Cofilin Cross-bridges Adjacent Actin Protomers and Replaces part of the Longitudinal F-actin Interface D. S. Kudryashov 1 * , V. E. Galkin 2 , A. Orlova 2 , M. Phan 1 E. H. Egelman 2 and E. Reisler 1 1 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA 2 Department of Biochemistry University of Virginia Health Sciences Center, Charlottesville VA 22908–0733, USA ADF/cofilins are abundant actin binding proteins critical to the survival of eukaryotic cells. Most ADF/cofilins bind both G and F-actin, sever the filaments and accelerate their treadmilling. These effects are linked to rearrangements of interprotomer contacts, changes in the mean twist, and filament destabilization by ADF/cofilin. Paradoxically, it was reported that under certain in vitro and in vivo conditions cofilin may stabilize actin filaments and nucleate their formation. Here, we show that yeast cofilin and human muscle cofilin (cofilin-2) accelerate the nucleation and elongation of ADP-F-actin and stabilize such filaments. Moreover, cofilin rescues the polymerization of the assembly incompetent tethramethyl rhodamine (TMR)-actin and T203C/C374S yeast mutant actin. Filaments of cofilin-decorated TMR-actin and unlabeled actin are indistinguishable, as revealed by electron microscopy and three-dimensional reconstruction. Our data suggest that ADF/cofilins play an active role in establishing new interprotomer interfaces in F-actin that substitute for disrupted (as in TMR- actin and mutant actin) or weakened (as in ADP-actin) longitudinal contacts in filaments. q 2006 Elsevier Ltd. All rights reserved. Keywords: actin; cofilin; polymerization; electron microscopy; image reconstruction *Corresponding author Introduction The ability of most living cells to support self- locomotion relies on the controlled polymerization/ depolymerization of actin at the leading edge of the cell. 1 Proteins of the actin depolymerizing factor (ADF)/cofilin family contribute prominently to this process. 2–4 The biological role of ADF/cofilins is related to their ability to sever actin filaments and accelerate their depolymerization from the pointed ends. 5,6 Initially the destabilization of F-actin was thought to be the main activity of ADF/cofilins, and hence the general name ADF. More recent work revealed that in vivo, ADF/cofilins appear to increase the level of F-actin in the cell, enhance membrane protrusion, stabilize invadopodia, and accelerate directional cell migration. 7–9 Although it is well documented that cofilin may accelerate the polymeri- zation of actin by increasing the steady-state concentration of ATP-G-actin, 5 this mechanism alone does not explain the increase in overall fraction of F-actin upon cofilin activation. The apparent paradox may result from the fact that in most cells G-actin is present at concentrations well above its critical concentration for polymerization, 10 but the assembly is restricted because capping proteins block the elongation of filaments. Under such conditions, the severing activity of ADF/cofilins is the dominant factor, yielding uncapped ends and thus stimulating the polymerization process. Furthermore, actin freshly polymerized at the uncapped barbed ends supports the Arp2/3-induced branch formation, 7,11 increasing the nucleation of new filaments. In addition to promoting new filament growth or branching, which is commonly linked to the filament severing, direct nucleation of actin filaments by cofilin has been postulated in several studies. 12,13 Thus, for example, ADF/cofilin was observed to improve greatly filament nucleation from ADP- actin. 13 ADF/cofilin-ADP-actin complexes can also assemble at the barbed ends of filaments with a rate 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. Abbreviations used: ADF, actin depolymerizing factor; ANP, N-(4-azido-2-nitrophenyl) putrescine; Cc, critical concentration for polymerization; EM, electron microscopy; SD, subdomain of actin; TC/CS, T203C/ C374S yeast mutant actin; TMR, tetramethyl rhodamine- maleimide. E-mail address of the corresponding author: dkudryas@ucla.edu doi:10.1016/j.jmb.2006.02.029 J. Mol. Biol. (2006) 358, 785–797