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. 2018 Jul 31;115(31):7973-7978.
doi: 10.1073/pnas.1807895115. Epub 2018 Jul 16.

Essential Nucleotide- And Protein-Dependent Functions of Actb/β-actin

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Free PMC article

Essential Nucleotide- And Protein-Dependent Functions of Actb/β-actin

Xiaobai Patrinostro et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.

Keywords: auditory hair cell; gene edited; stereocilia; β-actin; γ-actin.

Conflict of interest statement

Conflict of interest statement: D.F.V. is a named inventor on several patents involving the use of transcription activator-like effector nucleases (TALENs) to create targeted genome modifications.

Figures

Fig. 1.
Fig. 1.
Actbc-g transcript is synthesized from the edited Actb locus and correlates with a twofold increase in γ-actin protein expression. (AC) Calculated quantity of isoactin transcript via qRT-PCRs in brain (n = 4 mice), lung tissues (n = 4 mice), and MEFs (n = 3 embryos). The x axis denotes actin isoform; the y axis denotes transcript in pmol. (DF) Representative Western blots in brain, lung, and MEFs. (GI) Calculated relative isoactin protein expression in WT, heterozygous, and homozygous Actbc-g mice brain (n = 4 mice), lung (n = 4 mice), and MEFs (n ≥ 3 embryos). The x axis denotes actin isoform; the y axis denotes relative protein expression which was normalized to GAPDH and relative to a WT sample. Two-way ANOVA with Bonferroni posttest was performed. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2.
Fig. 2.
Actbc-g MEFs display cell proliferation and random migration rates not different from WT. (A) Representative immunofluorescence images of WT MEFs and Actbc-g MEFs. γ-Actin is labeled in red, β-actin is labeled in green, nucleus is labeled in blue, and the merged image is on the Right. (Scale bar: 50 μm.) (B) MEF growth curve analysis, cultured from 0 to 9 d. n = 3 embryos. (CF) Calculated average velocity, linear distance, total distance, and directionality from random migration assay. n ≥ 4 embryos, n ≥ 40 cells per genotype. (G and H) G-to F-actin ratio in WT and Actbc-g MEFs. The x axis denotes actin isoform; the y axis denotes G- to-F-actin ratio. n = 3 embryos. (I and J) Representative and calculated relative isoactin protein expression in WT, homozygous Actbc-g MEFs for SRF and MRTF-A. n = 3 embryos. The x axis denotes actin isoform; the y axis denotes relative protein expression which was normalized to GAPDH and relative to a WT sample. Error bars are SD.
Fig. 3.
Fig. 3.
Actbc-g mice suffer from progressive hearing loss. (A) ABR of 6-wk-old WT and homozygous Actbc-g mice. n = 3 mice. (B) ABR of 6-mo-old WT and homozygous Actbc-g mice. n = 4 mice. The x axis denotes defined frequency in kilohertz; the y axis denotes threshold (in decibels) sound that elicits an ABR. Student t test. *P < 0.05, ***P < 0.001. Error bars are SD.
Fig. 4.
Fig. 4.
β-Actin and γ-actin colocalized in developing OHC stereocilia. (A) Representative immunofluorescent images of the OHC in P7 and P21 C57BL/6 mice. β-Actin is labeled in green, γ-actin is labeled in red. (B) Representative immunofluorescent images of the IHC in P7 and P21 C57BL/6 mice. β-Actin is labeled in green, γ-actin is labeled in red. (C) Calculated fluorescent intensity of β- and γ-actin normalized to the shaft in the OHC. n = 3 mice, n ≥ 40 stereocilia. (D) Calculated fluorescent intensity of β- and γ-actin normalized to the shaft in the IHC. (Scale bar: 1 µm.) n = 3 mice, n ≥ 40 stereocilia. Error bars are SEM.
Fig. 5.
Fig. 5.
Actbc-g mice show evidence of stereocilia degeneration. Representative scanning electron microcopy images of (A) 6-wk-old and (B) 6-mo-old OHC stereocilia from the middle turn of the cochlea. (Scale bar: 1 µm.) (C) Cartoon image of the stereocilia. (D) Length (in micrometers) of stereocilia in row 3 of OHC bundles in 6-wk-old and 6-mo-old WT and homozygous Actbc-g mice. n = 4 mice, n ≥ 200 stereocilia. Number of stereocilia in row 3 of OHC bundles in 6-wk-old and 6-mo-old WT and homozygous Actbc-g mice. n = 4 mice, n ≥ 28 cells. One-way ANOVA with Tukey’s posttest was performed. ***P < 0.001. Error bars are SD.

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