Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 11 (8), 255-71

Cells Lacking β-Actin Are Genetically Reprogrammed and Maintain Conditional Migratory Capacity

Affiliations

Cells Lacking β-Actin Are Genetically Reprogrammed and Maintain Conditional Migratory Capacity

Davina Tondeleir et al. Mol Cell Proteomics.

Abstract

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.

Figures

Fig. 1.
Fig. 1.
Deletion of β-actin induces actin isoform switching. A, Increased expression of γ-cytoplasmic actin in the β-actin−/− embryonic stem cells. Two-dimensional gel-electrophoresis followed by Western blotting of β-actin−/− and β-actin+/+ ES cell lysates, probed with a pan-actin antibody. B, Western blot with a pan-actin antibody, after two-dimensional gel-electrophoresis of lysates from E10.5 WT and β-actin KO embryos, shows increased levels of both α- and γ-actin isoforms. C–E, Immunohistochemistry of E10.5 WT (C), β-actin KO (D), and KI (E) embryos. β-actin staining is ubiquitous in WT and KI embryos with more intense signals in the tail (Tl), the limb buds (Hlb and Flb) and the olfactory bulb (Ob). β-actin KO embryos lack staining for β-actin as expected. The γ-cytoplasmic actin pattern is similar to that of β-actin in WT, but more intense in β-actin KO embryos. α-SMA is expressed in the WT in somites (S), heart (Ht), and around the dorsal aorta (Da), whereas in the β-actin KO it is ectopically expressed at high levels throughout the entire embryo. In the β-actin KI α-SMA is still enriched in several regions such as the head, trunk, and tail region. F, Western blot with actin isoform specific antibodies of wild-type WT (WT10), heterozygous (HET8), and homozygous β-actin KO (KO1 and KO2) and β-actin KI (KI) MEFs reveals increased expression of α- and γ-SMA and γ-cytoplasmic actin in KO1 and KO2. Total actin levels, measured with pan-actin antibody, remain constant. GAPDH staining was used as loading control for each Western blot (data not shown). G, WT10 MEFs treated with shRNA against β-actin (sh +) display increased α-SMA expression relative to control (c) treated WT10 MEFs. An untreated KO1 cell line (-) was used as positive control for α-SMA staining, α-tubulin is a loading control and M marker are proteins.
Fig. 2.
Fig. 2.
Impaired migration of β-actin KO MEFs because of decreased protrusion length and persistence. A, Random cell migration analysis reveals a decrease in the percentage of migrating KO cells (N >78 cells) (* p < 0.05.; ** p < 0.01 (B–E)). Kymograph analysis of β-actin KO MEFs shows a significant reduction in the length (C) and persistence (D) of protrusions compared with WT cells is measured. In addition, length and persistence of protrusions and retractions are the same in β-actin KO cells (gray bars: protrusion; black bars: retraction; N >78). In contrast, the lamellipodial protrusion and retraction rates are not significantly different between KO and WT MEFs (E). (F–G) Stably re-introducing the β-actin coding sequence with only the ZIP-code (ZIP) or with its full-length 3′UTR (UTR) in the KO1 cells did not affect the expression level of α-SMA, although β-actin expression was similar to WT untransfected cells (G). It also did not restore cell migration. The hull area, which is a measure of the area covered by the cell trajectories during the four hour recording (27), was invariable in the vector control cells (V), the cells expressing β-actin with ZIP-code and the cells expressing β-actin with 3′UTR (F). As control in the migration experiment WT and β-actin KI cells were used (Number of cells analyzed 190<N>353).
Fig. 3.
Fig. 3.
Differential protein expression profiling reveals reprogramming of β-actin KO MEFs into a myogenic phenotype. (A, B) Proteins with at least a twofold change in expression level in both KO1 and KO2, relative to WT10 cells (supplemental Table S2B), were subjected to IPA8.0 (for settings see legend supplemental Fig. S3D–G): the ten highest ranked physiological system and development functions (A) and canonical pathways (B) are shown (details in supplemental Table S3B and S3D, all functions and signaling pathways without metabolic pathways are in supplemental Fig. 3D and 3E). C, KEGG pathway analysis of the intersection of up-regulated proteins. The p value for each pathway was calculated via the hyper geometric test (see supplemental Materials and Methods). Pathways with p value > 0.975 are overrepresented (red), whereas pathways with p value < 0.025 are underrepresented (green). D, Software Tool for Researching Annotations of Proteins (STRAP) indicates an enrichment of cytoskeletal proteins in the intersection of the KO cells whereas there is a reduction of nuclear genes.
Fig. 4.
Fig. 4.
Scheme of the Rho signaling canonical pathway identified in Ingenuity Pathway Analysis of the proteins with altered expression in both KO cell lines. To this IPA pathway were added: ARHGDI (Rho-GDI1), ARHGDI2 (Rho-GDI2), ARHGEF1 (GEFh1), ARHGEF10 (GEF10) as these had altered expression in one or both cell lines based on proteomics or qRT-PCR (supplemental Fig. S4). Appropriate interactions were made using the connect option or the path explorer option. The figure was overlaid with the mass spectroscopy data: red represents up-regulated and green represents down-regulated proteins, groups of proteins are indicated with double circles.
Fig. 5.
Fig. 5.
Lack of β-actin leads to reprogramming of cells resulting in transcriptional and post-translational effects leading to actin reorganization. A, Increased levels of MLC2 and phospho-MLC2 in KO-MEFs as assessed by Western blotting. The protein bands were quantified and the values relative to WT (set at 1) to assess the fold expression (upper panel) or the fold increase in phosphorylation (lower panel) are given below the blots. B, Cellular reprogramming influences cell morphology (see also supplemental Fig. S5D). KO-MEFs display robust phalloidin stained F-actin stress fibers and form many large vinculin positive focal adhesions. C, FAs were measured and the number of FAs and supermature FAs per cell was calculated. FAs are considered supermature when their surface is larger than 8 μm2 (number of cells analyzed 10<N>21). D, β-actin KO cells spread and adhere faster to collagen as monitored by xCelligence technology (Roche) (for adhesion curves on other substrates, see supplemental Fig. S5A, B). E, Adhesion rates were calculated from the various slopes in D and supplemental Fig. S5A-B. F, β-actin KO1 MEFs display increased contractility in a wrinkling silicone substrates assay (24).
Fig. 6.
Fig. 6.
Scheme of the TGFβ1 dependent transcriptional and post-translational alterations in the β actin-KO cells (full lines direct interaction, activation, or inhibition, striped lines represent indirect interaction, activation, or inhibition). Proteins in bold have up-regulated expression or increased phosphorylation (P-protein). Proteins typical for smooth muscle cells are transcriptionally up-regulated and by their involvement in stress fiber and focal adhesion formation contribute to increased contractility. Focal adhesion protein Hic-5 functions in an autocrine TGFβ1 activity loop (58) and cross communicates with ROCK because its overexpression induces ROCK dependent stress fiber formation and because its TGFβ1 induced expression is dependent on Rho-ROCK (59). Activity of Rho-ROCK downstream of TGFβ1 results in increased phosphorylation and activation of LIM kinases. Phosphorylation of their downstream targets ADF/cofilin is known to result in decreased actin turnover. Increased phosphorylation of MLC2 (by ROCK) activates actomyosin contraction whereas increased phosphorylation of MYPT1 (a subunit of MLCP) reduces inhibition of contractility (cross), Rho-GDI1 and 2 (ARHGDIA, ARHGDIB), negative regulators of Rho are downregulated (italics), whereas GEFH1 en GEF10 (AHRGEF2, AHRGEF10), activators of Rho are up-regulated (see supplemental Fig. S4). Other indirect activators of Rho that are up-regulated are p120catenin and SDRP. The model predicts increased and sustained TGFβ1 activation, increased contractility and that inhibiting ROCK could ameliorate the impaired migration.
Fig. 7.
Fig. 7.
Increased ROCK and TGFβ activity in β-actin KO MEFs. A, Western blotting with phospho-MYPT1 antibodies showed increased phosphorylation of MYPT1 in the KO cells. Treatment with ROCK inhibitor Y-27632 abolishes phosphorylation of this protein in all cell lines used, in agreement with increased ROCK activity in the untreated KO cells. Probing with a pan actin antibody was used as loading control. B, The KO1 cells, that have the most severe migration defect, have increased TGFβ activity in coculture using a reporter assay (26). Fold activation relative to WT cells is given, error bars respresent S.E. and the value for KO1 cells is statistically significant different from WT (p < 0.001) in a t test. C, Smooth muscle marker proteins are up-regulated in KO1 and KO2. Western blots for SM22, calponin, tropomyosin2, LPP, and FilaminA confirm the mass spectrometry results (for mRNA levels see supplemental Fig. S5). GAPDH or total actin were used to ensure equal loading (not shown).
Fig. 8.
Fig. 8.
The impaired cell migration that can be conditionally relieved by inhibiting ROCK or contractility. A, Phalloidin staining of KO cells treated with Y-27632, show disassembly of the stress fibers and formation of protrusions. B, Random 2D cell migration was monitored after treating the cells without or with ROCK inhibitor Y-27632 treatment for 24h or with or without blebbistatin. Treatment with either agents significantly increases migration (note that blebbistatin-treated KO cells migrate further than untreated WT cells). C, Treatment with ROCK results in more migrating cells (* p < 0.05).

Similar articles

See all similar articles

Cited by 34 articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback