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Myeloid cell leukemia-1 (Mcl-1) is involved in the regulation of mitochondrial fission and fusion. This report aims to explore whether Mcl-1 can interact with mitochondrial fission factor (Mff) and regulate Mff-mediated mitochondrial fragmentation and apoptosis. Fluorescence images of living cells coexpressing YFP-Mff and CFP-Mcl-1 showed that Mcl-1 markedly inhibited Mff-mediated mitochondrial fragmentation and apoptosis, suggesting that Mcl-1 played a key role in inhibiting mitochondrial fission. The cells coexpressing YFP-Mff and CFP-Mcl-1 exhibited consistent fluorescence resonance energy transfer (FRET) efficiency with that of the cells coexpressing CFP-Mcl-1 and YFP, demonstrating that Mcl-1 did not directly bind to Mff on mitochondria. Collectively, Mcl-1 inhibits Mff-mediated mitochondrial fission and apoptosis not via directly binding to Mff on mitochondria.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail‐anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl‐xl, an antiapoptotic protein, on the action of Mff by using live‐cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl‐xl. Microscopic imaging and live‐cell fluorescence resonance energy transfer analysis demonstrated that Bcl‐xl reconstructs the Mff network from punctate distribution of higher‐order oligomers to filamentous distribution of lower‐order oligomers. Live‐cell fluorescence resonance energy transfer two‐hybrid assay showed that Bcl‐xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl‐xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria., Mitochondrial fission factor (Mff)‐mediated mitochondrial fission is positively correlated with the self‐oligomerization of Mff. Bcl‐xl directly interacts with Mff to prevent Mff‐mediated mitochondrial fission and apoptosis. Bcl‐xl interacts with Mff to form heterogenous hexamers with 1 : 2 stoichiometry in cytoplasm and heterogenous tetramers with 1 : 1 stoichiometry on the mitochondrial membrane, respectively.

We here used fluorescence imaging to explore the effect of co-overexpression of Mcl-1 and Bak/BH3-only proteins on mitochondrial morphology. The cells co-expressing CFP-Mcl-1 and YFP-Bak/BimL/Puma/tBid showed co-localization of Mcl-1 with Bak/Puma/BimL/tBid and also showed the inhibitory action of Mcl-1 on the Bak-, BimL-, Puma- or tBid-mediated cell death. Co-expression of Mcl-1 and Bak but not BH3-only proteins induced time-dependent mitochondrial swelling. Fluorescence resonance energy transfer (FRET) imaging proved the direct binding of Mcl-1 to Bak, BimL, Puma and tBid, respectively. In addition, Mcl-1 prevented Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Moreover, Mcl-1-Bak complex exhibited a good co-localization with mitochondria, and co-expression of Mcl-1 and Bak for more than 24 h not only induced mitochondrial swelling but also impaired mitochondrial membrane potential. Collectively, co-expression of Mcl-1 and Bak but not BH3-only proteins significantly induced mitochondrial swelling and subsequent loss of mitochondrial membrane potential.

Bcl-2 family proteins, as central players of apoptotic program, participate in the regulation of mitochondrial network. Here, quantitative live-cell Fluorescence Resonance Energy Transfer (FRET) two-hybrid assay was used to confirm the homo-/hetero-oligomerization of mitofusins (MFN2 and MFN1), and also demonstrate the binding of MFN2 to MFN1 with 1:1 stoichiometry. FRET two-hybrid assay for living cells co-expressing CFP-labeled Bcl-XL (anti-apoptotic Bcl-2 family protein) and YFP-labeled MFN2/MFN1 demonstrate the binding of MFN2/MFN1 to Bcl-XL with 1:1 stoichiometry. Neither MFN2 nor MFN1 bind with monomeric Bax in healthy cells, but both MFN2 and MFN1 combine with punctate Bax (pro-apoptotic Bcl-2 family protein) during apoptosis. Oligomerized Bak (pro-apoptotic Bcl-2 family protein) can only associates with MFN1 but not MFN2. Moreover, co-expression of Bcl-XL and MFN2/MFN1 exhibited the same anti-apoptotic ability as the expression of Bcl-XL alone to staurosporine-induced apoptosis, indicating the full anti-apoptotic ability of Bcl-XL complexed with MFN2 or MFN1. However, knockdown of MFN2 but not MFN1 reduced mitochondrial aggregation induced by overexpressed Bcl-XL, indicating that MFN2 but not MFN1 mediates Bcl-XL-induced mitochondrial aggregation.

This report aims to explore how Bcl-xL, a Bcl-2 family protein, regulates PINK1/Parkin-dependent mitophagy. Compared with the Hela cells expressing Parkin alone, co-expression of Bcl-xL significantly inhibited CCCP (Carbonyl cyanide 3- chlorophenylhydrazone)-induced mitochondrial Parkin accumulation and mitophagy. Western blotting analysis illustrated that over-expressed Bcl-xL inhibited CCCP-induced decrease of mitochondrial proteins in Parkin over-expressed cells. Fluorescence loss in photobleaching (FLIP) analyses demonstrated that Bcl-xL inhibited the CCCP-induced translocation of Parkin into mitochondria not by retrotranslocating Parkin from mitochondria to cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed in Hela cells that Bcl-xL physically bound with Parkin to form oligomer in cytoplasm, and that Bcl-xL also directly interacted with PINK1 on mitochondria. analysis for HEK293 T cells verified that endogenous Bcl-xL interacted with both endogenous Parkin and PINK1. Collectively, Bcl-xL inhibits PINK1/Parkin- dependent mitophagy by preventing the accumulation of Parkin on mitochondria via two regulation ways: directly binds to Parkin in cytoplasm to prevent the translocation of Parkin from cytoplasm to mitochondria and directly binds to PINK1 on mitochondria to inhibit the Parkin from cytoplasm to mitochondria by PINK1.

Bax oligomerization is essential for triggering mitochondrial outer membrane permeabilization (MOMP) in many apoptotic programs. However, it is controversial whether Bax dimer is sufficient to trigger MOMP. In this report, multiple Gaussian function-based FRET analysis (Multi-Gaussian FRET analysis) was used to dissect the dimerization and then tetramerization of Bax in relation to MOMP. Multi-Gaussian FRET analysis on the time-lapse FRET images of single living cells co-expressing CFP-Bax and YFP-Bax revealed that formation of mitochondrial Bax homodimers preceded MOMP within 3 min and Bax dimer transformed into tetramer within 6 min concomitantly with complete MOMP within 10 min, providing direct evidence in support of the sufficient ability of Bax dimers to trigger MOMP at least in natural cells.

BCL-XL, an anti-apoptotic BCL-2 family protein, potently inhibits BAK oligomerization and the formation of toxic mitochondrial pores in response to cellular stress. This report aims to explore which form of mitochondrial monomeric and oligomerized BAK can be retrotranslocated by BCL-XL. Fluorescence imaging of living cells co-expressing CFP-BCL-XL and YFP-BAK showed that BCL-XL markedly inhibited mitochondrial BAK oligomerization and resulted in partial cytosolic BAK distribution. Live-cell fluorescence resonance energy transfer (FRET) analyses showed that BAK auto-oligomerized on mitochondria and BCL-XL physically sequestrated monomeric BAK to prevent BAK oligomerization. Fluorescence loss in photobleaching (FLIP) analyses showed that BCL-XL retrotranslocated the monomeric BAK from mitochondria into cytosol, whereas monomeric BAK reduced the retrotranslocation rate of BCL-XL. Live-cell time-lapse imaging and FLIP experiments in living cells with BAK oligomers displayed that BCL-XL did not depolymerize or retrotranslocate the oligomerized BAK. Collectively, BCL-XL retrotranslocates monomeric instead of oligomerized BAK from mitochondria into cytosol.

The present study aims to explore the neuro-protective effects of purified Sparassis crispa polysaccharides against L-glutamic acid (L-Glu)-induced differentiated PC12 (DPC12) cell damages and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix with (1→3)-linked Rha in the backbone, and (1→2) linkages and (1→6) linkages in the side bone. Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to L-Glu exposure effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) and reduced L-Glu-induced apoptosis (by Hoechst staining). SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca(2+) influx and prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore, SCWEA normalized expression of anti-apoptotic proteins in L-Glu-explored DPC12 cells. These results suggested that SCWEA protects against L-Glu-induced neuronal apoptosis in DPC12 cells and may be a promising candidate for treatment against neurodegenerative disease.