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 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 High-precision 3D inkjet technology for live cell bioprinting

2019 ◽  
Vol 5 (2) ◽  
pp. 27 ◽  
Author(s):  
Daisuke Takagi ◽  
Waka Lin ◽  
Takahiko Matsumoto ◽  
Hidekazu Yaginuma ◽  
Natsuko Hemmi ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Cell Number ◽  
Live Cell ◽  
Drop On Demand ◽  
Long Time ◽  
Spatial Positioning ◽  
Different Cell Types ◽  
Membrane Vibrations

In recent years, bioprinting has emerged as a promising technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. In the present study, we present the development of an inkjet-based bioprinting system to arrange multiple cells and materials precisely into structurally organized constructs. A novel inkjet printhead has been specially designed for live cell ejection. Droplet formation is powered by piezoelectric membrane vibrations coupled with mixing movements to prevent cell sedimentation at the nozzle. Stable drop-on-demand dispensing and cell viability were validated over an adequately long time to allow the fabrication of 3D tissues. Reliable control of cell number and spatial positioning was demonstrated using two separate suspensions with different cell types printed sequentially. Finally, a process for constructing stratified Mille-Feuille-like 3D structures is proposed by alternately superimposing cell suspensions and hydrogel layers with a controlled vertical resolution. The results show that inkjet technology is effective for both two-dimensional patterning and 3D multilayering and has the potential to facilitate the achievement of live cell bioprinting with an unprecedented level of precision.

LSP1 modulates leukocyte populations in resting and inflamed peritoneum

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1827-1835 ◽  
Author(s):  
Jenny Jongstra-Bilen ◽  
Virginia L. Misener ◽  
Chunjie Wang ◽  
Hedy Ginzberg ◽  
Anna Auerbach ◽  
...  
Keyword(s):  
Specific Protein ◽  
Negative Regulator ◽  
Actin Binding ◽  
Wild Type ◽  
Peripheral Tissues ◽  
Lymphocytic Cell ◽  
Peritoneal Mesothelium ◽  
Leukocyte Populations ◽  
Kinetics Of

Abstract Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1−/−) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen.Lsp1−/− mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5−macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased inLsp1−/− mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1−/−mice are similar to those of wt mice andLsp1−/−mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, theLsp1−/−mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1− neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

scholarly journals CbfA, the C-Module DNA-Binding Factor, Plays an Essential Role in the Initiation of Dictyostelium discoideum Development

2004 ◽  
Vol 3 (5) ◽  
pp. 1349-1358 ◽  
Author(s):  
Thomas Winckler ◽  
Negin Iranfar ◽  
Peter Beck ◽  
Ingo Jennes ◽  
Oliver Siol ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Density ◽  
Dictyostelium Discoideum ◽  
Ectopic Expression ◽  
Development Program ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Wild Type ◽  
Gene Encoding ◽  
Multicellular Development

ABSTRACT We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.

scholarly journals Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

2000 ◽  
Vol 191 (5) ◽  
pp. 859-870 ◽  
Author(s):  
Melanie Allen ◽  
Linne Svensson ◽  
Marsha Roach ◽  
John Hambor ◽  
John McNeish ◽  
...  
Keyword(s):  
Map Kinase ◽  
Es Cells ◽  
Interleukin 1 ◽  
Embryonic Stem ◽  
Wild Type ◽  
Antiinflammatory Drugs ◽  
Gene Encoding ◽  
Significant Difference ◽  
Induced Apoptosis

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.

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 Disruption of the ech42 (Endochitinase-Encoding) Gene Affects Biocontrol Activity in Trichoderma harzianum P1

1999 ◽  
Vol 12 (5) ◽  
pp. 419-429 ◽  
Author(s):  
S. L. Woo ◽  
B. Donzelli ◽  
F. Scala ◽  
R. Mach ◽  
G. E. Harman ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Trichoderma Harzianum ◽  
Pythium Ultimum ◽  
Single Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
Culture Filtrates ◽  
In Vitro Antifungal Activity ◽  
Better Than

The biocontrol strain P1 of Trichoderma harzianum was genetically modified by targeted disruption of the single-copy ech42 gene encoding for the secreted 42-kDa endochitinase (CHIT42). Stable mutants in which ech42 was interrupted, and unable to produce CHIT42, were obtained and characterized. These mutants lacked the ech42 transcript, the protein, and endochitinase activity in culture filtrates, and they were unable to clear a medium containing colloidal chitin. Other chitinolytic and glucanolytic enzymes expressed during mycoparasitism were not affected by the disruption of ech42. The disrupted mutant D11 grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42 disruptant culture filtrates against Botrytis cinerea and Rhizoctonia solani was reduced about 40%, compared with wild type; antifungal activity was fully restored by adding an equivalent amount of CHIT42 as secreted by P1. The mutant exhibited the same biocontrol effect against Pythium ultimum as strain P1, but the antagonism against B. cinerea on bean leaves by the mutant was significantly reduced (33% less biocontrol), compared with strain P1. Conversely, the endochitinase-deficient mutant performed better than the wild type (16% improvement of survival) in biocontrol experiments in soil infested with the soilborne fungus R. solani. These results indicate that the antagonistic interaction between the T. harzianum strain and various fungal hosts is based on different mechanisms.

scholarly journals The GTPase-Activating Protein FgGyp1 Is Important for Vegetative Growth, Conidiation, and Virulence and Negatively Regulates DON Biosynthesis in Fusarium graminearium

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiaojia Zheng ◽  
Zhi Yu ◽  
Yanping Yuan ◽  
Danli Sun ◽  
Yakubu Saddeeq Abubakar ◽  
...  
Keyword(s):  
Vegetative Growth ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Negative Regulator ◽  
Rab Gtpase ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Replacement Method

Ypt1 is a small Rab GTPase in yeast, Gyp1 functions at the Golgi as a negative regulator of Ypt1. Gyp1 homologs are conserved in filamentous fungi. However, the roles of Gyp1 in phytopathogenic fungi are still unclear. Herein, we investigated the functions of FgGyp1 in the wheat pathogen Fusarium graminearum by live-cell imaging, genetic, and pathological analyses. Targeted gene replacement method was used to delete FgGYP1 in F. graminearum. Phenotypic analyses showed that FgGyp1 is critically important not only for the vegetative growth of F. graminearum but also its conidiation. The mutant’s vegetative growth was significantly reduced by 70% compared to the wild type PH-1. The virulence of FgGYP1 deletion mutant was significantly decreased when compared with the wild type PH-1. We further found that FgGyp1 negatively regulates DON production of the fungus. Live-cell imaging clearly demonstrated that FgGyp1 mainly localizes to the Golgi apparatus. Moreover, the TBC domain, C-terminal, and N-terminal regions of FgGyp1 are found to be indispensable for its biological functions and normal localization. The Arg357 residue of FgGyp1 is essential for its functions but dispensable for the normal localization of the protein, while the Arg284 residue is not required for both the functions and normal localization of the protein. Furthermore, we showed that FgGyp1 essentially hydrolyzes the GTP-bound FgRab1 (activated form) to its corresponding GDP-bound (inactive) form in vitro, suggesting that FgGyp1 is a GTPase-activating protein (GAP) for FgRab1. Finally, FgGyp1 was found to be important for FgSnc1-mediated fusion of secretory vesicles from the Golgi with the plasma membrane in F. graminearum. Put together, these data demonstrate that FgGyp1 functions as a GAP for FgRab1 and is important for vegetative growth, conidiation and virulence, and negatively regulates DON biosynthesis in F. graminearum.

scholarly journals The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues

2021 ◽  
Author(s):  
Marina Spoerrer ◽  
Delf Kah ◽  
Richard C Gerum ◽  
Barbara Reischl ◽  
Danyil Huraskin ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Contractile Function ◽  
Three Dimensional ◽  
Mechanical Damage ◽  
Underlying Mechanism ◽  
Intermediate Filament Protein ◽  
Wild Type ◽  
Age Related ◽  
Contractile Forces

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

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