Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro.
ABSTRACT: Actin filaments assemble into a variety of networks to provide force for diverse cellular processes . 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 . 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 ?-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.
Project description:The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.
Project description:Tropomyosins, a family of actin-associated proteins, bestow actin filaments with distinct biochemical and physical properties which are important for determining cell shape and regulating many cellular processes in eukaryotic cells. Here, we used RNA-seq to investigate the effect of four tropomyosin isoforms on gene expression in undifferentiated and differentiated rat B35 neuroblastoma cells. In undifferentiated cells, overexpression of tropomyosin isoforms Tpm1.12, Tpm2.1, Tpm3.1, and Tpm4.2 differentially regulates a vast number of genes, clustering into several gene ontology terms. In differentiated cells, tropomyosin overexpression exerts a much weaker influence on overall gene expression. Our findings are particularly compelling because they demonstrate that tropomyosin-dependent changes are attenuated once the cells are induced to follow a defined path of differentiation.Sequence data for public availability are deposited in the European Nucleotide Archive under the accession number PRJEB24136.
Project description:The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.
Project description:The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.
Project description:Muscle contraction, cytokinesis, cellular movement, and intracellular transport depend on regulated actin-myosin interaction. Most actin filaments bind one or more isoform of tropomyosin, a coiled-coil protein that stabilizes the filaments and regulates interactions with other actin-binding proteins, including myosin. Isoform-specific allosteric regulation of muscle myosin II by actin-tropomyosin is well-established while that of processive myosins, such as myosin V, which transport organelles and macromolecules in the cell periphery, is less certain. Is the regulation by tropomyosin a universal mechanism, the consequence of the conserved periodic structures of tropomyosin, or is it the result of specialized interactions between particular isoforms of myosin and tropomyosin? Here, we show that striated muscle tropomyosin, Tpm1.1, inhibits fast skeletal muscle myosin II but not myosin Va. The non-muscle tropomyosin, Tpm3.1, in contrast, activates both myosins. To decipher the molecular basis of these opposing regulatory effects, we introduced mutations at conserved surface residues within the six periodic repeats (periods) of Tpm3.1, in positions homologous or analogous to those important for regulation of skeletal muscle myosin by Tpm1.1. We identified conserved residues in the internal periods of both tropomyosin isoforms that are important for the function of myosin Va and striated myosin II. Conserved residues in the internal and C-terminal periods that correspond to Tpm3.1-specific exons inhibit myosin Va but not myosin II function. These results suggest that tropomyosins may directly impact myosin function through both general and isoform-specific mechanisms that identify actin tracks for the recruitment and function of particular myosins.
Project description:The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities.
Project description:Tropomyosin is an actin binding protein that regulates actin filament dynamics and its interactions with actin binding proteins such as myosin, tropomodulin, formin, Arp2/3 and ADF-cofilin in most eukaryotic cells. Tropomyosin is the prototypical two-chained, ?-helical coiled coil protein that associates end-to-end and binds to both sides of the actin filament. Each tropomyosin molecule spans four to seven actin monomers in the filament, depending on the size of the tropomyosin. Tropomyosins have a periodic heptad repeat sequence that is characteristic of coiled coil proteins as well as additional periodicities required for its interaction with the actin filament, where each periodic repeat interacts with one actin molecule. This review addresses the role of periodic features of the Tm molecule in carrying out its universal functions of binding to the actin filament and its regulation and the specific features that may determine the isoform specificity of tropomyosins.
Project description:Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F-Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.
Project description:Tropomyosins are coiled-coil proteins that bind actin filaments and regulate multiple cytoskeletal functions, including actin network dynamics near the leading edge of motile cells. Previous work demonstrated that tropomyosins inhibit actin nucleation by the Arp2/3 complex and prevent filament disassembly by cofilin. We find that the Arp2/3 complex and cofilin, in turn, regulate the binding of tropomyosin to actin filaments. Using fluorescence microscopy, we show that tropomyosin (non-muscle Drosophila Tm1A) polymerizes along actin filaments, starting from "nuclei" that appear preferentially on ADP-bound regions of the filament, near the pointed end. Tropomyosin fails to bind dendritic actin networks created in vitro by the Arp2/3 complex, in part because the Arp2/3 complex blocks pointed ends. Cofilin promotes phosphate dissociation and severs filaments, generating new pointed ends and rendering Arp2/3-generated networks competent to bind tropomyosin. Tropomyosin's attraction to pointed ends reflects a strong preference for conformations localized to that region of the filament and reveals a basic molecular mechanism by which lamellipodial actin networks are insulated from the effects of tropomyosin.
Project description:Cellular actin networks exhibit diverse filamentous architectures and turnover dynamics, but how these differences are specified remains poorly understood. Here, we used multicolor total internal reflection fluorescence microscopy to ask how decoration of actin filaments by five biologically prominent Tropomyosin (TPM) isoforms influences disassembly induced by Cofilin alone, or by the collaborative effects of Cofilin, Coronin, and AIP1 (CCA). TPM decoration restricted Cofilin binding to pointed ends, while not interfering with Coronin binding to filament sides. Different isoforms of TPM provided variable levels of protection against disassembly, with the strongest protection by Tpm3.1 and the weakest by Tpm1.6. In biomimetic assays in which filaments were simultaneously assembled by formins and disassembled by CCA, these TPM isoform-specific effects persisted, giving rise to filaments with different lengths and treadmilling behavior. Together, our data reveal that TPM isoforms have quantitatively distinct abilities to tune actin filament length and turnover.