Organization of F-Actin by Avian Smooth Muscle Synaptopodin 2 (Fesselin)

Abstract

Tropomodulin, a tropomyosin-dependent actin filament capping protein, consists of two structurally and functionally different domains. Tropomodulin lacking its C-terminal domain can cap actin filaments with an affinity close to the full-length protein in vitro. To investigate the functional properties of the C-terminal domain in live cells, truncated GFP-tagged tropomodulin was expressed in rat cardiac myocytes. Three fragments were analyzed: Tmod1(1-159) that lacks the entire C-terminal domain, Tmod1(1-320) and Tmod1(1-349). GFP-Tmod1(1-159) did not assemble well at the pointed ends of the filaments (~80% of the cells demonstrated a diffuse distribution, while ~20% showed faint, inconsistent assembly). Together, the in vitro and live cell studies indicate that the C-terminal domain is not required for capping actin filaments but is important for specifically targeting tropomodulin to the pointed ends of the actin filaments in sarcomeres. In the cells where GFP-Tmod1(1-320) was expressed most (~70%) of the assembly was faint and inconsistent. Tmod1(1-320) lacks the 39 C-terminal residues that include both the C-terminal helix that is not a part of LRR fold, and the tropomyosin independent actin filament capping site. The precise location of the tropomyosin independent actin filament capping site is not known, although removal of 15 residues from the C-terminus destroys the actin filament capping ability of Tmod1 in the absence of tropomyosin. GFP-Tmod1(1-349) is missing the ten C-terminal residues which discriminates Tmod1 from other tropomodulin isoforms; this fragment consistently assembled at the thin filament pointed ends, comparable to the cells expressing wild type GFP-Tmod1. Based on these data we suggest that both the LRR fold (residues 160-320) and residues 321-349 are important for regulating tropomodulin's pointed end capping activity though the specific role each of these regions play in this phenomenon may be different.

Synaptopodin family of natively unfolded, actin binding proteins: physical properties and potential biological functions

“…Avian synaptopodin 2 also increased the elongation rate by 3-fold. Synaptopodin 2 increased the critical concentration from 0.3 to 0.49 μM suggesting growth at the pointed ends of actin filaments (Schroeter et al 2010).…”

Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches

“…Another protein is synaptopodin 2, which belongs to the synaptopodin family whose members have been shown to stimulate actin polymerization and bundling of actin filaments. Synaptopodin 2, also known as myopodin or fesselin is known to localize in the Z‐bands of striated and heart muscle cells . It is known to be an F‐actin‐binding protein, and interacts with the myofilament‐associated proteins filamin C and α‐actinin .…”

Avian Synaptopodin 2 (Fesselin) Stabilizes Myosin Filaments and Actomyosin in the Presence of ATP

“…Binding to F-actin results in the formation of actin aggregates (1) or bundles (8). These bundles are ordered with a uniform polarity (9). Fesselin also binds to G-actin and stimulates actin polymerization (10).…”

“…8 These bundles are ordered with a uniform polarity. 9 Fesselin also binds to G-actin and stimulates actin polymerization. 10 Ca 2+ -calmodulin inhibits the ability of fesselin to stimulate Gactin polymerization.…”