A Lim protein involved in the progression of cytokinesis and regulation of the mitotic spindle

Schneider, Natalie; Weber, Igor; Faix, Jan; Prassler, Josef; Müller‐Taubenberger, Annette; Köhler, Jana; Burghardt, Emmanuel; Gerisch, Günther; Marriott, Gerard

doi:10.1002/cm.10139

Cited by 56 publications

(61 citation statements)

References 59 publications

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“…The D. discoideum strain LimE-GFP (20) was cultivated in HL5 medium (Formedium) at 22°C on polystyrene Petri dishes (Primaria, Falcon, BD Becton Dickinson) or shaken in suspension at 150 rpm. For preparation of an experiment, cells were starved in shaking phosphate buffer (PB, 2 g KH 2 PO 4 and 0.36 g Na 2 HPO 4 ·2H 2 O per 1 L, pH 6) for 6 h, at a density of ∼2 × 10 6 cells/ mL (40).…”

Section: Methodsmentioning

confidence: 99%

“…As a marker for filamentous actin, we used DdLimEΔcoil-GFP (LimE-GFP), the fluorescently tagged version of a Dictyostelium Limdomain protein with truncated coiled-coil domain (17,18). It is known to colocalize with F-actin (19,20). Due to its excellent fluorescence properties and low cytosolic background levels, LimE-GFP became a popular in vivo marker for F-actin structures.…”

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confidence: 99%

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Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

Westendorf

Negrete

Bae

et al. 2013

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

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The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments.Dictyostelium discoideum | microfluidics | caged cAMP | delay-differential equation T he actin cytoskeleton provides the basis for shape dynamics and motility of eukaryotic cells. Essential biological processes like wound healing, embryonic morphogenesis, or cancer metastasis rely on the rapid rearrangement of the actin cytoskeleton in response to external chemical cues (1). Many of the underlying actin-driven processes have been investigated in cells of the social amoeba Dictyostelium discoideum. Under starvation, the singlecelled amoeba expresses a chemotactic signaling system to aggregate into a multicellular structure, mediated by the chemoattractant cAMP. The corresponding receptor signaling pathway and the downstream cytoskeletal machinery show remarkable similarities to motile cells of higher organisms, in particular neutrophils (2), making Dictyostelium one of the most popular models for eukaryotic cell motility and chemotaxis (3, 4).In earlier studies, it was observed that the actin system of chemotactic Dictyostelium cells shows a complex, nonmonotonic response when exposed to a sudden increase in the extracellular chemoattractant concentration (5-7). Between 5 and 10 s after the stimulus, a first maximum in the filamentous actin content is observed, followed by a second, less intense but prolonged maximum that starts about 30 s after the stimulus and...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

Westendorf

Negrete

Bae

et al. 2013

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

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“…To investigate the relation between F-actin distribution and the localization of sGC during chemotaxis, the filamentous actin binding protein LimE⌬coil (Schneider et al, 2003) was C-terminally fused to mKO (Karasawa et al, 2004) and used as an F-actin probe in the various cell strains expressing the GFP-tagged sGC mutants. The cells were placed in a cAMP gradient and visualized by confocal microscopy.…”

Section: Distribution Of Filamentous Actin and Sgcmentioning

confidence: 99%

Guanylyl Cyclase Protein and cGMP Product Independently Control Front and Back of ChemotaxingDictyosteliumCells

Veltman

Haastert

2006

MBoC

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Chemotaxis of amoeboid cells is driven by actin filaments in leading pseudopodia and actin-myosin filaments in the back and at the side of the cell to suppress pseudopodia. In Dictyostelium, cGMP plays an important role during chemotaxis and is produced predominantly by a soluble guanylyl cyclase (sGC). The sGC protein is enriched in extending pseudopodia at the leading edge of the cell during chemotaxis. We show here that the sGC protein and the cGMP product have different functions during chemotaxis, using two mutants that lose either catalytic activity (sGC⌬cat) or localization to the leading edge (sGC⌬N). Cells expressing sGC⌬N exhibit excellent cGMP formation and myosin localization in the back of the cell, but they exhibit poor orientation at the leading edge. Cells expressing the catalytically dead sGC⌬cat mutant show poor myosin localization at the back, but excellent localization of the sGC protein at the leading edge, where it enhances the probability that a new pseudopod is made in proximity to previous pseudopodia, resulting in a decrease of the degree of turning. Thus cGMP suppresses pseudopod formation in the back of the cell, whereas the sGC protein refines pseudopod formation at the leading edge. INTRODUCTIONChemotaxis is a vital process in a variety of organisms, ranging from bacteria to vertebrates. Prokaryotes use chemotaxis to move toward high nutrient concentrations or away from unfavorable conditions, whereas in eukaryotes chemotaxis is also involved in embryogenesis, wound healing, and the immune response. Chemotaxis is achieved by coupling gradient sensing to basic cell movement. Prokaryotes are too small to sense spatial gradients and therefore rely on temporal changes of the chemoattractant concentration to achieve chemotaxis. They do this by adjusting their tumbling frequency in response to temporal changes of the chemoattractant concentration (Szurmant and Ordal, 2004;Wadhams and Armitage, 2004). Eukaryotic cells are typically large enough to be able to measure a spatial gradient. The difference in receptor occupation between each side of the cell leads to an internal polarization. A pseudopod is extended at the side with the highest receptor occupation and at the same time, pseudopod formation at all other sides is repressed, resulting in directional cell migration (Devreotes and Janetopoulos, 2003;Postma et al., 2004).Dictyostelium is a eukaryotic organism that is widely used to study chemotaxis (Williams and Harwood, 2003;Parent, 2004;Affolter and Weijer, 2005). Starved Dictyostelium cells periodically secrete cAMP. Through relay of the cAMP signal by neighboring cells, concentric cAMP waves are generated. Starved Dictyostelium cells are chemotactically sensitive to cAMP and by movement in the direction of the origin of the cAMP waves, the cells are able to aggregate into groups of up to 100,000 cells. The chemotactic response of Dictyostelium is optimized for the dynamic cAMP waves that coordinate both aggregation and multicellular development. Cells show a much stronger chemotact...

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“…LimED-mRFP, the fluorescently tagged version of a Dictyostelium Lim-domain protein with truncated coiledcoil domain, is a well-established marker for filamentous actin. 29 Together with the GFP-tagged version of myosin II, the intracellular acto-myosin distribution was directly recorded in live cells by fluorescence microscopy. Images were taken by dual-color confocal laser scanning microscopy, so that both labels were simultaneously followed in the same cell.…”

Section: Resultsmentioning

confidence: 99%

Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells

et al. 2013

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Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. Insight, innovation, integrationSecond messengers like cGMP play a central role in the regulatory pathways of living cells. Here, we demonstrate that an increase in intracellular cGMP can trigger not only myosin II but also actin responses in motile amoeboid cells. In previous studies, an increase in intracellular cGMP was induced indirectly via membrane receptor stimulation. In the present work, we employ, for the first time, the direct light-induced release of cGMP from a caged precursor to raise the cytosolic cGMP level in Dictyostelium cells that carry fluorescent markers for filamentous actin and myosin II. Based on this advanced combination of live cell photo-uncaging and multi-color confocal microscopy, we could identify a link between cGMP and actin dynamics in motile amoeboid cells.

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A Lim protein involved in the progression of cytokinesis and regulation of the mitotic spindle

Cited by 56 publications

References 59 publications

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

Guanylyl Cyclase Protein and cGMP Product Independently Control Front and Back of ChemotaxingDictyosteliumCells

Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells

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