scholarly journals Migration and bidirectional phototaxis in Dictyostelium discoideum slugs lacking the actin cross-linking 120 kDa gelation factor.

1997 ◽  
Vol 200 (24) ◽  
pp. 3213-3220 ◽  
Author(s):  
E Wallraff ◽  
H G Wallraff
Keyword(s):  
Dictyostelium Discoideum ◽  
Incident Light ◽  
Actin Binding ◽  
Cross Linking ◽  
Alternative Possibility ◽  
Wild Type ◽  
Oblique Angle ◽  
Mutant Strains ◽  
Cross Linker ◽  
Actin Capping

Three mutant strains of Dictyostelium discoideum, lacking different actin-binding proteins, were tested for behavioural deficits in the multicellular pseudoplasmodium (slug) stage. Two strains, defective in the production of either -actinin (an actin cross-linker) or severin (an actin capping and severing protein), did not show changes in slug behaviour. Slugs of the mutant lacking another actin cross-linker, the 120 kDa gelation factor (ABP-120), however, migrated shorter distances in darkness as well as in horizontally directed light. More remarkably, they migrated at an angle of approximately 45 degrees to the left or right of the incident light, whereas wild-type slugs migrated on fairly straight paths towards the light. We discuss the hypothesis that this bidirectional oblique-angle phototaxis is due to changes in the optical properties of the pseudoplasmodia. Normally, in wild-type slugs, a lens effect causes stronger stimulation on the side distal to the incident light. We propose that in the mutant the lens quality is reduced, so that at small angles between the slug axis and the rays of light the proximal side is stimulated more intensely. As a result, the intended symmetrical stimulation is achieved at a certain angle to the left or right of the incident light. We assume that the absence of ABP-120 alters the shape of the lens and/or enhances internal light scattering via degradation of intercellular coherence; however, intracellular attenuation of light remains an additional or alternative possibility.

scholarly journals Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

2015 ◽  
Vol 26 (6) ◽  
pp. 1084-1097 ◽  
Author(s):  
Wei-Chi Lin ◽  
Liang-Chen Wang ◽  
Te-Ling Pang ◽  
Mei-Yu Chen
Keyword(s):  
Cell Migration ◽  
Dictyostelium Discoideum ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Protein Domain ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Protein G ◽  
Wild Type ◽  
Actin Binding Protein

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.

scholarly journals Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

1996 ◽  
Vol 7 (5) ◽  
pp. 803-823 ◽  
Author(s):  
D Cox ◽  
D Wessels ◽  
D R Soll ◽  
J Hartwig ◽  
J Condeelis
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Cell Lines ◽  
Binding Proteins ◽  
Chemical Mutagenesis ◽  
Actin Binding ◽  
Direct Result ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Binding Proteins

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

scholarly journals The Spectraplakin Short Stop Is an Actin–Microtubule Cross-Linker That Contributes to Organization of the Microtubule Network

2010 ◽  
Vol 21 (10) ◽  
pp. 1714-1724 ◽  
Author(s):  
Derek A. Applewhite ◽  
Kyle D. Grode ◽  
Darby Keller ◽  
Alireza Dehghani Zadeh ◽  
Kevin C. Slep ◽  
...  
Keyword(s):  
Cross Talk ◽  
Function Analysis ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Cross Linking ◽  
Cell Periphery ◽  
Cellular Functions ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Cross Linker

The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin–microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure–function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH2-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk.

scholarly journals Why Are Two Different Cross-linkers Necessary for Actin Bundle Formation In Vivo and What Does Each Cross-link Contribute?

1998 ◽  
Vol 143 (1) ◽  
pp. 121-133 ◽  
Author(s):  
Lewis G. Tilney ◽  
Patricia S. Connelly ◽  
Kelly A. Vranich ◽  
Michael K. Shaw ◽  
Gregory M. Guild
Keyword(s):  
Potassium Iodide ◽  
Actin Filaments ◽  
Cross Linking ◽  
Wild Type ◽  
Cross Link ◽  
Actin Bundle ◽  
Cross Linker ◽  
Three Phases

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus ∼700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.

scholarly journals A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity.

1981 ◽  
Vol 88 (2) ◽  
pp. 396-409 ◽  
Author(s):  
E J Luna ◽  
V M Fowler ◽  
J Swanson ◽  
D Branton ◽  
D L Taylor
Keyword(s):  
Membrane Proteins ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Lipid Vesicles ◽  
Binding Activity ◽  
Actin Binding ◽  
Insoluble Residue ◽  
Cross Linking ◽  
Membrane Cytoskeleton ◽  
Triton X

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre-extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat-denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat-denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X-100 extraction of isolated D. discoideum membranes results in a Triton-insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.

scholarly journals Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum

2019 ◽  
Vol 30 (4) ◽  
pp. 453-466 ◽  
Author(s):  
Petros Batsios ◽  
Hellen C. Ishikawa-Ankerhold ◽  
Heike Roth ◽  
Michael Schleicher ◽  
Catherine C. L. Wong ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Three Dimensional ◽  
Model Organism ◽  
Live Cell ◽  
Actin Binding ◽  
Spectrometric Analysis ◽  
Wild Type ◽  
Gene Encoding ◽  
Adhesion Sites

The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.

scholarly journals Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum.

1984 ◽  
Vol 4 (3) ◽  
pp. 514-519 ◽  
Author(s):  
B A Murray ◽  
S Wheeler ◽  
T Jongens ◽  
W F Loomis
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Post Translational Modification ◽  
Group Vi ◽  
Two Dimensional Gel Electrophoresis ◽  
Mutant Strains ◽  
Type Strains ◽  
Cell Cell

We isolated two independent mutations in Dictyostelium discoideum that result in the absence of the antigenic determinant recognized by monoclonal antibody E28D8. This antibody reacts with a post-translational modification on the surface glycoprotein gp80 and several other proteins. Both of the mutations occur in the same locus, modB, which was mapped to linkage group VI. The modB mutations result in sufficient alteration of gp80 that it is absent or unrecognizable by two-dimensional gel electrophoresis. Strains carrying modB mutations exhibit "contact sites A"-mediated cell-cell adhesion although more weakly than do wild-type strains and develop to fruiting bodies carrying viable spores. Although gp80 has been implicated in the mechanism of cell-cell adhesion in D. discoideum, it is clear from the behavior of these mutant strains that the determinant on gp80 recognized by E28D8 is not necessary for either morphogenesis or reduced EDTA-resistant adhesion.

scholarly journals Role of fascin in filopodial protrusion

2006 ◽  
Vol 174 (6) ◽  
pp. 863-875 ◽  
Author(s):  
Danijela Vignjevic ◽  
Shin-ichiro Kojima ◽  
Yvonne Aratyn ◽  
Oana Danciu ◽  
Tatyana Svitkina ◽  
...  
Keyword(s):  
Rna Interference ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Cellular Localization ◽  
Fluorescence Recovery After Photobleaching ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Organization ◽  
Cross Linker

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

scholarly journals Effects of actin filament cross-linking and filament length on actin-myosin interaction.

1985 ◽  
Vol 101 (5) ◽  
pp. 1850-1857 ◽  
Author(s):  
T R Coleman ◽  
M S Mooseker
Keyword(s):  
Atpase Activity ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Filament Length ◽  
Maximum Enhancement ◽  
Actomyosin Atpase ◽  
Cross Linker

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration-dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2-25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.

Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum

1984 ◽  
Vol 4 (3) ◽  
pp. 514-519
Author(s):  
B A Murray ◽  
S Wheeler ◽  
T Jongens ◽  
W F Loomis
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Post Translational Modification ◽  
Group Vi ◽  
Two Dimensional Gel Electrophoresis ◽  
Mutant Strains ◽  
Type Strains ◽  
Cell Cell

We isolated two independent mutations in Dictyostelium discoideum that result in the absence of the antigenic determinant recognized by monoclonal antibody E28D8. This antibody reacts with a post-translational modification on the surface glycoprotein gp80 and several other proteins. Both of the mutations occur in the same locus, modB, which was mapped to linkage group VI. The modB mutations result in sufficient alteration of gp80 that it is absent or unrecognizable by two-dimensional gel electrophoresis. Strains carrying modB mutations exhibit "contact sites A"-mediated cell-cell adhesion although more weakly than do wild-type strains and develop to fruiting bodies carrying viable spores. Although gp80 has been implicated in the mechanism of cell-cell adhesion in D. discoideum, it is clear from the behavior of these mutant strains that the determinant on gp80 recognized by E28D8 is not necessary for either morphogenesis or reduced EDTA-resistant adhesion.

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