Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue‐specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed‐end capping activity of Tmods. Tmods are defined by a TM‐regulated/Pointed‐End Actin Capping (TM‐Cap) domain in their unstructured N‐terminal portion, followed by a compact, folded Leucine‐Rich Repeat/Pointed‐End Actin Capping (LRR‐Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods' functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod‐based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. © 2012 Wiley Periodicals, Inc

This paper introduces a new, easy-to-use method of fluorescence single-molecule speckle microscopy for actin with nanometer-scale accuracy. This new method reveals that actin flows in front of mature focal adhesions (FAs) are fast and biased toward FAs, suggesting that mature FAs are actively engaged in pulling and remodeling the local actin network.

Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1-4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1-3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second ␣-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second ␣-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this ␣-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second ␣-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat ␤-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal ␣6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N-and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.Dynamic assembly and disassembly of actin filaments are essential for establishing functional actin networks to execute various cellular phenomena. Regulation of actin dynamics at the filament ends, where polymerization and depolymerization occur, is crucial for rearrangement of the actin cytoskeleton. Although the concentration of monomeric actin in cells is in excess of the critical concentrations for assembly at both barbed and pointed ends, actin polymerization occurs predominantly at fast growing barbed ends (1, 2). Indeed, the actin barbed end-binding drug cytochalasin D inhibits many cellular phenomena, including cell migration, cell adhesion, and endocytosis (3-5). Six actin-nucleating proteins, including Arp2/3, formins, spire, cordon-bleu, leiomodin 2 (Lmod2), and JMY, have been described and play important roles in enhancing actin polymerization from barbed ends of filaments in vivo (6 -11). It appears that each of these proteins has a unique mechanism for actin nucleation. Arp2 and Arp3 subunits of the Arp2/3 complex are thought to template a new actin filament from the side of a preexisting filament and to anchor the pointed end of the growing filament (12). Formin homology 2 domains of formin family proteins form homodimers and stabilize an actin dimer that resembles the actin short pitch dimer in the filam...

Actin-depolymerizing factor (ADF)/cofilin enhances the turnover of actin filaments by two separable activities: filament severing and pointed-end depolymerization. Multicellular organisms express multiple ADF/cofilin isoforms in a tissue-specific manner, and the vertebrate proteins are grouped into ADFs and cofilins on the basis of their biochemical activity. A recent comparative study has shown that ADF has greater severing and depolymerizing activities than cofilin [Chen, H., Bernstein, B. W., Sneider, J. M., Boyle, J. A., Minamide, L. S., and Bamburg, J. R. (2004) Biochemistry 43, 7127-7142]. Here, we show that the two Caenorhabditis elegans ADF/cofilin isoforms exhibit different activities for severing and depolymerizing actin filaments. The ADF-like non-muscle isoform UNC-60A had greater activities to cause net depolymerization and inhibit polymerization than the cofilin-like muscle isoform UNC-60B. Surprisingly, UNC-60B exhibited much stronger severing activity than UNC-60A, which was the opposite of what was observed for vertebrate counterparts. Moreover, UNC-60B induced much faster pointed-end depolymerization of rabbit muscle actin than UNC-60A, while UNC-60A caused slightly faster depolymerization of C. elegans actin than UNC-60B. These results suggest that cofilin-like UNC-60B is kinetically more efficient in enhancing actin turnover than ADF-like UNC-60A, while ADF-like UNC-60A is suitable for maintaining higher concentrations of monomeric actin. These functional differences might be specifically adapted for different actin dynamics in muscle and non-muscle cells.

Background: Tropomodulins (Tmods) cap pointed ends of actin filaments in a tropomyosin (TM)-dependent manner. Results: Tmod1 and Tmod3 similarly cap actin filaments with diverse TM and actin isoforms, but only Tmod3 sequesters ␤-and ␥ cyto -actin monomers. Conclusion: Isoform-specific actin monomer sequestration by Tmod3 may provide a mechanism for actin remodeling in TM-deficient regions of cells. Significance: Defining the actin-regulatory activities of Tmods illuminates cytoskeletal dynamics.

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal beta-propeller, whereas one residue is located in the C-terminal beta-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

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The somatic gonad of the nematode Caenorhabditis elegans contains a myoepithelial sheath, which surrounds oocytes and provides contractile forces during ovulation. Contractile apparatuses of the myoepithelial-sheath cells are non-striated and similar to those of smooth muscle. We report the identification of a specific isoform of actin depolymerizing factor (ADF)/cofilin as an essential factor for assembly of contractile actin networks in the gonadal myoepithelial sheath. Two ADF/cofilin isoforms, UNC-60A and UNC-60B, are expressed from the unc-60 gene by alternative splicing. RNA interference of UNC-60A caused disorganization of the actin networks in the myoepithelial sheath. UNC-60B, which is known to function in the body-wall muscle, was not necessary or sufficient for actin organization in the myoepithelial sheath. However, mutant forms of UNC-60B with reduced actin-filament-severing activity rescued the UNC-60A-depletion phenotype. UNC-60A has a much weaker filament-severing activity than UNC-60B, suggesting that an ADF/cofilin with weak severing activity is optimal for assembly of actin networks in the myoepithelial sheath. By contrast, strong actin-filament-severing activity of UNC-60B was required for assembly of striated myofibrils in the body-wall muscle. Our results suggest that an optimal level of actin-filament-severing activity of ADF/cofilin is required for assembly of actin networks in the somatic gonad.