Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins

Carman, Peter J.; Barrie, Kyle R.; Domínguez, Roberto

doi:10.1016/j.jbc.2021.101154

Cited by 12 publications

(9 citation statements)

References 94 publications

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“…Construct His-FLAG-actin was obtained using overlap extension PCR to add the sequence encoding for a His-FLAG-TEV affinity/cleavage tag (MAHHHHHH-SG-DYKDDDDK-TS-ENLYFQ) N-terminally to the α-actin cDNA [lacking the first two N-terminal residues that are removed posttranslationally ( 10 )]. The construct was inserted between the SalI and BamHI sites of vector pJCX4 (Addgene ID: 170756) ( 47 , 48 ). A SpeI site was silently inserted into the TEV-encoding sequence, and other actin isoforms (β cytoplasmic and γ smooth) were inserted between the SpeI and BamHI sites.…”

Section: Methodsmentioning

confidence: 99%

A solution to the long-standing problem of actin expression and purification

Ceron

Carman

Rębowski

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.

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Section: Methodsmentioning

confidence: 99%

A solution to the long-standing problem of actin expression and purification

Ceron

Carman

Rębowski

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…A recent study showed that different mammalian Tpm isoforms associate with actin filaments with different dynamics, and compete with other actin-binding proteins in an isoform-specific manner in vitro (Gateva et al, 2017). Further, tropomyosins contain post-translational modifications (Carman et al, 2021). In fission yeast, phosphorylation of S125 in Cdc8 was shown to significantly reduce its apparent actin Cdc12 from its roles in contractile ring assembly (Bohnert et al, 2013;Willet et al, 2018), but no studies show direct phosphorylation of For3 (of 35 publications mentioning For3 listed on PubMed.gov).…”

Section: Discussionmentioning

confidence: 99%

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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“…These Tpms contain an acetylation-mimic Met-Ala-Ser sequence in the N-terminus. Tpms purified from mammalian cells that harbor native post-translational modifications, however, display slightly different affinities on actin filaments (Carman et al, 2021). Thus, also the dynamics of native Tpms on actin filaments may exhibit small differences from the ones produced in E. coli .…”

Section: Discussionmentioning

confidence: 99%

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

Selvaraj

Kokate

Reggiano

et al. 2022

Preprint

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SUMMARYThe 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 major groove of the actin filament, providing an explanation for their incapability to co-polymerize on actin filaments. The structures and biochemical work 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-catalysed severing.

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Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins

Cited by 12 publications

References 94 publications

A solution to the long-standing problem of actin expression and purification

A solution to the long-standing problem of actin expression and purification

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

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

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