Analysis of Actin Assembly by In Vitro TIRF Microscopy

Breitsprecher, Dennis; Kiesewetter, Antje K; Linkner, Joern; Faix, Jan

doi:10.1007/978-1-60761-198-1_27

Cited by 21 publications

(17 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Actin polymerization and bundle assembly were analyzed by TIRFM using a Zeiss Axiovert 200M inverted microscope equipped with a ϫ100/1.46-NA Alpha Plan-Apochromat TIRF objective. A 1:1 mixture of cold and Alexa Fluor 488-labeled actin (1 M; Invitrogen) with 3 M MLP or 250 nM fascin was prepared in fluorescence buffer (100 mM DTT, 0.5% methylcellulose, 20 g/ml catalase, 100 g/ml glucose oxidase, and 15 mM glucose) supplemented with 1ϫ standard polymerization F buffer (50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, and 10 mM imidazole, pH 7.0), flowed into a visualization chamber coated with 2.5 nM N-ethylmaleimide (NEM)-myosin (43), and immediately imaged. An excitation ray (488 nm) was provided by an argon laser, and emission light was collected with a 525/50 BP filter.…”

Section: Methodsmentioning

confidence: 99%

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

Hoffmann

Moreau

Moes

et al. 2014

Molecular and Cellular Biology

View full text Add to dashboard Cite

The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking.

show abstract

Section: Methodsmentioning

confidence: 99%

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

Hoffmann

Moreau

Moes

et al. 2014

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…Myosin was purified and chemically inactivated with N-ethylmaleimide according to Ref. 29. Actin was labeled in random surface lysines with atto488 or atto532, or it was purchased from Hypermol (Bielefeld, Germany).…”

Section: Methodsmentioning

confidence: 99%

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics

Crevenna

Naredi‐Rainer

Schönichen

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: pH plays important roles in cellular morphogenesis, but how actin polymer dynamics are affected by pH is not well understood. Results: Actin filament nucleation and elongation are strongly enhanced at acidic pH due to a reduction of charge repulsion. Conclusion: Actin filament dynamics in vitro and in vivo are strongly influenced by electrostatics. Significance: Cellular pH homeostasis will impact directly on actin dynamics.

show abstract

“…For TIRF microscopy, glass flow chambers of ,50 ml were prepared as previously described (Breitsprecher et al, 2009). Chambers were perfused with 2.5 nM NEM-myosin in myosin buffer (10 mM imidazole, pH 7.0, 0.5 M KCl, 10 mM MgCl 2 ).…”

Section: Tirf Microscopy and Vaem Live Cell Imagingmentioning

confidence: 99%

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Hoffmann

Moes

Dieterle

et al. 2013

Journal of Cell Science

View full text Add to dashboard Cite

Crosslinking of actin filaments into bundles is critical for the assembly/stabilization of specific cytoskeletal structures. Relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM domain-containing (LIM) proteins define a novel and evolutionary-conserved family of actin bundlers whose actin-binding and -crosslinking activities primarily rely on their LIM domains. Using TIRF microscopy, we describe real-time formation of actin bundles induced by tobacco NtWLIM1 in vitro. We show that NtWLIM1 binds to single filaments and subsequently promotes their interaction and zippering into tight bundles of mixed polarity. NtWLIM1-induced bundles grew by both elongation of internal filaments and addition of preformed fragments at their extremities. Importantly, these data are highly consistent with the modes of bundle formation and growth observed in transgenic Arabidopsis plants expressing a GFP fused Arabidopsis AtWLIM1 protein. Using two complementary live cell imaging approaches, a close relationship between NtWLIM1 subcellular localization and self-association was established. Indeed, both BiFC and FLIM-FRET data revealed that, although unstable NtWLIM1 complexes can sporadically form in the cytosol, stable complexes concentrate along the actin cytoskeleton. Remarkably, the disruption of the actin cytoskeleton significantly impaired NtWLIM1 self-association. In addition, biochemical analyses support that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di/oligomeric state. Based on our data we propose a model in which actin binding promotes the formation/stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.

show abstract

Analysis of Actin Assembly by In Vitro TIRF Microscopy

Cited by 21 publications

References 26 publications

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Contact Info

Product

Resources

About