Relative importance of β_cyto- and γ_cyto-actin in primary mouse embryonic fibroblasts

Abstract

Both gene- and transcript-targeted ablations of Actb expression demonstrate that βcyto-actin is more disruptive than γcyto-actin to primary fibroblast function. This is evident via a decrease in cell proliferation and cellular ATP and an increase in myofibroblast differentiation signaling and protein expression in Actb-null primary cells.

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms

“…Consistent with our measurements of oxygen consumption in isolated gastrocnemius muscles (Figure 8) and primary mouse embryonic fibroblasts [36], there was no difference between aged Actb-msCT and Actb-msKO mice in any measured parameter of whole body respiratory function (Figure 9). …”

“…Mitochondrial associated membranes (MAMs) are a functionally important interface between mitochondria and the sarco-/endo-plasmic reticulum involved in mitochondrial respiration, Ca 2+ crosstalk, cell death signaling, lipid metabolism, and mitochondrial dynamics [30–34]. Therefore, we biochemically isolated MAM, mitochondrial, and endoplasmic reticulum fractions from wildtype mouse liver [35] and performed western blot analysis to identify the fractions containing β cyto - and γ cyto -actin immunoreactivity using previously established antibodies specific to each isoform (Figure 4) [24,12,36,37]. As expected, both γ cyto - and β cyto -actin were present in the cytosolic fraction (verified by tubulin immunoreactivity), weakly present in the crude mitochondrial fraction (identified by cytochrome C), and most enriched in the isolated MAM fraction verified by the presence of FACL4, a lipid enzyme localized to the MAM [38,39].…”

“…Both the endoplasmic reticulum and actin have recently been shown to play key roles in mitochondrial fission in many cell types [25,26,41], however, mitochondrial dynamics are challenging to measure in adult skeletal muscle fibers [42]. Therefore, we imaged mitochondria in primary mouse embryonic fibroblasts (MEFs) homozygous for floxed Actb or Actg1 alleles which were treated with adenovirus-5 CRE to knockout the target gene or adenovirus-5 GFP as a control [36]. MEFs ablated for either β cyto -actin, γ cyto -actin, or both actins displayed individual mitochondria which were significantly larger by surface area compared to mitochondrial size in control MEFs (Figure 5 A–G).…”

“…On the seventh (Actb L/L , Actg1 L/L MEFs) or ninth (Actb L/L /Actg1 L/L MEFs) day post infection cells showed complete knockout of protein by western blot as previously published [36]. Once knockout was confirmed by western blot, MEFs were stained with 0.01nM MitoTracker CMXRos (ThermoFisher Scientific M7512; Waltham, MA, USA) in Opti-MEM with 10%FBS for 15 min.…”

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels

“…In many organisms, reduction or loss of a cytoplasmic actin isoform is compensated for by increased expression of other cytoplasmic or smooth muscle isoforms. In mammalian cells, knock out or depletion of β‐actin leads to increased expression of cytoplasm γ‐actin and smooth muscle actins (Patrinostro et al, ; Shagieva et al, ; Tondeleir et al, ). Depletion of cytoplasmic γ‐actin also leads to increased expression of smooth muscle actin (Patrinostro et al, ).…”