Gelsolin Family Of Proteins Research Articles

Actin Polymerization Dynamics - Insights from In vitro TIRF Microscopy

Actin elongation is a bi-molecular reaction between monomeric actin (G-actin) and filamentous actin (F-actin), in the first approximation. It can be controlled by changing the ability of either G-actin or F-actin to participate in the reaction. Either of the two mechanisms alone is not sufficient to maintain a large pool of G-actin ready to polymerize in a signal-controlled fashion [1]. Mammalian cells have hundreds of actin-binding proteins (ABP) which bind either or both the forms of actin. Profilin sequesters G-actin and makes them pre-disposed towards F-actin barbed-end addition, cofilin severs F-actin and deploymerizes it into G-actin. On the other hand, capping protein (CP) caps the barbed-end and stops further elongation of F-actin [2]. Gelsolin-family of proteins [3] sever F-actin as well as cap filaments. In vitro TIRF microscopy [4] has been used to monitor real-time actin dynamics in the presence of ABPs [5], [6]. Representative results on some ABPs which alter actin assembly will be presented. [1] Pantaloni et al, Science (2001) 292: 1502-06 [2] J.A. Cooper et al, Int. Rev. Cell Mol. Biol. (2008) 267: 183-206 [3] P. Silacci et al, Cell Mol Life Sci (2004) 61: 2614-2623 [4] J.R. Kuhn et al, Biophys. J (2005) 88:1387-1402 [5] S. Nag et al, Proc. Natl. Acad. Sci. (2009) 106:13713-13718 [6] M. H.-Valladares et al, Nat Struct Mol Biol (2010) 17: 497-503

Single-Molecule Study of Actin Filament Severing by Gelsolin using Total Internal Reflection Fluorescence Microscopy

Gelsolin-family proteins are regulators of actin polymerization dynamics.[1] To understand the mechanism of actin filament (F-actin) severing by gelsolin at the single-molecule level an in vitro total internal reflection fluorescence (TIRF) microscopy[2] assay has been developed. Preliminary TIRF data confirm that gelsolin sever F-actin.[3] These real-time microscopic observations are in concert with the structural and biochemical studies that have established activation of gelsolin by calcium ions. [3],[4],[5] To further ascertain a) if gelsolin preferentially binds to F-actin rather than sequestering actin monomers (G-actin), b) if it preferentially binds and severs ADP-F-actin or ATP-F-actin, and c) if gelsolin or cofilin, a member of actin depolymerizing factor (ADF), sever gelsolin-capped F-actin better than native filaments, dual color TIRFM assays are undertaken. Results will be discussed in the context of structural and biochemical data on gelsolin-family of proteins.[1] P. Silacci et al, Cell Mol Life Sci 2004, 61: 2614-2623[2] J.R. Kuhn, T. D. Pollard, Biophys. J 2005. 88:1387-1402[3] S. Nag et al, Proc. Natl. Acad. Sci. 2009, 106 :1371313718[4] R.C. Robinson et al, Science 1999. 286: 1939-1942[5] K. Narayan et al, Febs Lett. 2003. 552:82-85

Caenorhabditis elegans Gelsolin-like Protein 1 Is a Novel Actin Filament-severing Protein with Four Gelsolin-like Repeats

The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins.

Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins.

gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.