Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 15;28(6):771-782.
doi: 10.1091/mbc.E16-07-0503. Epub 2017 Jan 11.

Relative Importance of β cyto- And γ cyto-actin in Primary Mouse Embryonic Fibroblasts

Affiliations
Free PMC article

Relative Importance of β cyto- And γ cyto-actin in Primary Mouse Embryonic Fibroblasts

Xiaobai Patrinostro et al. Mol Biol Cell. .
Free PMC article

Abstract

The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin.

Figures

FIGURE 1:
FIGURE 1:
Adenoviral Cre efficiently ablated βcyto- and γcyto-actin in primary MEFs. (A, B) Representative images of βcyto-actin CT and βcyto-actin KO cells at 7 dpi. Scale bar, 20 µm. (C–F) Representative qRT-PCR analysis of βcyto- and γcyto-actin transcript amount (picomoles) in ActbL/L, Actg1L/L, and ActbL/L Actg1L/L MEFs. (G–I) Representative relative Western blot analysis of MEF lysates probed with βcyto-actin and γcyto-actin antibodies; GAPDH served as loading control.
FIGURE 2:
FIGURE 2:
βcyto-actin deficient MEFs were growth impaired. (A–C) Growth curve analysis of ActbL/L, Actg1L/L, and ActbL/LActg1L/L MEFs treated with either Ad5-GFP or Ad5-Cre (n = 3, hand counted in duplicate). *p < 0.05, ***p < 0.001. Two-way ANOVA with Bonferroni posttest; error bars are SEM.
FIGURE 3:
FIGURE 3:
βcyto-Actin–deficient MEFs displayed lower ATP levels but maintained ETC protein abundance. (A) ATP levels for CT and KO MEFs determined through a luciferase assay at 7 dpi. Results were normalized to cell number and relative to the paired embryo control (6 ≥ n ≥ 3). (B, C) Western blot and relative quantification of protein levels of succinate dehydrogenase complex, subunit A, flavoprotein (SDHA) for complex II; ubiquinol-cytochrome C reductase core protein I (UQCR) for complex III; mitochondrially encoded cytochrome C oxidase I (MTCO1) for complex IV; and ATP5a ATP synthase, H+ transporting, mitochondrial F1 complex, α-subunit (ATP5A) for the F1/F0 ATPase; GAPDH served as loading control. Levels were normalized to GAPDH and relative to the paired embryo control, which was set at 1 (n = 3). (D) Oxygen consumption rate of CT and KO MEFs at 7 dpi. Levels normalized to background. ***p < 0.001. One sample t test; error bars are SEM.
FIGURE 4:
FIGURE 4:
αsm-Actin transcript was up-regulated in βcyto-actin–ablated MEFs. (A–C) qRT-PCR analysis of six mouse actin isoforms in CT and KO MEFs at 5 dpi (n = 3, in triplicate). Calculated transcript amount (picomoles) were calculated based on the standard curve, amplified in parallel. *p < 0.05, ***p < 0.001. Two-way ANOVA with Bonferroni posttest; error bars are SEM.
FIGURE 5:
FIGURE 5:
αsm-Actin protein was up-regulated in cytoplasmic ablated MEFs. (A) Representative Western blot of CT and KO MEF lysates blotted with αsm-actin, βcyto-actin, and γcyto-actin antibodies; GAPDH served as loading control. (B–D) Quantitative Western blot analysis of CT and KO MEF lysates (n = 3). Calculated protein concentrations (nanograms/microliter lysate) were determined based on the standard curve blotted in parallel. *p < 0.05, **p < 0.01, and ***p < 0.001. Two-way ANOVA with Bonferroni posttest; error bars are SEM.
FIGURE 6:
FIGURE 6:
Unequal Actb/Actg1 transcript and protein ratios in primary MEFs. (A–C) Calculated transcript and protein ratios between βcyto- and γcyto-actin in CT MEFs (n = 3). Calculations were based on the qRT-PCR and quantitative Western blot data; γcyto-actin was set at 1. ***p < 0.001. One-sample t test; error bars are SEM.
FIGURE 7:
FIGURE 7:
αsm-Actin protein was up-regulated in siRNA-mediated βcyto-actin knockdown MEFs. (A) Representative Western blot of CT and βcyto- and/or γcyto-actin KD MEF lysates at 3 dpi for single KD and 4 dpi for dKD blotted with αsm-actin, βcyto-actin, and γcyto-actin antibodies; GAPDH served as loading control. (B–D) Quantitative Western blot analysis of control and KD MEF lysates (n = 3). Calculated protein concentrations (nanograms/microliter lysate) were determined based on the standard curve, blotted in parallel. ***p < 0.001. Two-way ANOVA with Bonferroni posttest; error bars are SEM.
FIGURE 8:
FIGURE 8:
The βcyto- and/or γcyto-actin–ablated MEFs displayed increased stress fiber thickness. (A–F) Representative images of phalloidin-stained actin filaments in Ad5-GFP– or Ad5-Cre–treated primary ActbL/L, Actg1L/L, and ActbL/LActg1L/L MEFs imaged at 7, 7, and 9 dpi, respectively. Scale bar, 20 µm. (G) Representative line scans of actin fluorescence across the dKO cells in E and F. Arrows denote sample valley and peak. (H) Quantification of differences between valley:peak ratios for all genotypes (n ≥ 8). *p < 0.05, ***p < 0.001. Two-way ANOVA; error bars are SEM.
FIGURE 9:
FIGURE 9:
Caldesmon smooth muscle isoform protein expression was up-regulated in βcyto-actin–deficient MEFs. (A) Representative Western blot analysis of CT and KO MEFs blotted with caldesmon (Cald1sm), CNN1, Sm22α, MLC2, and pMLC2; GAPDH served as loading control. (B–D) Relative protein expression normalized to GAPDH and relative to the paired embryo control. *p < 0.05, ***p < 0.001. One-sample t test, error bars are SEM.
FIGURE 10:
FIGURE 10:
SRF activity but not protein was up-regulated in βcyto-actin–ablated MEFs. (A) Representative Western blot analysis of CT and KO MEFs blotted with MRTF-A and SRF; GAPDH served as loading control. (B, C) Relative protein expression normalized to GAPDH and relative to the paired embryo control (n = 3). (D) Calculated fold increase in SRF activity, via luciferase assay, in KO over CT MEFs (n = 3). *p < 0.05, **p < 0.01. One-sample t test; error bars are SEM.

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

References

    1. Almuzzaini B, Sarshad AA, Rahmanto AS, Hansson ML, Von Euler A, Sangfelt O, Visa N, Farrants AKO, Percipalle P. In β-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects. FASEB J. 2016;30:2860–2873. - PubMed
    1. Belyantseva IA, Perrin BJ, Sonnemann KJ, Zhu M, Stepanyan R, McGee J, Frolenkov GI, Walsh EJ, Friderici KH, Frideman TB, et al. Gamma-actin is required for cytoskeletal maintenance but not development. Proc Natl Acad Sci USA. 2009;106:9703–9708. - PMC - PubMed
    1. Bergeron SE, Zhu M, Thiem SM, Friderici KH, Rubenstein P A. Ion-dependent polymerization differences between mammalian beta- and gamma-nonmuscle actin isoforms. J Biol Chem. 2010;285:16087–16095. - PMC - PubMed
    1. Bunnell TM, Burbach BJ, Shimizu Y, Ervasti JM. β-Actin specifically controls cell growth, migration, and the G-actin pool. Mol Biol Cell. 2011;22:4047–4058. - PMC - PubMed
    1. Bunnell TM, Ervasti JM. Delayed embryonic development and impaired cell growth and survival in Actg1 null mice. Cytoskeleton. 2010;67:564–572. - PMC - PubMed

LinkOut - more resources

Feedback