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First published online August 20, 2008
doi: 10.1242/10.1242/jcs.023911

Journal of Cell Science 121, 2913-2920 (2008)
Published by The Company of Biologists 2008
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Mitochondrial shuttling of CAP1 promotes actin- and cofilin-dependent apoptosis

Changhui Wang1,*,{ddagger}, Guo-Lei Zhou1,*,§, Srilakshmi Vedantam1, Peng Li2 and Jeffrey Field1,§

1 Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
2 Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China


Figure 1
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  		Fig. 1. CAP1 translocates to mitochondria upon treatment to induce apoptosis. (A) STS stimulates CAP1 translocation to mitochondria. HeLa cells were treated with 1 µM STS for 4 hours, mitochondria were isolated by sucrose gradient centrifugation and mitochondria or cytosol fractions were subjected to western blot. Cytochrome c (Cyto c) images were from the same gel, although mitochondrial and cytosol results were scanned separately because a longer exposure was required to detect signals in the cytosol. (B) STS but not LA stimulated translocation of CAP1 to mitochondria. HeLa cells were treated with STS, LA and TNF{alpha}, mitochondria were isolated as above and CAP1 and cofilin were detected. (C) Treatment with etoposide also induces CAP1 translocation to mitochondria. HeLa cells were treated for 24 hours with etoposide concentrations ranging from 1 µM to 100 µM, mitochondria were purified and subjected to western blots with CAP1. (D) Time course of CAP1 translocation to the mitochondria. HeLa cells were treated with STS (1 µM) in the presence of 50 µM zVAD-fmk, where indicated. Gradient-purified mitochondrial (top) and cytosolic (bottom) samples were subjected to western blots to detect CAP1. HSP60 and actin were used as loading standards.

 

Figure 2
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  		Fig. 2. CAP1 translocates to the outer mitochondrial membrane. (A) CAP1 translocates to mitochondria upon apoptosis induction with STS in NIH3T3 cells. Cells were plated on coverslips overnight and stained with 50 nM MitoTracker Red CMXROS for 1 hour followed by treatment for 1 hour with (+) or without (–) 1 µM STS. Fixed and permeabilized cells were incubated with mouse anti-CAP1 antibody for 2 hours and then incubated for 1 hour with anti-mouse FITC conjugate. The slides were analyzed using a 40x objective lens by fluorescence microscopy. Scale bar: 10 µm. (B) Western blots of NIH 3T3 cell lysate with anti-CAP1 antibody demonstrate specificity for the antibody. (C) Confocal imaging of CAP1 reveals punctate staining at the mitochondria in treated cells. NIH3T3 cells were treated and processed as described above. Fluorescence images were collected at similar focal planes by confocal microscopy with a 63x objective lens; two independent STS-treated cells are shown. Scale bars: 10 µm. (D) Mitochondrial CAP1 is proteinase sensitive. HEK293T cells were transfected with GFP-CAP1 plasmid and treated with STS for 2 hours. Gradient-purified mitochondria were digested with proteinase K as indicated and then subjected to western blot analysis.

 

Figure 3
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  		Fig. 3. CAP1 is directed to mitochondria through the N-terminal domain. (A) GFP fusion constructs. (B) Translocation of GFP fusion CAP1 functional domains to mitochondria after apoptosis induction. HEK293T cells were transfected with the indicated CAP1constructs. 24 hours after transfection, cells were treated with 1 µM STS for 2 hours. Gradient-purified mitochondria were subjected to western blotting with anti-GFP. VDAC blotting was used to normalize total proteins from mitochondrial fractions.

 

Figure 4
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  		Fig. 4. Knockdown of CAP1 and its interaction with cofilin. (A) CAP1 knockdown in HeLa cells. Stable cell lines S2-2 and S3-2 are from two independent shRNA constructs, S2 and S3, which target different sequences within CAP1 (see Materials and Methods). Control cell lines include HeLa cells harboring empty vector or a scrambled shRNA S2. Purified CAP1 from pig platelet was used as a positive control and MAPK was blotted to normalize total protein. CAP1 levels in the cell lysates were detected by western blotting. (B) CAP1 knockdown does not affect cofilin translocation to mitochondria during apoptosis induction. HeLa wild-type or CAP1-knockdown cells (S3-2) were treated with 1 µM STS for 2 hours. Sucrose-gradient-purified mitochondria were isolated and analyzed by western blot. HSP60 was monitored to normalize total mitochondrial protein. (C) Mitochondrial cofilin stimulates CAP1 translocation to mitochondria. GFP-tagged CAP1 constructs, FL-CAP, NT-CAP and {Delta}NTCAP1 were cotransfected with mitochondria-targeted cofilin (M-cof) into COS-7 cells and stained with MitoTracker. Cofilin was stained with anti-HA monoclonal antibody 12CA5. Images were viewed on a fluorescence microscope with a 40x objective lens. Arrows indicate cells with localization of GFP fusion CAP1 (domains) to mitochondria.

 

Figure 5
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  		Fig. 5. CAP1 interacts with cofilin to promote apoptosis. (A) Transient expression of CAP1 shRNA in HeLa cells reduces STS-induced apoptosis. HeLa control and CAP1-knockdown cell lines were cotransfected with GFP and ShRNA constructs (1:1). After 24 hours, cells were treated with 1 µM STS for 2 hours and labeled with Hoechst 33342. GFP-positive cells were counted. Cells with DNA fragmentation and nuclear collapse were scored as apoptotic cells. ~200 cells were counted for each treatment and the experiment was done three times with similar results. Percentage of apoptotic cells are shown as mean ± s.e.m. (B) CAP1 knockdown reduces cofilin-induced apoptosis. CAP1-knockdown HeLa cells were transfected with mitochondrial cofilin along with GFP. 24 hours after transfection, cells were stained with Hoechst 33342 and scored for chromosome condensation. Approximately 100 transfected cells were scored for each of three fields and the experiment was repeated three times with similar results. Data are presented as mean ± s.e.m. (C) Overexpression of CAP1 stimulates cofilin-induced apoptosis. HEK293T cells were cotransfected with GFP-fused FL-CAP1, NT-CAP1 or CT-CAP1 along with either M-cofilin or wild-type cofilin (0.4 µg and 0.8 µg, respectively, per well). Cells were allowed to express proteins for 18 hours and then fixed cells were stained with 1 µg/ml Hoechst 33342 for 10 minutes. Apoptosis was identified by nuclear condensation and DNA fragmentation under fluorescence microscopy in GFP-positive cells. At least 300 GFP-positive cells were scored for each of three fields and experiment was repeated three times with similar results. Data are presented as mean ± s.e.m.

 

Figure 6
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  		Fig. 6. CAP1 knockdown reduces cytochrome c release and ROS accumulation. (A) CAP1-knockdown cells have reduced cytochrome c release when apoptosis is induced. HeLa stable cell lines harboring vector (vector) and CAP1 shRNA (S3-2) treated with STS (1 µM). After 4 hours of treatment, mitochondria were isolated by sucrose gradient centrifugation and subjected to western blots for CAP1 and cytochrome c. VDAC was monitored to normalize total protein. (B) Effect of CAP1 knockdown on ROS accumulation. HeLa wild-type cells, stable cell lines with scrambled S2 or CAP1 shRNA were cultured overnight and treated with 1 µM STS for 1 hour, cells were washed and stained with fresh 10 µM H2DCF-DA (Ros; Invitrogen) for 15 minutes and then examined under an inverted fluorescence microscope. All images were taken with the same exposure time.

 

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© The Company of Biologists Ltd 2008