A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

Bonello, Teresa; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock‐DeGregori, Sarah E.; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

doi:10.1038/srep19816

Cited by 30 publications

(36 citation statements)

References 43 publications

(46 reference statements)

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“…Surprisingly, neither anti‐Tpm compound ATM‐1001 nor ATM‐4015 altered cellular morphologies, raising the question why the anticipated mesenchymal transition was not observed. The answer may be found in recent analyses which have revealed that while (TR100) perturbs Tpm3.1 regulated actin filament depolymerisation (Bonello et al, ), Tpm3.1 still associates with the actin filaments in the presence of the drug. This therefore may explain why anti‐Tpm3.1 drugs do not phenocopy all effects of genetic deletion.…”

Section: Discussionmentioning

confidence: 99%

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac‐mediated multicellular invasion

2018

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The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitises neuroblastoma cells to inhibition of Rac-mediated multicellular invasion.

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Section: Discussionmentioning

confidence: 99%

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac‐mediated multicellular invasion

2018

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“…Recombinant human non-muscle cofilin 1, WT, and the phosphomimetic S3D were expressed and purified from E. coli as described for WT cofilin (4), and final concentrations were determined spec-troscopically (54). Recombinant human tropomyosin Tpm3.1 (55) was purified as described (56). A 1 mM phalloidin stock (Thermo Scientific) was prepared in methanol.…”

Section: Protein Expression Purification and Labelingmentioning

confidence: 99%

Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

Elam

Cao

Kang

et al. 2017

Journal of Biological Chemistry

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Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification ( substitution with Asp or phosphorylation) "undocks" and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilin's ability to weaken longitudinal filament subunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filament mechanical properties and promote severing.

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“…Myosin Va and Tpm3.1 are widely-expressed in cells and are implicated in disease states including metastatic cancer, melanoma and type 2 diabetes, though we do not yet know if they function together in cells (Alves et al, 2013;Bonello et al, 2016;Chen et al, 2012;Kee et al, 2015). The myosin and tropomyosin pairs were selected such that they form both physiologically-relevant (striated muscle myosin II/Tpm1.1, myoVa/Tpm3.1) and non-physiologically-relevant (striated muscle myosin II/Tpm3.1, myoVa/Tpm1.1) pairs.…”

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Distinct sites in tropomyosin specify shared and isoform‐specific regulation of myosins II and V

et al. 2018

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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.

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A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

Cited by 30 publications

References 43 publications

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac‐mediated multicellular invasion

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac‐mediated multicellular invasion

Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

Distinct sites in tropomyosin specify shared and isoform‐specific regulation of myosins II and V

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