce the reliance on the use of animal tissue and whole-animal experiments, has recently included the use of Dictyostelium. In addition to not feeling pain and thus being relatively free of ethical constraints, Dictyostelium provides a range of distinct methodological advantages for researchers that has led to a number of breakthroughs. These methodologies include using cell behavior (cell movement and shape) as a rapid indicator of sensitivity to poorly characterized medicines, natural products, and other chemicals to help understand the molecular mechanism of action of compounds. Here, we outline a general approach to employing Dictyostelium as a 3Rs research model, using cell behavior as a readout to better understand how compounds, such as the active ingredient in chilli peppers, capsaicin, function at a cellular level. This chapter helps scientists unfamiliar with Dictyostelium to rapidly employ it as an advantageous model system for research, to reduce the use of animals in research, and to make paradigm shift advances in our understanding of biological chemistry.


Subject(s)
Dictyostelium/drug effects , Dictyostelium/genetics , Pharmacogenetics , Research , Animal Testing Alternatives , Capsaicin/pharmacology , Cell Movement/drug effects , Cells, Cultured , Dictyostelium/metabolism , Drug Resistance , Microscopy , Molecular Imaging , Mutation , Pharmacogenetics/methods , Signal Transduction , Time-Lapse Imaging
2.
Proc Natl Acad Sci U S A ; 111(32): 11703-8, 2014 Aug 12.
Article in English | MEDLINE | ID: mdl-25074921

ABSTRACT

Two motors can drive extension of the leading edge of motile cells: actin polymerization and myosin-driven contraction of the cortex, producing fluid pressure and the formation of blebs. Dictyostelium cells can move with both blebs and actin-driven pseudopods at the same time, and blebs, like pseudopods, can be orientated by chemotactic gradients. Here we ask how bleb sites are selected and how the two forms of projection cooperate. We show that membrane curvature is an important, yet overlooked, factor. Dictyostelium cells were observed moving under agarose, which efficiently induces blebbing, and the dynamics of membrane deformations were analyzed. Blebs preferentially originate from negatively curved regions, generated on the flanks of either extending pseudopods or blebs themselves. This is true of cells at different developmental stages, chemotaxing to either folate or cyclic AMP and moving with both blebs and pseudopods or with blebs only. A physical model of blebbing suggests that detachment of the cell membrane is facilitated in concave areas of the cell, where membrane tension produces an outward directed force, as opposed to pulling inward in convex regions. Our findings assign a role to membrane tension in spatially coupling blebs and pseudopods, thus contributing to clustering protrusions to the cell front.


Subject(s)
Cell Surface Extensions/physiology , Chemotaxis/physiology , Pseudopodia/physiology , Actins/metabolism , Animals , Biophysical Phenomena , Cell Polarity/physiology , Cyclic AMP/metabolism , Dictyostelium/physiology , Folic Acid/metabolism , Fundulidae , Models, Biological , Protozoan Proteins/metabolism
3.
J Cell Biol ; 204(6): 1027-44, 2014 Mar 17.
Article in English | MEDLINE | ID: mdl-24616222

ABSTRACT

Blebs and F-actin-driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.


Subject(s)
Dictyostelium/physiology , Pseudopodia/physiology , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Chemotaxis , Cyclic AMP/physiology , Dictyostelium/ultrastructure , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Multimerization , Protozoan Proteins/metabolism , Pseudopodia/ultrastructure , Second Messenger Systems
4.
J Cell Sci ; 126(Pt 23): 5465-76, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24006265

ABSTRACT

Detection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylthiourea. This approach identified a G-protein-coupled receptor mutant, grlJ(-), which showed a significantly increased tolerance to phenylthiourea in growth, survival and movement. This mutant was not resistant to a structurally dissimilar potent bitter tastant, denatonium benzoate, suggesting it is not a target for at least one other bitter tastant. Analysis of the cell-signalling pathway involved in the detection of phenylthiourea showed dependence upon heterotrimeric G protein and phosphatidylinositol 3-kinase activity, suggesting that this signalling pathway is responsible for the cellular effects of phenylthiourea. This is further supported by a phenylthiourea-dependent block in the transient cAMP-induced production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in wild-type but not grlJ(-) cells. Finally, we have identified an uncharacterized human protein γ-aminobutyric acid (GABA) type B receptor subunit 1 isoform with weak homology to GrlJ that restored grlJ(-) sensitivity to phenylthiourea in cell movement and PIP3 regulation. Our results thus identify a novel pathway for the detection of the standard bitter tastant phenylthiourea in Dictyostelium and implicate a poorly characterized human protein in phenylthiourea-dependent cell responses.


Subject(s)
Dictyostelium/physiology , Phenylthiourea/chemistry , Phosphatidylinositol 3-Kinase/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, GABA-B/genetics , Taste/physiology , Cell Movement , Cell Survival , Cyclic AMP/metabolism , Gene Deletion , Gene Expression Regulation , Genetic Complementation Test , Humans , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol Phosphates/metabolism , Quaternary Ammonium Compounds/chemistry , Receptors, G-Protein-Coupled/metabolism , Receptors, GABA-B/metabolism , Signal Transduction , Taste Buds/metabolism
5.
Dev Biol ; 382(2): 496-503, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23876427

ABSTRACT

Convergent extension (CE) is a conserved morphogenetic movement that drives axial lengthening of the primary body axis and depends on the planar cell polarity (PCP) pathway. In Drosophila epithelia, a polarised subcellular accumulation of PCP core components, such as Dishevelled (Dvl) protein, is associated with PCP function. Dvl has long been thought to accumulate in the mediolateral protrusions in Xenopus chordamesoderm cells undergoing CE. Here we present a quantitative analysis of Dvl intracellular localisation in Xenopus chordamesoderm cells. We find that, surprisingly, accumulations previously observed at mediolateral protrusions of chordamesodermal cells are not protrusion-specific but reflect yolk-free cytoplasm and are quantitatively matched by the distribution of the cytoplasm-filling lineage marker dextran. However, separating cell cortex-associated from bulk Dvl signal reveals a statistical enrichment of Dvl in notochord-somite boundary-(NSB)-directed protrusions, which is dependent upon NSB proximity. Dvl puncta were also observed, but only upon elevated overexpression. These puncta showed no statistically significant spatial bias, in contrast to the strongly posteriorly-enriched GFP-Dvl puncta previously reported in zebrafish. We propose that Dvl distribution is more subtle and dynamic than previously appreciated and that in vertebrate mesoderm it reflects processes other than protrusion as such.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Mesoderm/metabolism , Phosphoproteins/metabolism , Xenopus Proteins/metabolism , Xenopus/metabolism , Animals , Cell Polarity , Dishevelled Proteins , Drosophila Proteins , Embryo, Nonmammalian/metabolism , Xenopus/embryology
6.
Yeast ; 30(4): 145-56, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23447405

ABSTRACT

Ras signalling is central to fundamental and diverse cellular processes. In higher eukaryotes ras signalling is highly complex, involving multiple isoforms, regulatory proteins and effectors. As a consequence, the study of ras activity in mammalian systems presents a number of technical challenges. The model organism Schizosaccharomyces pombe has previously proved a key system for the study of human signalling components and provides an ideal model for the study of ras, as it contains just one ras protein (Ras1p), which is non-essential and controls a number of downstream processes. Here we present data demonstrating the quantitative analysis of three distinct Ras1-related signalling outputs, utilizing the three most abundant human ras isoforms, H-Ras, N-Ras and K-Ras4B, in Sz. pombe. Further, we have characterized the localization of these three human ras isoforms in Sz. pombe, utilizing quantitative image analysis techniques. These data indicate that all three human ras isoforms are functional in fission yeast, displaying differing localization patterns which correlate strongly with function in the regulation of pheromone response and cell shape. These data demonstrate that such yeast strains could provide powerful tools for the investigation of ras biology, and potentially in the development of cancer therapies.


Subject(s)
Schizosaccharomyces/genetics , ras Proteins/metabolism , Gene Expression , Humans , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , Schizosaccharomyces/metabolism , ras Proteins/genetics
7.
Crit Care Med ; 36(12): 3171-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19020432

ABSTRACT

OBJECTIVES: This study investigates the possibility of a relationship between oversedation and mortality in mechanically ventilated patients. The presence of burst suppression, a pattern of severely decreased brain wave activity on the electroencephalogram, may be unintentionally induced by heavy doses of sedatives. Burst suppression has never been studied as a potential risk factor for death in patients without a known neurologic disorder or injury. DESIGN: Post hoc analysis of a prospectively observational cohort study. SETTING: Medical intensive care units of a tertiary care, university-based medical center. PATIENTS: A total of 125 mechanically ventilated, adult, critically ill patients. MEASUREMENTS AND MAIN RESULTS: A validated arousal scale (Richmond Agitation-Sedation Scale) was used to measure sedation level, and the bispectral index monitor was used to capture electroencephalogram data. Burst suppression occurred in 49 of 125 patients (39%). For analysis, the patients were divided into those with burst suppression (49 of 125, 39%) and those without burst suppression (76 of 125, 61%). All baseline variables were similar between the two groups, with the overall cohort demonstrating a high severity of illness (Acute Physiology and Chronic Health Evaluation II scores of 27.4 +/- 8.2) and 98% receiving sedation. Of those with burst suppression, 29 of 49 (59%) died within 6 months compared with 25 of 76 (33%) who did not demonstrate burst suppression. Using time-dependent Cox regression to adjust for clinically important covariates (age, Charlson comorbidity score, baseline dementia, Acute Physiology and Chronic Health Evaluation II, Sequential Organ Failure Assessment, coma, and delirium), patients who experienced burst suppression were found to have a statistically significant higher 6-month mortality [Hazard's ratio = 2.04, 95% confidence interval, 1.12-3.70, p = 0.02]. CONCLUSION: The presence of burst suppression, which was unexpectedly high in this medical intensive care unit population, was an independent predictor of increased risk of death at 6 months. This association should be studied prospectively on a larger scale in mechanically ventilated, critically ill patients.


Subject(s)
Critical Illness/mortality , Deep Sedation/adverse effects , Electroencephalography , Respiration, Artificial , Female , Humans , Length of Stay , Male , Middle Aged , Prospective Studies
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