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Single Molecule imaging and Kinetic Analysis of Cooperative Cofilin–Actin Filament Interactions

Significance Statement

Cooperativity is essential for biomolecules to regulates their enzymatic activity or affinity for a ligand. However, the dynamic interactions that endow proteins with cooperativity in solution have not been directly imaged and analyzed. Cofilin is an important actin binding protein, which accelerates actin dynamics by severing and depolymerizing actin filaments, and the binding of cofilin to actin filaments is positively cooperative (1, 2); one cofilin binding facilitates another cofilin binding Cofilin–Actin . Thus, the cooperative binding leads to formation of the cofilin cluster which is essential for the actin filament severing (3). Direct observation of the cooperative cofilin binding at the single molecule level will help to elucidate the molecular process in the cofilin cluster formation and the filament severing. In this study, the interaction between cofilin and actin filaments was analyzed using super-high-resolution optical microscopy with 18 nm spatial resolution for the first time, by which we directly observed each cofilin binding along an actin filament very precisely (see details in the original paper). We observed a cofilin binding was frequently followed by another cofilin binding in the vicinity of the first one. The mean distance of these two bindings was 65 nm, which corresponds to 24 actin protomers; “protomer” denotes an actin monomer in the filament. In other words, the chance to find an  additional cofilin binding was high in the vicinity of the initial binding, and it declined along the actin filament with a space constant of 65 nm. The most likely scenario of the consecutive f cofilin bindings and following cluster formation based on our observation is shown in the figure. The initial cofilin binding is designated by initial (“i”)-cofilin, which cooperatively facilitates additional (“a”)-cofilin bindings in its vicinity. Many cofilin molecules bind in a similar (i.e., cooperative) manner, and form a loosely packed cofilin cluster. This cluster presumably promotes a severing as mentioned in a recent study (3).

References

  1. McGough A, Pope B, Chiu W, & Weeds A (1997) Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function. J Cell Biol 138(4):771-781.
  2. Ressad F, et al. (1998) Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation. J Biol Chem 273(33):20894-20902.
  3. McCullough BR, et al. (2011) Cofilin-linked changes in actin filament flexibility promote severing. Biophys J 101(1):151-159.
  4. De La Cruz EM (2005) Cofilin binding to muscle and non-muscle actin filaments: isoform-dependent cooperative interactions. J.Mol.Biol. 346(2):557-564.

Single Molecule imaging and Kinetic Analysis of Cooperative Cofilin–Actin Filament Interactions. Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

Proc Natl Acad Sci U S A. 2014 ;111(27):9810-5. Hayakawa1, S. Sakakibara2, M. Sokabe1,2, H. Tatsumi2

1Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, 65 Tsurumai, Nagoya 466-8550, Japan

2Department of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Nagoya 466-8550, Japan

Abstract

The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics. The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin. In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a singlemoleculeimaging technique. The on-rate of cofilin binding to the actin filament was estimated to be 0.06 µM(-1)⋅s(-1) when the cofilin concentration was in the range of 30 nM to 1 µM. A dwell time histogram of cofilin bindings decays exponentially to give an off-rate of 0.6 s(-1). During long-term cofilin binding events (>0.4 s), additional cofilin bindings were observed in the vicinity of the initial binding site. The on-rate for these events was 2.3-fold higher than that for noncontiguous bindings. Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event. By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin. Neither decreasing the temperature nor increasing the viscosity of the test solution altered the on-rates, off-rates, or the cooperative parameter (ω) of the binding. These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca. 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments.

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