1. Biochemistry and Chemical Biology
  2. Cell Biology
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Diverse functions of homologous actin isoforms are defined by their nucleotide, rather than their amino acid sequence

  1. Pavan Vedula
  2. Satoshi Kurosaka
  3. Nicolae Adrian Leu
  4. Yuri I Wolf
  5. Svetlana A Shabalina
  6. Junling Wang
  7. Stephanie Sterling
  8. Dawei W Dong
  9. Anna Kashina  Is a corresponding author
  1. University of Pennsylvania, United States
  2. National Institutes of Health, United States
  3. Perelman School of Medicine, University of Pennsylvania, United States
Research Article
Cite as: eLife 2017;6:e31661 doi: 10.7554/eLife.31661
5 figures, 1 table and 6 additional files

Figures

Figure 1 with 2 supplements
β -coded γ -actin (Actbc-g) mice exhibit no phenotypic changes compared to control.

(A) CRISPR/Cas9 editing strategy used to generate Actbc-g mouse. (B) photos of Actbc-g E12.5 mouse embryos, with genotypes indicated. (C) photos of Actbc-g mice after gene editing, alone (top left) and next to age-matched (top right) and littermate wild type (WT) (bottom). Three mice from two different litters are shown. (D) H&E-stained sagittal sections of the heads (top) and bodies (bottom) of littermate P0 wild type (WT) and Actbc-g mice. Scale bar, 1 mm.

https://doi.org/10.7554/eLife.31661.003
Figure 1—figure supplement 1
Generation of Actbc-g mouse.

Top left, genotyping strategy: editing of the N-terminal codons of the β-actin gene abolishes an EcoRV restriction site, enabling the screening of the edited gene variants by EcoRV digestion of the PCR-generated DNA fragments corresponding to the 5′ of the actin sequence. Top right, PCR products before (top) and after (bottom) EcoRV digestion. Bottom, western blots of wild type, heterozygous, and Actbc-g mouse tail lysates with antibodies to β- and γ- actin.

https://doi.org/10.7554/eLife.31661.004
Figure 1—figure supplement 2
Sequencing result for wild type Actb and the edited Actbc-g alleles.

Screen shots from the Chromas sequence viewing software.

https://doi.org/10.7554/eLife.31661.005
Figure 2 with 5 supplements
Actb gene editing abolishes β-actin protein from multiple organs and is accompanied by up-regulation of γ-actin without changing the total actin levels.

Western blot analysis showing images (left) and quantifications (right) of whole tissue lysates from wild type (Actb+/+) and Actbcg mice. Fluorescence images obtained from the Odyssey gel imager are shown. For quantification, total fluorescence from the 43 kDa actin band was normalized to the loading control and to the actin level in the first lane for each blot. Error bars represent SEM, n = 3.

https://doi.org/10.7554/eLife.31661.006
Figure 2—figure supplement 1
Actin levels in Actbc-g mice are similar to control.

Western blots of wild type, heterozygous, and Actbc-g mouse brain lysates probed with antibodies to β- and γ-actin and total actin (pan-actin). Mouse genotypes are indicated on top of each lane, and the antibodies used are listed below each blot.

https://doi.org/10.7554/eLife.31661.007
Figure 2—figure supplement 2
2D gel distribution of actin isoforms in wild type (top) and Actbc-g (bottom) mouse tissue lysates.
https://doi.org/10.7554/eLife.31661.008
Figure 2—figure supplement 3
Generation of Actg1c-b mouse.

Top, genotyping strategy: editing of the N-terminal codons of theγ- actin gene generates an EcoRV restriction site, enabling the screening of the edited gene variants by EcoRV digestion of the PCR-generated DNA fragments corresponding to the beginning of the actin sequence. Bottom, PCR products before (top) and after (bottom) EcoRV digestion.

https://doi.org/10.7554/eLife.31661.009
Figure 2—figure supplement 4
γ-coded β-actin (Actg1c-b mice exhibit no phenotypic changes compared to control.

Top, CRISPR/Cas9 editing strategy used to generate Actg1c-b mouse. Bottom left, photos of Actg1c-b mouse after gene editing, to age-matched wild type. Bottom right, H and E-stained sagittal sections of the heads (top) and bodies (bottom) of littermate wild type (WT) and Actg1c-b mice. Scale bar, 1 mm.

https://doi.org/10.7554/eLife.31661.010
Figure 2—figure supplement 5
Partial editing of the γ-actin gene to encode β-actin-like protein abolishes γ-actin protein from multiple organs.

Western blots with the actin antibodies indicated on the left using tissue homogenates from wild type control and Actg1c-b mouse.

https://doi.org/10.7554/eLife.31661.011
Mouse embryonic fibroblasts derived from Actbc-g mice have normal actin cytoskeleton, despite complete lack of β-actin.

Top, quantification of total F-actin detected by Phalloidin-AlexaFluor488 staining of wild type (Actb+/+) and Actbc-g primary mouse embryonic fibroblasts. Numbers were averaged from 69 cells in WT and 76 cells in Actbc-g, obtained from two different primary cultures independently derived from two different littermate embryos for each set. Bottom, representative images of both cell types stained with Phalloidin-AlexaFluor488 or antibodies to both actin isoforms as indicated.

https://doi.org/10.7554/eLife.31661.012
Mouse embryonic fibroblasts show no major changes in morphology and actin distribution.

Representative images of wild type (WT) and Actbc-g primary mouse embryonic fibroblasts stained with antibodies to both actin isoforms as indicated.

https://doi.org/10.7554/eLife.31661.013
Figure 5 with 2 supplements
Mouse embryonic fibroblasts derived from Actbc-g mice migrate at normal rates.

Left, phase contrast images of the first (0′) and last (600′) frame taken from a representative time lapse videos of the WT and Actbc-g cells at the edge of a monolayer migrating into an infinite scratch wound. Overlay of the two frames is shown in the bottom row. Scale bar, 100 µm. Right top, quantification of the cell migration rate as μm2/min, WT: n = 28, Actbc-g: n = 29 averaged from two independently derived primary cultures for each set. See Supplemental Videos 1 and 2. Right bottom, quantification of cell directionality in single cell migration assays (calculated as persistence over time, WT: n = 49, Actbc-g: n = 50) suggests that single cell migration was not affected in Actbc-g cells.

https://doi.org/10.7554/eLife.31661.014
Figure 5—video 1
Time lapse video showing migration of control fibroblasts into the infinite scratch wound.
https://doi.org/10.7554/eLife.31661.015
Figure 5—video 2
Time lapse video showing migration of Actbc-g fibroblasts into the infinite scratch wound.
https://doi.org/10.7554/eLife.31661.016

Tables

Table 1
Severity of the actin isoform knockout phenotypes and their cross-compensation for each other correlate with their ribosome density.

See (Perrin and Ervasti, 2010) for the references on the isoform knockout data. * From heterozygotes (since homozygous knockout is embryonic lethal) and knockout MEFs. Tissue specific upregulation of different isoforms See (Bunnell and Ervasti, 2010).

https://doi.org/10.7554/eLife.31661.017
NameGene symbolNCBI accession number, proteinNCBI accession number, mRNAComposite ribosome densityMouse knockout phenotypeOther actin isoforms upregulated upon knockout
β-cytoplasmic actinActbNP_031419NM_0073931351.607Early embryonic lethalityActa2; some Actg1*
α-smooth muscle actinActa2NP_031418NM_00739253.781Viable, with vascular contractility and blood pressure defectsActa1
α-skeletal actinActa1NP_033736NM_00960610.267Muscle weakness; postnatal lethalityActa2 and Actc1
α-cardiac actinActc1NP_033738NM_0096083.872Perinatal lethalityActa2 and Acta1
γ-cytoplasmic actinActg1NP_033739NM_0096091.289Viable, with growth defects and progressive deafnessActa2, Actb, Acta1, and Actc1
γ-enteric smooth muscle actinActg2NP_033740NM_0096100.377UnknownUnknown
Table 1—source data 1

Composite ribosome profiling data for the actin gene family, plotted in logarithmic scale.

Bottom panel shows the coarse curves for the data on top.

https://doi.org/10.7554/eLife.31661.018
Table 1—source data 2

Ribosome profiling data for the individual members of the actin family, plotted in logarithmic scale.

https://doi.org/10.7554/eLife.31661.019
Table 1—source data 3

Predictions of the secondary structures for β− and γ− actin coding sequences.

Plot shows distance in nucleotides (x axis, 0 indicates the first ATG of the coding sequence) versus free energy (y axis).

https://doi.org/10.7554/eLife.31661.020
Table 1—source data 4

Predictions for the initial regions of the β− and γ− actin coding sequence, compared to their codon-switched versions.

Plot shows distance in nucleotides (x axis, 0 indicates the first ATG of the coding sequence) versus free energy (y axis). β-coded γ− actin mRNA is predicted to have a more relaxed structure around the translation initiation site, while being indistinguishable throughout the rest of the sequence.

https://doi.org/10.7554/eLife.31661.021

Additional files

Supplementary file 1

Mouse families of homologous protein isoforms showing the highest differences in ribosome densities between the members of each family.

Families are grouped by function, starting with the most abundant ones.

https://doi.org/10.7554/eLife.31661.022
Supplementary file 2

Human families of homologous protein isoforms showing the highest differences in ribosome densities between the members of each family.

Families are grouped by function, starting with the most abundant ones.

https://doi.org/10.7554/eLife.31661.023
Supplementary file 3

Zebrafish families of homologous protein isoforms showing the highest differences in ribosome densities between the members of each family.

Families are grouped by function, starting with the most abundant ones.

https://doi.org/10.7554/eLife.31661.024
Supplementary file 4

Drosophila families of homologous protein isoforms showing the highest differences in ribosome densities between the members of each family.

Families are grouped by function, starting with the most abundant ones.

https://doi.org/10.7554/eLife.31661.025
Supplementary file 5

C.elegans families of homologous protein isoforms showing the highest differences in ribosome densities between the members of each family.

Families are grouped by function, starting with the most abundant ones.

https://doi.org/10.7554/eLife.31661.026
Transparent reporting form
https://doi.org/10.7554/eLife.31661.027

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