Fesselin is a proline-rich actin binding protein that has recently been isolated from smooth muscle [Leinweber, B. D., Fredricksen, R. S., Hoffman, D. R., and Chalovich, J. M. (1999) J. Muscle Res. Cell Motil. 20,[539][540][541][542][543][544][545]. Fesselin is similar to synaptopodin [Mundel, P., Heid, H. W., Mundel, T. M., Krüger, M., Reiser, J., and Kriz, W. (1997) J. Cell Biol. 139, 193-204] in terms of its size, isoelectric point, and sequence although synaptopodin is not present in smooth muscle. The function of fesselin is unknown. Evidence is presented here that fesselin accelerates the polymerization of actin. Fesselin was effective on actin isolated from either smooth or skeletal muscle at low ionic strength and in the presence of 100 mM KCl. At low ionic strength, fesselin decreased the time for 50% polymerization to about 1% of that in the absence of fesselin. The lag phase characteristic of the slow nucleation process of polymerization was eliminated as the fesselin concentration was increased from very low levels. Fesselin did not alter the critical concentration for actin but did increase the rate of elongation by ≈3-fold. The increase in elongation rate constant is insufficient to account for the total increase in polymerization rate. It is likely that fesselin stabilizes the formation of actin nuclei. Time courses of actin polymerization at varied fesselin concentrations and varied actin concentrations were simulated by increasing the rate of nucleation and both the forward and reverse rate constants for elongation.Fesselin is an actin binding protein that has recently been isolated from turkey gizzard muscle (1). Fesselin is noteworthy because of its potent ability to bundle actin and because of its similarity to synaptopodin, a protein found in telencephalic dendrites and renal podocytes but not in smooth muscle (2). Fesselin has in common with synaptopodin a similar pI (9.3), a similar mobility on SDS gels (103 and 79 kDa for fesselin and 110 kDa for synaptopodin), a high proline content, and regions of sequence homology. Antibodies directed against synaptopodin decorate actin filaments in cells forming a punctate pattern (2); this pattern is lost following depolymerization of actin with cytochalasin B. Preliminary studies with fesselin also indicate that fesselin co-localizes with α-actinin on actin filaments (3). The functions of fesselin and synaptopodin are unknown.The effect of fesselin on actin polymerization was examined because of its ability to bind to and bundle actin filaments and its high isoelectric point. The isoelectric point was a consideration because cationic substances tend to polymerize actin (4,5). Actin binding and polymerizing proteins are often basic (6), and charge neutralization may be a factor in the function of these proteins (5).The present study provides evidence that fesselin increases the rate of actin polymerization. This was seen by increases in the apparent rates of increase of both pyrene fluorescence and † Funded by NIH Grant AR35216 and a Facult...

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and alpha-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.

An analysis of the primary structure of the actin binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 1 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582-586]. We isolated synaptopodin 2 from rabbit stomach and showed that it shared several key functions with fesselin. Both fesselin and synaptopodin 2 bound to Ca2+-calmodulin, α–actinin, and smooth muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca2+-calmodulin dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of α-actinin, to polymerize actin in a Ca2+-calmodulin dependent manner, or to bind to Ca2+-calmodulin, or myosin. These properties are consistent of the proposed function of synaptopodin 2 in organizing the cytoskeleton.

Fesselin or avian synaptopodin 2 is a member of the synaptopodin family of actin binding proteins. Fesselin promotes G-actin polymerization and the formation of large actin complexes that can be collected by centrifugation at low speed. Because of the potential role of fesselin in some cancers and its effects on actin we further investigated the effect of fesselin on actin. Fesselin initiated actin polymerization at a variety of conditions including the virtual absence of salt. Actin filaments formed at low salt in the presence of fesselin were similar to filaments polymerized in the presence of 100 mM KCl. In both cases the filaments were long and straight with a common orientation. Highly ordered actin bundles formed with increasing times of incubation. Blockers of actin growth at the barbed end (cytochalasin D and CapZ) did not prevent fesselin from polymerizing actin. Low concentrations of fesselin increased the critical concentration of actin. Both observations are consistent with preferential growth at the pointed end of actin filaments. These results indicate a role of fesselin in organizing cellular actin. These and other results indicate that fesselin is part of a cellular actin organizing center.

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.