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The Actin-Family Protein Arp4 Is a Novel Suppressor for the Formation and Functions of Nuclear F-Actin.
Yamazaki S
,
Gerhold C
,
Yamamoto K
,
Ueno Y
,
Grosse R
,
Miyamoto K
,
Harata M
.
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The crosstalk between actin and actin-related proteins (Arps), namely Arp2 and Arp3, plays a central role in facilitating actin polymerization in the cytoplasm and also in the nucleus. Nuclear F-actin is required for transcriptional regulation, double-strand break repair, and nuclear organization. The formation of nuclear F-actin is highly dynamic, suggesting the involvement of positive and negative regulators for nuclear actin polymerization. While actin assembly factors for nuclear F-actin have been recently described, information about inhibitory factors is still limited. The actin-related protein Arp4 which is predominantly localized in the nucleus, has been previously identified as an integral subunit of multiple chromatin modulation complexes, where it forms a heterodimer with monomeric actin. Therefore, we tested whether Arp4 functions as a suppressor of nuclear F-actin formation. The knockdown of Arp4 (Arp4 KD) led to an increase in nuclear F-actin formation in NIH3T3 cells, and purified Arp4 potently inhibited F-actin formation in mouse nuclei transplanted into Xenopus laevis oocytes. Consistently, Arp4 KD facilitated F-actin-inducible gene expression (e.g., OCT4) and DNA damage repair. Our results suggest that Arp4 has a critical role in the formation and functions of nuclear F-actin.
Figure 1. Knockdown of Arp4 increased nuclear F-actin. (A) Confocal microscopy images of NIH3T3 cells transfected with control siRNA (control), Arp4-1 siRNA (Arp4 KD-1), or Arp4-2 siRNA (Arp4 KD-2). Nuclear F-actin formation was detected by expressing the nuclear actin probe nAC-mCherry. Nuclei were labeled using DAPI (blue). Bar = 10 μm; (B) Quantifications of nuclear F-actin formation in control cells (control+nAC-mCherry) and Arp4 KD cells (Arp4 KD-1 or Arp4 KD-2+nAC-mCherry) cells. For the quantification, 309 control cells, 308 Arp4 KD-1 cells, and 306 Arp4 KD-2 cells were analyzed. Data shown are mean ± S.D. (n ≥ 3); (C) Quantitative RT-PCR analysis of Arp4, actin, exportin 6 (Exp6), and importin 9 (Ipo9) mRNAs in control and Arp4 KD-1 cells. Results shown are values relative to those in the control cells.
Figure 2. Injection of purified Arp4 suppresses F-actin formation and Oct4 expression in mouse nuclei transplanted into Xenopus oocytes. (A) A schematic diagram of the experimental strategy used to visualize the nuclear F-actin formed in the transplanted nuclei. Dissected GVs were alive in mineral oil and were sufficiently transparent to permit observation of interior structures by confocal microscopy; (B) The polymerization of actin in the transplanted nuclei was suppressed by injected Arp4-mCherry in a concentration-dependent manner; (C) Levels of mouse Oct4, Gapdh, Sox2, and Utf1 mRNAs were quantified in the transplanted nuclei after injecting Arp4-mCherry into oocytes. The mRNA levels (mean ± SEM, n = 3) shown are relative to those in the control nuclei.
Figure 3. Effects of Arp4 KD on gene expression. (A) Expression levels of OCT4, SOX2, UTF1, GDF3, and LEFTY1 mRNAs in Arp4 KD cells (Arp4-1 siRNA) were quantified and are shown relative to their respective levels in the control cells expressing control siRNA; (B) The expression level of OCT4 in control non-treated NIH3T3 cells, Arp4 KD cells, cells ectopically expressing NLS-actin (NLS-actin), and Arp4 KD cells ectopically expressing NLS-actin (Arp4 KD+NLS-actin). Values shown are relative to their respective levels in control cells; (C) The SRF-RE reporter plasmid was introduced into NIH3T3 cells expressing control-siRNA (control) or Arp4-1 siRNAs (Arp4 KD), and SRF responsive luciferase activity in each sample was measured as described in the Materials and Methods section. The plot shows activity relative to that in the control cells, the value for which was assigned as 1.0; (D) Quantitative RT-PCR analysis of mRNAs of SRF-targeting genes IGF-1 and ACTA2 in control and Arp4 KD cells. The expression level of each gene was normalized with respect to that of the GAPDH gene. Data shown are mean ± S.D. (n ≥ 3).
Figure 4. Arp4 is involved in β-catenin-dependent gene expression through suppressing nuclear F-actin formation. (A) NIH3T3 cells expressing siArp4-1 (Arp4 KD) and nAC-mCherry were immunostained with a specific β-catenin antibody. The arrows show colocalization of nuclear F-actin and β-catenin. Bar = 5 μm; (B) Signal intensities of β-catenin in the nucleus and cytoplasm were measured in control or Arp4 KD cells, and the relative amount of nuclear β-catenin was shown. Box plot, the amount of β-catenin in the nucleus relative to that in the cytoplasm and n = 52; (C) A TCF/LEF reporter plasmid construct was introduced into the control or Arp4 KD cells, and TCF/LEF responsive luciferase activity was measured in each sample. Luciferase activity shown is relative to that in the control cells; (D) Quantitative RT-PCR analysis of mRNAs of TCF/LEF targeting genes, CCND1, AXIN2, and TCF7. The expression level of each gene in control siRNA and Arp4 KD cells was normalized with respect to that of the GAPDH gene; (E) The binding of endogenous β-catenin to its targeting sites in OCT4 and CCND1 genes was analyzed by quantitative ChIP assay, using an antibody specific for β-catenin. The values shown are relative to those in control cells. Data shown are mean ± SD (n ≥ 3).
Figure 5. The effect of Arp4-KD in DNA double-strand break repair. Control siRNA (control or cont) or siArp4-1 (Arp4 KD) was introduced into NIH3T3 cells, and cells were incubated for 24 h. The cells were, then, treated with zeocin (50 or 100 µg/mL) for 1 h and subjected to γH2A.X staining a specific antibody and DAPI staining. The cells were observed under a fluorescent microscope (A), and the relative fluorescence intensity of γH2A.X to that of DAPI was determined using Fiji image analysis software (B). Box plot, the signal intensity of γH2A.X relative to that of DAPI and n > 88.
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