Analysis of Random Migration of Dictyostelium Amoeba in Confined and Unconfined Environments

Litschko, Christof; Damiano-Guercio, Julia; Brühmann, Stefan; Faix, Jan

doi:10.1007/978-1-4939-7701-7_24

Cited by 9 publications

(6 citation statements)

References 8 publications

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“…Cells that contacted each other or divided were excluded from analyses. Mean square displacements and cell speed were calculated in Excel (Microsoft) using a customized macro 80 . Lamellipodium protrusion was determined based on kymographs generated from time-lapse movies recording advancing lamellipodia at 5 s intervals over a time period of at least 10 min using a UPlan FI ×100/1.30NA oil immersion objective (Olympus).…”

Section: Methodsmentioning

confidence: 99%

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

et al. 2022

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Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.

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Section: Methodsmentioning

confidence: 99%

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

et al. 2022

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“…Single cell tracking was performed in ImageJ by MTrackJ. Analyses of cell speed and cell trajectories, turning angles, and mean square displacements were performed in Excel (Microsoft, Redmond, WA) using a customized macro (Litschko et al, 2018). Cells that contacted each other or divided were excluded from analyses.…”

Section: Live Cell Imagingmentioning

confidence: 99%

Loss of Ena/VASP Interferes with Lamellipodium Architecture, Motility and Integrin-Dependent Adhesion

et al. 2019

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Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.

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“…Thus, we measured the size of plaques formed by WT and mutant cells when grown on a dense lawn of Klebsiella aerogenes as the only nutrient source. Impaired growth on bacterial lawns as seen with the double mutant can occur due to the poor uptake of bacteria, but it can also result from defects in cell division ( 45 ) or migration ( 28 , 46 ). However, the double mutant exhibited no noticeably defects in cytokinesis of cells cultivated in contact with the substrate and random migration rate as compared to WT and the single knockout (KO) mutants ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

Convergence of Ras- and Rac-regulated formin pathways is pivotal for phagosome formation and particle uptake in Dictyostelium

Körber

Junemann

Litschko

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Macroendocytosis comprising phagocytosis and macropinocytosis is an actin-driven process regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, nonprotrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction in F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization.

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Analysis of Random Migration of Dictyostelium Amoeba in Confined and Unconfined Environments

Cited by 9 publications

References 8 publications

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

Loss of Ena/VASP Interferes with Lamellipodium Architecture, Motility and Integrin-Dependent Adhesion

Convergence of Ras- and Rac-regulated formin pathways is pivotal for phagosome formation and particle uptake in Dictyostelium

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