Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro.

Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with α-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin IIa (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIIa, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.

Gateva G ，Kremneva E ，Reindl T ，Kotila T ，Kogan K ，Gressin L ，Gunning PW ，Manstein DJ ，Michelot A ，Lappalainen P ... -  《-》

Regulation of actin filament turnover by cofilin-1 and cytoplasmic tropomyosin isoforms.

Tropomyosin and cofilin are actin-binding proteins which control dynamics of actin assembly and disassembly. Tropomyosin isoforms can either inhibit or enhance cofilin activity, but the mechanism of this diverse regulation is not well understood. In this work mechanisms of actin dynamics regulation by four cytoskeletal tropomyosin isoforms and cofilin-1 were studied with the use of biochemical and fluorescent microscopy assays. The recombinant tropomyosin isoforms were products of two genes: TPM1 (Tpm1.6 and Tpm1.8) and TPM3 (Tpm3.2 and Tpm3.4). Tpm1.6/1.8 bound to F-actin with higher apparent binding constants and lower cooperativities than Tpm3.2/3.4. In consequence, subsaturating concentrations of cofilin-1 removed 50% of Tpm3.2/3.4 from F-actin. By contrast, 2 and 5.5 molar excess of cofilin-1 over actin was required to dissociate 50% of Tpm1.6/1.8. All tropomyosins inhibited the rate of spontaneous polymerization of actin, which was reversed by cofilin-1. Products of TPM1 favored longer filaments and protected them from cofilin-induced depolymerization. This was in contrast to the isoforms derived from TPM3, which facilitated depolymerization. Tpm3.4 was the only isoform, which increased frequency of the filament severing by cofilin-1. Tpm1.6/1.8 inhibited, but Tpm3.2/3.4 enhanced cofilin-induced conformational changes leading to accelerated release of rhodamine-phalloidin from the filament. We concluded that the effects were executed through different actin affinities of tropomyosin isoforms and cooperativities of tropomyosin and cofilin-1 binding. The results obtained in vitro were in good agreement with localization of tropomyosin isoforms in stable or highly dynamic filaments demonstrated before in various cells.

Ostrowska Z ，Robaszkiewicz K ，Moraczewska J 《BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS》

Tropomyosin isoforms differentially modulate the regulation of actin filament polymerization and depolymerization by cofilins.

The specific functions of actin filaments located in the contractile and cytoskeletal compartments of muscle cells depend on the stability and dynamic polymerization/depolymerization of filaments. Tropomyosins and cofilins control the length and dynamic rearrangement of the filaments, although the mechanisms regulating actin dynamics are not well understood. In the present study, we used in vitro assays to examine the regulation of two cofilin isoforms, constitutive cofilin-1 and muscle cofilin-2, by the muscle homodimer Tpm1.1, muscle heterodimer Tpm1.1/Tpm2.2, and the cytoskeletal Tpm3.1. Depolymerization from the pointed end induced by the muscle-specific cofilin-2 was inhibited by all tropomyosins, whereas the muscle isoforms were most effective. By contrast, depolymerization by cofilin-1 was inhibited by Tpm3.1 and Tpm1.1, but not by Tpm1.1/Tpm2.2. Polymerization of G-actin was inhibited by cofilin-2, whereas cofilin-1 had no effect. All three tropomyosins switched on the inhibiting activity of cofilin-1; however, Tpm3.1 and Tpm1.1 were much more efficient. Cofilin-2-induced inhibition of polymerization was affected neither by Tpm1.1, nor by Tpm3.1, but partly relieved by Tpm1.1/Tpm2.2. Cofilins removed tropomyosin isoforms from the filament with different efficiencies, which correlated with the cooperativities of cofilin binding to the F-actin/tropomyosin complex. Because neither zero-length, nor long-arm cross-linking between tropomyosin and cofilin isoforms was observed, the effects of tropomyosin isoforms on the activities of cofilins were executed allosterically. The results reveal that isoform-specific interactions with actin filament permit tropomyosins to discriminate between cofilin isoforms and to differentially regulate their activities.

Robaszkiewicz K ，Ostrowska Z ，Marchlewicz K ，Moraczewska J ... -  《-》

Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms.

The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the actin filament major groove, providing an explanation for their incapability to co-polymerize on actin filaments. We also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalyzed severing.

Selvaraj M ，Kokate SB ，Reggiano G ，Kogan K ，Kotila T ，Kremneva E ，DiMaio F ，Lappalainen P ，Huiskonen JT ... -  《Cell Reports》

Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin.

Robaszkiewicz K ，Śliwinska M ，Moraczewska J 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》