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. 2018 Apr 24;115(17):4399-4404.
doi: 10.1073/pnas.1718336115. Epub 2018 Mar 26.

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility

Adrian Drazic  1   2 , Henriette Aksnes  1   2 , Michaël Marie  1   2 , Malgorzata Boczkowska  3 , Sylvia Varland  1   2 , Evy Timmerman  4   5   6 , Håvard Foyn  1 , Nina Glomnes  1   7 , Grzegorz Rebowski  3 , Francis Impens  4   5   6 , Kris Gevaert  4   6 , Roberto Dominguez  3 , Thomas Arnesen  8   2   9
Affiliations

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility

Adrian Drazic et al. Proc Natl Acad Sci U S A. .

Abstract

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Keywords: N-terminal acetylation; NAA80; NAT; actin; cell motility.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NAA80 Nt-acetylates β- and γ-actin. (A) NAA80-V5 was immunoprecipitated from HeLa cells and used in Nt-acetylation assays with [14C]-Ac-CoA and peptides representing a variety of N termini. (B) NAA80-eGFP expressed in HeLa and HAP1 cells shows a cytosolic distribution. (C) Phosphorescence imaging shows a distinct 43-kDa band in NAA80 KO1 cell lysates incubated with [14C]-Ac-CoA and purified recombinant MBP-NAA80, suggesting the incorporation of [14C]-Ac at the N terminus of actin. (D) The isoform and Nt-acetylation specificity of Ac-β-actin and Ac-γ-actin antibodies is confirmed in dot blot assays with peptides representing the unacetylated and acetylated N termini of β- and γ-actin. (E) NAA80 expression is required for actin Nt-acetylation. Immunoblot analysis of HAP1 control and NAA80 KO1 cells transfected with empty V5 plasmid, wild-type NAA80-V5, or the catalytically inactive mutant NAA80mut-V5. The samples were probed with Ac-β-actin, Ac-γ-actin, pan-actin, V5, and GAPDH antibodies. (F) Actin acetylation is eliminated in NAA80-KO cells and restored upon reexpression of active NAA80. HAP1 control cells were transfected with an empty eGFP vector, and NAA80 KO1 cells were additionally transfected with NAA80-eGFP or NAA80mut-eGFP and stained with Ac-β-actin or Ac-γ-actin antibodies and analyzed by IF. The general presence of F-actin in the analyzed cells was visualized by Rhodamine phalloidin staining (Bottom Row). Of note, the cells shown here are from separate samples not subjected to antibody labeling. (Scale bars, 10 µm.)
Fig. 2.
Fig. 2.
Actin Nt-acetylation affects cell motility. (A) Representative images of HAP1 control (Ctrl) and NAA80 KO1 cells in the wound-healing assay showing the degree of gap closure after 18 h. (Scale bar, 150 μm.) (B) Quantification of gap size in wound-healing assays relative to the size at t0. (C) Average cell-front velocity calculated from B. (D) Chemotaxis assay showing the percentage of cells that migrated from a 1% FBS chamber to a 10% FBS chamber (see also Fig. S4). (E) Random migration assay showing the percentage of cells migrating from a 10% FBS chamber to a 10% FBS chamber. ***P ≤ 0.001, two-sided Student’s t test. Data are shown as the means ± SEM.
Fig. 3.
Fig. 3.
Effects of actin Nt-acetylation on cytoskeletal morphology. (A and B) Representative micrographs illustrating a differential morphological phenotype at the level of filopodia between HAP1 control (Ctrl) (A) and NAA80 KO1 (B) cells where the absence of NAA80 promotes filopodia formation. (C and D) For both cell lines, the number (C) and length (D) of filopodia were determined. (E) G/F-actin ratios from HAP1 control (blue) and NAA80 KO1 (orange) cells. (F and G) Confocal images (Upper) and stimulated emission depletion microscopy (STED) zoom-in frames (Lower) of lamellipodia. The number of cells containing at least one clearly distinguishable lamellipodium supported by filopodia/microspikes was determined. Since HAP1 cells typically grow in clusters, only cells at the borders of clusters (i.e., with a front facing away from the cluster) were considered, as indicated by the numbering. Lamellipodia-positive cells are indicated by green numbers. (H) Quantification of lamellipodia-positive cells. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, Student’s t test. Data are shown as the means ± SEM.
Fig. 4.
Fig. 4.
Actin Nt-acetylation affects filament elongation and depolymerization. (A) Cytoplasmic actin (a mixture of β and γ isoforms) purified from wild-type and NAA80-KO cells was analyzed by SDS/PAGE and Western blotting using isoform- and Nt-acetylation–specific Ac-β-actin and Ac-γ-actin antibodies. Nt-acetylated α-skeletal actin (15) is shown as reference. (B) The polymerization rate of β/γ-actin alone is unchanged with or without Nt-acetylation. Data are shown as the average curve from five independent experiments (as indicated), with SD error bars in lighter color (Ac-actin, blue; non–Ac-actin, red). (C) The barbed-end elongation rate of β/γ-actin filament seeds is ∼2.2-fold faster for Ac-actin (blue) than for non–Ac-actin (red). The polymerization of Ac-actin (black) and non–Ac-actin (gray) in the absence of seeds is shown as control. (D) The depolymerization rate of β/γ-actin filament seeds is ∼1.7-fold faster for Ac-actin (blue) than for non–Ac-actin (red). (E) The concentration dependence of polymerization rates of actin assembly by Arp2/3 complex (nucleation and branching) shows no difference for Ac-actin (blue) vs. non–Ac-actin (red) (see also Fig. S7B). (F) The concentration dependence of polymerization rates of actin assembly induced by mDia2 (nucleation and barbed-end capping) is unchanged with or without Nt-acetylation (see also Fig. S7C). (G) The concentration dependence of polymerization rates of actin assembly induced by mDia1 (nucleation and barbed-end elongation) is >twofold faster for Ac-actin (blue) than for non–Ac-actin (red) (see also Fig. S7D). (H) The elongation rate of filament seeds by mDia1 from profilin1-actin is ∼35% faster for Ac-actin (blue) than for non–Ac-actin (red). For both actins, the elongation rate of mDia1 from profilin1-actin is faster than the elongation rate of β/γ-actin seeds alone (shown as controls). (I) In contrast, barbed-end elongation by mDia1 from profilin2-actin is unchanged for non–Ac-actin (red) and is reduced ∼40% for Ac-actin (blue) relative to the elongation rate of β/γ-actin seeds. Rates are reported as the mean ± SEM of the number of independent experiments indicated in each panel.
Fig. 5.
Fig. 5.
NAA80 expression is evolutionarily linked to a unique processing mechanism of actin’s N terminus. (A) Processing pathway of animal actins, including an initial step of cotranslational acetylation catalyzed by NatB, which is shared with other eukaryotic proteins beginning with Met-Asp/Met-Glu. (B) Taxonomic tree showing the coevolution of NAA80 and an acidic actin N terminus. For each species, filled red circles indicate the presence of an acidic N terminus in mature actin, and filled blue circles indicate the expression of Naa80. The full names of the organisms are given in Table S2.
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Comment in

  • NATure of actin amino-terminal acetylation.
    Rubenstein PA, Wen KK. Rubenstein PA, et al. Proc Natl Acad Sci U S A. 2018 Apr 24;115(17):4314-4316. doi: 10.1073/pnas.1803804115. Epub 2018 Apr 9. Proc Natl Acad Sci U S A. 2018. PMID: 29632202 Free PMC article. No abstract available.

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