Distinct actin–tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes

Reindl, Theresia; Giese, Sven; Greve, Johannes N.; Reinke, P.; Chizhov, Igor; Latham, Sharissa L.; Mulvihill, Daniel P.; Taft, Manuel H.; Manstein, Dietmar J.

doi:10.1016/j.isci.2022.104484

Cited by 17 publications

(27 citation statements)

References 107 publications

(154 reference statements)

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“…Given that we found no significant differences between muscle and S. pombe actin in motility assays employing Myo2 and Myo51, there may be key structural differences in how these two yeasts have diverged over ∼400 million years since their common ancestor (Sipiczki, 2000). Work from the Manstein laboratory has suggested that skeletal-α and cytoplasmic -β and -γ actins can have differential impacts on the kinetics of mammalian non-muscle myosin-II isoforms but not on Myosin-IC (Müller et al, 2013; Reindl et al, 2022), supporting our findings that some myosins are insensitive to actin isoform differences. Additionally, Tpm isoforms further differentially fine-tune class-specific myosin motor activities in their associated structures in mammalian cells (Reindl et al, 2022).…”

Section: Discussionsupporting

confidence: 88%

“…Work from the Manstein laboratory has suggested that skeletal-α and cytoplasmic -β and -γ actins can have differential impacts on the kinetics of mammalian non-muscle myosin-II isoforms but not on Myosin-IC (Müller et al, 2013; Reindl et al, 2022), supporting our findings that some myosins are insensitive to actin isoform differences. Additionally, Tpm isoforms further differentially fine-tune class-specific myosin motor activities in their associated structures in mammalian cells (Reindl et al, 2022). Intriguingly, recent work has suggested that sequence differences in actin ortholog at the DNA/gene level can have a greater impact on growth and survival of S. cerevisiae mutants than the protein sequence per se (Boiero Sanders et al, 2022).…”

Section: Discussionsupporting

confidence: 88%

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Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

Tang

Pollard

Homa

et al. 2022

Preprint

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It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds (Coulton et al., 2010). Such a mechanism would have a profound impact on how actin networks self-segregate to perform different cellular functions, but direct biochemical evidence for this interaction is lacking. To address this knowledge gap, we employed 3-color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving S. pombe actin, acetylated and unacetylated Tpm, formins Cdc12 and For3, and myosins Myo2 and Myo51. We show that acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. This result is consistent with acetylation promoting end-to-end polymerization of Tpm but inconsistent with unacetylated Tpm functioning as an independent variant, especially at low concentrations. In disagreement with the differential recruitment model, fluorescently labeled Tpm, with or without acetylation, showed no preferential binding to filaments assembled by different formins, nor did Tpm binding have any bias towards the growing formin-bound barbed ends of filaments. We also showed that despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with the two actins, consistent with the structural finding that myosin interacts with a conserved footprint on actin (Robert-Paganin et al., 2021). Although our in vitro findings do not support a direct formin-tropomyosin interaction, there remains the possibility that formins bias differential tropomyosin isoform recruitment through an undiscovered mechanism involving additional proteins.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 88%

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

Tang

Pollard

Homa

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Work from the Manstein laboratory has suggested that skeletal-α and cytoplasmic -β and -γ actins can have differential impacts on the kinetics of mammalian nonmuscle myosin-II isoforms but not on Myosin-IC (Müller et al, 2013;Reindl et al, 2022), supporting our findings that some myosins are insensitive to actin isoform differences. Additionally, Tpm isoforms further differentially fine-tune class-specific myosin motor activities in their associated structures in mammalian cells (Reindl et al, 2022).…”

Section: Our Recent In Vitro Characterization Of Fission Yeast Myosinssupporting

confidence: 87%

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

et al. 2023

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It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino‐terminal acetylation that specifies which formin‐mediated F‐actin networks it binds, but with no supporting biochemistry. To address this mechanism in vitro, we developed methods for S. pombe actin expression in Sf9 cells. We then employed 3‐color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl‐Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1–FH2‐domains of two S. pombe formins, nor did Tpm binding have any bias towards the growing formin‐bound actin filament barbed end. Although our in vitro findings do not support a direct formin–tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.

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“…The coiled-coil tropomyosin dimers form head-to-tail polymers along the major groove of actin filaments, and are only known to be functional when bound to actin filaments 38,39 . The four tropomyosin genes in mammals, TPM1, TPM2, TPM3 and TPM4, can generate >40 Tpm isoforms through alternative splicing, and the different isoforms provide specific functional features to the associated actin filaments [40][41][42][43] . Several Tpm isoforms localize to actin stress fibers and regulate their assembly.…”

Section: Tpm16 and Tpm32 Generate Specific Actin Filament Layers Alon...mentioning

confidence: 99%

Specialized actin nanoscale layers control focal adhesion turnover

Kumari

Ven

Chastney

et al. 2023

Preprint

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Focal adhesions (FAs) connect inner workings of the cell to the extracellular matrix to control cell adhesion, migration, and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. However, cellular processes rely on precisely-regulated FA turnover, but the molecular machineries that control FA assembly and disassembly have remained elusive. By using super-resolution iPALM microscopy, we identified two unprecedented nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments beneath the previously identified actin-regulatory layer are critical for adhesion maturation and controlled cell motility, whereas the Tpm3.2-actin filament layer towards the bottom of FA facilitates adhesion disassembly. Mechanistically, Tpm3.2 stabilizes KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Loss of Tpm3.2 leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during cell migration. Thus, FAs are composed of at least three distinct actin filament layers, each having specific roles in coupling of adhesion to the cytoskeleton, or in controlling adhesion dynamics. In a broader context, these findings demonstrate how distinct actin filament populations can co-exist and perform specific functions within a defined cellular compartment.

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Distinct actin–tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes

Cited by 17 publications

References 107 publications

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

Specialized actin nanoscale layers control focal adhesion turnover

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