Microfilament dynamics during cell movement and chemotaxis monitored using a GFP–actin fusion protein

Westphal, Monika; Jungbluth, Andreas; Heidecker, Manfred; Mühlbauer, Bettina; Heizer, Christina; Schwartz, Jean-Marc; Marriott, Gerard; Gerisch, Günther

doi:10.1016/s0960-9822(97)70088-5

Cited by 238 publications

(190 citation statements)

References 36 publications

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“…The cloning vector was p1AbBsr8 (39). A protein composed of GFP and the complete rod composed of repeats 1-6 (GFP-rod) was expressed from a pDEXbased vector (38). The constructs were generated by PCR and verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

Khaire

Müller

Blau-Wasser

et al. 2007

Journal of Biological Chemistry

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Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.

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Section: Methodsmentioning

confidence: 99%

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

Khaire

Müller

Blau-Wasser

et al. 2007

Journal of Biological Chemistry

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“…Nevertheless, to guard against this concern, only separate linear portions of the zigzag trajectories were included in the final analysis. The speed of polymerized MreB motion was computed to be 2.9 Ϯ 0.1 nm͞s by using a fit to the equation MSD ϭ 4Dt ϩ (Vt) 2 , where D is the diffusion coefficient and V is the speed.…”

Section: Polymerized and Unpolymerized Mreb Can Be Distinctly Visualimentioning

confidence: 99%

“…A quantitative understanding of the kinetic dynamics and ultrastructural architecture of actin's polymerized filaments has helped elucidate the mechanisms by which eukaryotic actin functions. For example, high-resolution imaging and the in vivo and in vitro dissection of the kinetics of its assembly have demonstrated how actin polymerization at the tips of a rigid, crosslinked actin meshwork can drive cell motility at the leading edge of Dictyostelium (1,2). In budding yeast, the polarized assembly of actin cables provides both a road and direction signs for the directed transport of proteins to the tip of growing buds (3).…”

mentioning

confidence: 99%

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus

Kim

Gitai

Kinkhabwala

et al. 2006

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

194

241

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The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.bacteria ͉ cytoskeleton ͉ single-molecule fluorescence I n both eukaryotic and prokaryotic cells, actin mediates essential cellular processes. A quantitative understanding of the kinetic dynamics and ultrastructural architecture of actin's polymerized filaments has helped elucidate the mechanisms by which eukaryotic actin functions. For example, high-resolution imaging and the in vivo and in vitro dissection of the kinetics of its assembly have demonstrated how actin polymerization at the tips of a rigid, crosslinked actin meshwork can drive cell motility at the leading edge of Dictyostelium (1, 2). In budding yeast, the polarized assembly of actin cables provides both a road and direction signs for the directed transport of proteins to the tip of growing buds (3).There are two known bacterial actin homologs, the widely conserved, chromosomally encoded MreB family of proteins and the plasmid-specific ParM family of proteins. ParM functions to partition plasmid DNA by polymerizing in between two sister plasmids, thereby generating a tension rod that physically pushes them apart (4). MreB is essential in most bacteria and has been shown to form a lengthwise spiral that contributes to cell shape, chromosome segregation, and polar protein localization in multiple species, including Caulobacter crescentus, Escherichia coli, and Bacillus subtilis (5-10). The mechanism by which MreB executes its functions remains largely unknown (11).In vitro studies of the dynamics of eukaryotic actin filament assembly have demonstrated th...

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“…Due to its general importance, considerable effort has been expended to visualize F-actin distribution and dynamics in living cells and tissues [Merrifield, 2004;Dormann and Weijer, 2006]. Three general approaches have been employed for this purpose: microinjection or expression of fluorescent G-actin [Kreis et al, 1982;Westphal et al, 1997]; microinjection of fluorescent phalloidin, a mushroom toxin that binds specifically to F-actin [Wulf et al, 1979;Wang, 1987;Planques et al, 1991] and; expression of fluorescent F-actin binding proteins or domains [Gerisch et al, 1995;Edwards et al, 1997;Kost et al, 1998]. While each of these approaches have provided essential information about the distribution and dynamics of actin in living cells, each has one or more important disadvantages.…”

Section: Introductionmentioning

confidence: 99%

Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

Burkel

Dassow

Bement

2007

Cell Motil. Cytoskeleton

453

485

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Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.

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Microfilament dynamics during cell movement and chemotaxis monitored using a GFP–actin fusion protein

Cited by 238 publications

References 36 publications

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus

Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

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