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. 2006 May 15;396(1):1-5.
doi: 10.1042/BJ20060241.

The Increase in Mitochondrial Association With Actin Precedes Bax Translocation in Apoptosis

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The Increase in Mitochondrial Association With Actin Precedes Bax Translocation in Apoptosis

Ho Lam Tang et al. Biochem J. .
Free PMC article

Abstract

Accumulating evidence indicates the potential role of actin cytoskeleton in facilitating the mitochondrial recruitment of various pro-apoptotic proteins from the cytosol to initiate apoptosis. In the present paper, we report the observation of the increase in mitochondrial association of actin in early apoptosis. Using cell fractionation and Western blot analysis, we found that mitochondrial accumulation of beta-actin occurred before the mitochondrial insertion of Bax and release of cytochrome c in apoptosis. The mitochondrial accumulation of beta-actin was observed with various apoptotic stimuli in various cell lines, suggesting that this is a general apoptotic phenomenon in mammalian systems. Using fluorescence microscopy, we have shown that an apoptotic induction triggered the reorganization of the F-actin (filamentous actin) network with an increase in the association with mitochondria, which was observed before mitochondrial fission and nuclear condensation. Perhaps actin could contribute to the initiation of apoptosis by enabling cytosolic pro-apoptotic proteins to be carried to mitochondria by the cytoskeleton-driven trafficking system.

Figures

Figure 1
Figure 1. Mitochondrial translocation of β-actin in STS-induced HEK-293T cells
Western blot analysis of β-actin and Bax in cytosolic (C), ER-containing (E) and mitochondria-enriched (M) fractions of HEK-293T cells treated with or without STS for 2 h. The mitochondrial marker Bak indicates that the majority of mitochondria were present in the mitochondrial fraction, while the ER marker calnexin shows that ER was present in both the ER-containing and mitochondrial fractions. β-Tubulin served as a loading control for the cytosolic fraction.
Figure 2
Figure 2. Mitochondrial translocation of β-actin in early apoptosis in STS-treated COS-7 cells and UV-exposed HeLa cells
(A) Western blot analysis of β-actin and Bax in cytosolic (C) and mitochondrial (M) fractions of COS-7 cells treated with STS for 0.5–16 h. Bak and β-tubulin served as loading controls for mitochondrial and cytosolic fractions respectively. (B) Western blot analysis of β-actin, Bax and cytochrome c (Cyt. c) in cytosolic and mitochondrial fractions of HeLa cells from 2 to 6 h after UV induction. (C) Quantification of mitochondrial translocation of β-actin and Bax, and cytochrome c release in the apoptotic HeLa cells induced with UV. The relative change in signal intensity was calculated as follows: relative change in signal intensity=(signal intensity at a given time−signal intensity at zero time)/(signal intensity at 6 h−signal intensity at zero time).
Figure 3
Figure 3. Increase in co-localization of F-actin and mitochondria at an early stage of STS-induced apoptosis in COS-7 cells as determined by fluorescence microscopy
(A) Monochromatic F-actin images of healthy and STS-treated (30 min) COS-7 cells. (B) F-actin, mitochondria and nuclei were stained with Phalloidin (green), MitoTracker (red) and H-33342 (blue) respectively. (C) Enlarged fluorescence images of the corresponding dotted boxes in (B). Images presented in (A)–(C) are representative of three experiments with similar results. (D) The fluorescent signal intensity of F-actin (green) and mitochondria (red) was quantified along the dotted line in (B). (E) Mean±S.D. correlation coefficient of the fluorescent signals of F-actin and mitochondria in healthy or STS-treated cells in three independent signal-quantification measurements [including the measurement in (D) and two extra measurements from two other cells]. The correlation coefficient was calculated as described previously [24]. *P<0.01; Student's t test.
Figure 4
Figure 4. Mitochondrial translocation of β-actin in response to various apoptotic stimuli in HeLa cells
HeLa cells were treated with H2O2 for 5 h, STS for 3 h, serum withdrawal (SW) for 5 days or UV for 3 h. The protein levels of β-actin in mitochondrial fraction of the apoptotic cells were detected by Western blot analysis. The β-actin level in the healthy cells served as a control.

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