Sawako Yamashiro

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities

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 α-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 α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-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 β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-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.

Total synthesis and biological evaluation of monorhizopodin and 16-epi-monorhizopodin.

For many years, polyketide natural products have provided the scientific community with a rich source of novel molecular architectures, many of which have become important therapeutics for clinical use.[1] In 1993, the polyketide rhizopodin was isolated from the myxobacterium Myxococcus stipitatus.[2] It was shown to display an interesting array of biological properties, including potent anti-tumor activity against a range of cancer cell lines in the low nanomolar range and the ability to inhibit the polymerization of actin.[2,3] Despite its original structural assignment as the 19-membered monomeric lactone 1a (monorhizopodin), recent studies revealed dimeric structure 2 to be the correct architecture of rhizopodin (Figure 1).[4] These molecules have started to attract attention from the synthetic community, although no total syntheses have been reported to date.[5]

Sarcomeric actin organization is synergistically promoted by tropomodulin, ADF/cofilin, AIP1 and profilin in C. elegans

Sarcomeric organization of thin and thick filaments in striated muscle is important for the efficient generation of contractile forces. Sarcomeric actin filaments are uniform in their lengths and regularly arranged in a striated pattern. Tropomodulin caps the pointed end of actin filaments and is a crucial regulator of sarcomere assembly. Here, we report unexpected synergistic functions of tropomodulin with enhancers of actin filament dynamics in Caenorhabditis elegans striated muscle. Pointed-end capping by tropomodulin inhibited actin filament depolymerization by ADF/cofilin in vitro. However, in vivo, the depletion of tropomodulin strongly enhanced the disorganization of sarcomeric actin filaments in ADF/cofilin mutants, rather than antagonistically suppressing the phenotype. Similar phenotypic enhancements by tropomodulin depletion were also observed in mutant backgrounds for AIP1 and profilin. These in vivo effects cannot be simply explained by antagonistic effects of tropomodulin and ADF/cofilin in vitro. Thus, we propose a model in which tropomodulin and enhancers of actin dynamics synergistically regulate elongation and shortening of actin filaments at the pointed end.

Essential role of ADF/cofilin for assembly of contractile actin networks in the C. elegans somatic gonad

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.

Tropomodulin Protects α-Catenin-Dependent Junctional-Actin Networks under Stress during Epithelial Morphogenesis

α-catenin is central to recruitment of actin networks to the cadherin-catenin complex, but how such networks are subsequently stabilized against stress applied during morphogenesis is poorly understood. To identify proteins that functionally interact with α-catenin in this process, we performed enhancer screening using a weak allele of the C. elegans α-catenin, hmp-1, thereby identifying UNC-94/tropomodulin. Tropomodulins (Tmods) cap the minus ends of F-actin in sarcomeres. They also regulate lamellipodia, can promote actin nucleation, and are required for normal cardiovascular development and neuronal growth-cone morphology. Tmods regulate the morphology of cultured epithelial cells, but their role in epithelia in vivo remains unexplored. We find that UNC-94 is enriched within a HMP-1-dependent junctional-actin network at epidermal adherens junctions subject to stress during morphogenesis. Loss of UNC-94 leads to discontinuity of this network, and high-speed filming of hmp-1(fe4);unc-94(RNAi) embryos reveals large junctional displacements that depend on the Rho pathway. In vitro, UNC-94 acts in combination with HMP-1, leading to longer actin bundles than with HMP-1 alone. Our data suggest that Tmods protect actin filaments recruited by α-catenin from minus-end subunit loss, enabling them to withstand the stresses of morphogenesis.

UNC-87, a calponin-related protein in C. elegans, antagonizes ADF/cofilin-mediated actin filament dynamics

Stabilization of actin filaments is critical for supporting actomyosin-based contractility and for maintaining stable cellular structures. Tropomyosin is a well-characterized ubiquitous actin stabilizer that inhibits ADF/cofilin-dependent actin depolymerization. Here, we show that UNC-87, a calponin-related Caenorhabditis elegans protein with seven calponin-like repeats, competes with ADF/cofilin for binding to actin filaments and inhibits ADF/cofilin-dependent filament severing and depolymerization in vitro. Mutations in the unc-87 gene suppress the disorganized actin phenotype in an ADF/cofilin mutant in the C. elegans body wall muscle, supporting their antagonistic roles in regulating actin stability in vivo. UNC-87 and tropomyosin exhibit synergistic effects in stabilizing actin filaments against ADF/cofilin, and direct comparison reveals that UNC-87 effectively stabilizes actin filaments at much lower concentrations than tropomyosin. However, the in vivo functions of UNC-87 and tropomyosin appear different, suggesting their distinct roles in the regulation of actomyosin assembly and cellular contractility. Our results demonstrate that actin binding via calponin-like repeats competes with ADF/cofilin-driven cytoskeletal turnover, and is critical for providing the spatiotemporal regulation of actin filament stability.

New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

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.

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.

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

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 β- and γcyto-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. Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-β/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.

Molecular viewing of actin polymerizing actions and beyond: Combination analysis of single-molecule speckle microscopy with modeling, FRAP and s-FDAP (sequential fluorescence decay after photoactivation)

Live‐cell single‐molecule imaging is a powerful tool to elucidate the in vivo biochemistry of cytoskeletal proteins. However, it is often somewhat difficult to interpret how a bulk population of the observed molecule might behave as a whole. We review our recent studies in which the combination of image analysis with modeling and bulk kinetics measurements such as FRAP (fluorescence recovery after photobleaching) clarified basic problems in the regulation of actin remodeling pathways.