Chunsun Jiang

Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide

The precise molecular mechanism underlying arsenic trioxide (As2O3)-induced apoptosis is a subject of extensive study. Here, we show that clinically relevant doses of As2O3 can induce typical apoptosis in IM-9, a multiple myeloma cell line, in a Bcl-2 inhibitable manner. We confirmed that As2O3 directly induced cytochrome c (cyto c) release from isolated mouse liver mitochondria via the mitochondrial permeability transition pore, and we further identified the voltage-dependent anion channel (VDAC) as a biological target of As2O3 responsible for eliciting cyto c release in apoptosis. First, pretreatment of the isolated mitochondria with an anti-VDAC antibody specifically prevented As2O3-induced cyto c release. Second, in proteoliposome experiments, VDAC by itself was sufficient to mediate As2O3-induced cyto c release, which could be specifically inhibited by Bcl-XL. Third, As2O3 induced mitochondria membrane potential (ΔΨm) reduction and cyto c release only in the VDAC-expressing, but not in the VDAC-deficient yeast strain. Finally, we found that As2O3 induced the increased expression and homodimerization of VDAC in IM-9 cells, but not in Bcl-2 overexpressing cells, suggesting that VDAC homodimerization could potentially determine its gating capacity to cyto c, and Bcl-2 blockage of VDAC homodimerization represents a novel mechanism for its inhibition of apoptosis.

One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1

The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.

A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase‐3 in vitro

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase‐3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl‐xL, cyclosporin A and z‐VAD.fmk. Moreover, caspase‐3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl‐xL‐ and cyclosporin A‐inhibitable manner. These results suggest that caspase‐3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.

Development of photosystems 2 and 1 during leaf growth in grapevine seedlings probed by chlorophyll a fluorescence transient and 820 nm transmission in vivo

Chlorophyll (Chl) a fluorescence transient and 820-nm transmission kinetic were investigated to explore the development of photosynthetic apparatus in grapevine leaves from emergence to full expansion. In this study, all leaves at various developing stages exhibited typical Chl a fluorescence transient. In newly initiating leaves, the maximum quantum yield of primary photochemistry (ϕP0) was slightly lower (<10 %) than that in fully expanded leaves. Nevertheless, the fluorescence rise from O to J step was clearly speeded up in young leaves compared with that in fully expanded leaves. Additionally, a distinct K step appeared in young leaves at high irradiances. With leaf development, the efficiency that a trapped exciton can move an electron into the electron transport chain further than QA− (Ψ0), the quantum yield of electron transport beyond QA (ϕE0), electron transport flux per excited cross section (ET0/CS0), the amount of active photosystem (PS) 2 reaction centres per excited cross section (RC/CS0), and the performance index on cross section basis (PICS) increased gradually and rapidly. Young leaves had strikingly lower amplitude of transmission at 820 nm. A linear relationship between Ψ0 and the transmission at 820 nm (I30/I0) was evident. Based on these data, we suggest that (1) the primary photochemistry of PS2 may be not the limiting step of the photosynthetic capacity during leaf growth under natural irradiance; (2) oxygen evolving complex (OEC) might be not fully connected to PS2 at the beginning of leaf growth; (3) though there are a few functional PS1 and PS2 at the early stages of leaf development, they match perfectly.

G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion

Mitofusins (Mfns) mediate the fusion of mitochondrial membranes. However, little is known about how Mfns are regulated to control mitochondrial fusion, which is a multistep process requiring tethering and docking of the outer membranes of two mitochondria. In this study, we report that guanine nucleotide binding protein-β subunit 2 (Gβ2), a WD40 repeats protein and a member of the β-subunits of the heterotrimeric G proteins, has a crucial function in mitochondrial fusion. Gβ2 was found to be enriched on the surface of mitochondria and interacted with mitofusin 1 (Mfn1) specifically. Gβ2 also regulated the mobility of Mfn1 on the surface of the mitochondrial membrane and affected the mitochondrial fusion. Depletion of endogenous Gβ2 resulted in mitochondrial fragmentation, which could be rescued by exogenous Gβ2. These findings have thus uncovered a novel role of Gβ2 in regulating mitochondrial fusion through its interaction with Mfn1.

Tolerance vs. avoidance: two strategies of soybean (Glycine max) seedlings in response to shade in intercropping

Intercropping is a sustainable agricultural practice used worldwide for highly efficient utilization of resources. However, short crops often grow under the shade of the canopy of tall crops in intercropping systems. Plants evolved two main strategies to deal with shade: avoidance and tolerance. Soybean (Glycine max), a legume crop, is often planted in intercropping. But little is known about a strategy that soybean may employ to deal with shade at seedling stage. Therefore, we determined morphological and physiological traits related to shade tolerance and shade avoidance in seedlings of two varieties. Generally, both varieties showed similar shade tolerance traits, such as increased specific leaf area and chlorophyll (Chl) content, and reduced photosynthetic capacity and the Chl a/b ratio. The light-limiting environment eliminated the benefits of shade tolerance traits for the carbon gain, which led to similar real-time photosynthesis and biomass in intercropping. By contrast, two varieties expressed different changes in shade avoidance traits. The variety Guixia 3 exhibited clear preference of shade avoidance that resulted in a high main stem, hypocotyl elongation, and biomass allocation towards the stem. The variety Gongxuan 1 showed those traits less. We suggested that the genetic variation occurs within soybean, thus the shade avoidance related traits might be important for variety selection for intercropping. Hence, the evaluation of performance should focus on shade avoidance in soybean genotypes in future experiments.

Effect of storage temperature on spore viability and early gametophyte development of three vulnerable species of Alsophila (Cyatheaceae)

To effectively preserve the vulnerable species of Alsophila, we studied the effects of varying the temperature and duration of storage on spore viability, early gametophyte development and the microstructure of brown spores of three Alsophila species. Spores of A. spinulosa (Wall. ex Hook.) Tryon and A. gigantea Wall. ex Hook. lost viability quickly when stored at room temperature and suffered from great loss when stored at –18°C from 6 to 12 months. Within 1 month, spore viability of A. spinulosa and A. gigantea stored at 4°C was higher than that of those stored in liquid nitrogen. In contrast, long-term storage in liquid nitrogen resulted in a comparatively small loss of viability for these two species. The spores of A. podophylla Hook. died within 3 months after storage at room temperature, 4°C and –18°C, and they died within 12 months when stored in liquid nitrogen. The spores of A. spinulosa and A. gigantea stored at room temperature, 4°C and –18°C, were prone to develop into abnormal gametophytes. These results suggest that storage of A. spinulosa and A. gigantea spores in liquid nitrogen is an effective method of preserving these vulnerable species. The reasons for the failure to preserve ephemeral A. podophylla spores by storage in liquid nitrogen are discussed.

Enhanced photosystem 2 thermostability during leaf growth of elm (Ulmus pumila) seedlings

We examined photosynthetic activities and thermostability of photosystem 2 (PS2) in leaves of elm (Ulmus pumila) seedlings from initiation to full expansion. During leaf development, net photosynthetic rate (PN) increased gradually and reached the maximum when leaves were fully developed. In parallel with the increase of PN, chlorophyll (Chl) content was significantly elevated. Chl a fluorescence measurements showed that the maximum quantum yield of PS2 (ϕPS2), the efficiency a trapped exciton, moved an electron into the electron transport chain further than QA− (Ψo), and the quantum yield of electron transport beyond QA (ϕEo) increased gradually. These results were independently confirmed by our low irradiance experiments. When subjected to progressive heat stress, the young leaves exhibited considerably lower ϕPS2 and higher minimal fluorescence (F0) than the mature leaves, revealing the highly sensitive nature of PS2 under heat in the newly initiating leaves. Further analysis showed that PS2 structure in the newly initiating leaves was strongly altered under heat, as evidenced by the increased fluorescence signals at the position of the K step. We therefore demonstrated an inhibition in the oxygen-evolving complex (OEC) in the young leaves. This resulted in decrease in amount of the functional PS2 reaction centres and relative increase in the PS2 reaction centres with inhibited electron transport at the acceptor side under heat. We suggest that the enhanced thermostability of PS2 during leaf development is associated with improved OEC stability.

Multi-Patterned Dynamics of Mitochondrial Fission and Fusion in a Living Cell

Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called “kiss and run” model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission.

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (Δψm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-XL, Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-XL sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.