LETTERS NATURE CELL BIOLOGY VOLUME 5 | NUMBER 12 | DECEMBER 2003 1083 Mitochondrial translocation of cofilin is an early step in apoptosis induction Boon Tin Chua 1 , Christiane Volbracht 1 , Kuan Onn Tan 2 , Rong Li 3 , Victor C. Yu 2,4 and Peng Li 1,5,6 Increasing evidence suggests that movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Here, we report identification of the actin-binding protein cofilin by a proteomic approach, as such a factor translocated from cytosol into mitochondria after induction of apoptosis. We found that after induction of apoptosis, cofilin was translocated to mitochondria before release of cytochrome c. Reduction of cofilin protein levels with small-interfering RNA (siRNA) resulted in inhibition of both cytochrome c release and apoptosis. Only dephosphorylated cofilin was translocated to mitochondria, and the cofilin S3D mutant, which mimicks the phosphorylated form, suppressed mitochondrial translocation and apoptosis. Translocation was achieved through exposure of an amino-terminal mitochondrial targeting signal in combination with carboxy-terminal sequences. When correctly targeted to mitochondria, cofilin induced massive apoptosis. The apoptosis-inducing ability of cofilin, but not its mitochondrial localization, was dependent on the functional actin-binding domain. Thus, domains involved in mitochondrial targeting and actin binding are indispensable for its pro-apoptotic function. Our data suggest that cofilin has an important function during the initiation phase of apoptosis. Mitochondria are the major organelles involved in the signal trans- duction and biochemical execution of apoptosis 1 . Many types of apoptotic signals converge at the level of mitochondria and induce the release of apoptogenic mitochondrial proteins that directly promote apoptosis. Cytochrome c is released into the cytoplasm 2 and in turn activates caspase-9 by forming a complex with Apaf-1 (ref. 3), activat- ing the execution caspase cascade. Bcl-2 family members are known to regulate the release of cytochrome c by controlling mitochondrial membrane integrity 4 . Translocation of pro-apoptotic Bcl-2 family proteins to mitochondria has been suggested to be important for initi- ating apoptotic signalling from the organelle 4 . Activation of these pro- apoptotic proteins can be achieved by post-transcriptional modifications such as dephosphorylation of BAD 5 . Direct signalling to the mitochondria through translocation is not restricted to Bcl-2 family members, as several other proteins (including the transcription factor TR3 (ref. 6) and the Peutz-Jegher gene product LKB1 (ref. 7)) have also been shown to translocate into the mitochondria, thereby inducing apoptosis. Although the role of mitochondria in controlling downstream apoptotic events such as caspase activation is relatively well characterized, mechanisms by which upstream apoptotic signals are transduced into mitochondria remain largely elusive. Intrigued by the observation that protein movement in or out of the mitochondria seems to be essential for regulation of apoptosis, we iso- lated mitochondria from staurosporine (STS)-treated and untreated HL60 cells and investigated their mitochondrial protein compositions using two-dimensional gel electrophoresis and silver staining. STS was used as it is a general kinase inhibitor that rapidly induces caspase dependant apoptosis. We observed a significant increase in distinct protein spots in mitochondrial fractions from STS-treated apoptotic cells, compared with untreated controls. Using mass spectrometric analysis, we identified one of the spots as cofilin (Fig. 1a, arrows). Cofilin is a member of the cofilin/actin depolymerizing factor (ADF) family, which regulates actin dynamics by increasing the rate of actin depolymerization and facilitating actin filament turnover 8 . Western blot analysis confirmed that cofilin was found in mitochondrial frac- tions only in the presence of STS (Fig. 1b, top), and this was concomi- tant with the disappearance of cofilin from cytosolic fractions (Fig. 1b, bottom). Translocation of cofilin was detected as early as 5 min after STS treatment (see Supplementary Information, Fig. S1a) and was not blocked by the broad-range caspase inhibitor zVAD-fmk (Fig. 1b, top) or by Bcl-x L in the presence of apoptotic stimuli (see Supplementary Information, Fig. S1c). Translocation of cofilin was further confirmed in SH-SY5Y cells by indirect immunofluorescence microscopy (see Supplementary Information, Fig. S1b). Mitochondrial translocation of cofilin did not occur through increased association of actin with mitochondria, as similar levels of actin were detected in mitochondrial fractions from control and apoptotic cells (Fig. 1b, top). The gradient- purified mitochondrial fraction was free of cytosolic protein contami- nation, as no cytoplasmic Grb2b protein was detected (Fig. 1b, top). An early accumulation of cofilin in mitochondrial fractions was 1 Laboratories of Apoptosis Regulation, 2 Mechanisms of Apoptosis in Mammalian Cells and 3 Proteomics, Institute of Molecular and Cell Biology, National Institute of Singapore, 30 Medical Drive, 117609, Singapore. 4 Adjunct staff of the Department of Pharmacology, National University of Singapore, 18 Medical Drive, Singapore, 119260. 5 Department of Biology, Hong Kong University of Science and Technology, Clear water bay, Kowloon, Hong Kong. 6 Correspondence should be addressed to P.L. (e-mail: bolipeng@ust.hk). Published online: 23 November 2003; DOI: 10.1038/ncb1070 ©2003 Nature Publishing Group