Co-localization of mitochondria with chloroplasts is a light-dependent reversible response

Co-localization of mitochondria with chloroplasts in plant cells has long been noticed as beneficial interactions of the organelles to active photosynthesis. Recently, we have found that mitochondria in mesophyll cells of Arabidopsis thaliana expressing mitochondrion-targeted green fluorescent protein (GFP) change their distribution in a light-dependent manner. Mitochondria occupy the periclinal and anticlinal regions of palisade cells under weak and strong blue light, respectively. Redistributed mitochondria seem to be rendered static through co-localization with chloroplasts. Here we further demonstrated that distribution patterns of mitochondria, together with chloroplasts, returned back to those of dark-adapted state during dark incubation after blue-light illumination. Reversible association of the two organelles may underlie flexible adaptation of plants to environmental fluctuations.Key words: Arabidopsis thaliana, blue light, chloroplast, green fluorescent protein, mesophyll cell, mitochondrion, organelle positioningHighly dynamic cell organelles, mitochondria, are responsible not only for energy production, but also for cellular metabolism, cell growth and survival as well as gene regulations.^1,2 Appropriate intracellular positioning and distribution of mitochondria contribute to proper organelle functions and are essential for cell signaling.^3,4 In plant cells operating photosynthesis, the co-localization of mitochondria with chloroplasts has been a well known phenomenon for a long period of time.^5,6,7 Physical contact of mitochondria with chloroplasts may provide a means to transfer genetic information from the organelle genome,⁸ as well as to exchange metabolite components; a process required for the maintenance of efficient photosynthesis.^9,10,11Using Arabidopsis thaliana stably expressing mitochondrion-targeted GFP,¹² we have recently examined a different aspect of mitochondria positioning. Although mitochondria in leaf mesophyll cells are highly motile under dark condition, mitochondria change their intracellular positions in response to light illumination.¹³ The pattern of light-dependent positioning of mitochondria seems to be essentially identical to that of chloroplasts.¹⁴ Mitochondria occupy the periclinal regions under weak blue light (wBL; 470 nm, 4 µmol m⁻²s⁻¹) and the anticlinal regions under strong blue light (sBL; 100 µmol m⁻²s⁻¹), respectively. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts in the periclinal regions with time period of wBL illumination clearly demonstrates that the co-localization of these two organelles is a light-induced phenomenon.¹³In the present study, to ask whether the light-dependent positioning of mitochondria is reversible or not, a time course of mitochondria redistribution was examined transferring the sample leaves from light to dark conditions. The representative results (Fig. 1) clearly show that mitochondria re-changed their positions within several hours of dark treatment. Immediately after dark adaptation, mitochondria in the palisade mesophyll cells were distributed randomly throughout the cytoplasm (Fig. 1A and ^{ref. 13}). Chloroplasts were distributed along the inner periclinal walls and the lower half of the anticlinal walls. On the contrary, mitochondria accumulated along the outer (Fig. 1B) and inner periclinal walls when illuminated with wBL. Chloroplast position was also along the outer and inner periclinal walls. Many of the mitochondria located near the chloroplasts lost their motility. When wBL-illuminated leaves were transferred back to dark condition, the numbers of mitochondria and chloroplasts present on the periclinal regions began to decrease within several hours (Fig. 1C). After 10 h dark treatment, distribution patterns of mitochondria as well as chloroplasts almost recovered to those of dark-adapted cells (Fig. 1D).Open in a separate windowFigure 1Distribution of mitochondria and chloroplasts on the outer periclinal regions of palisade mesophyll cells of A. thaliana under different light conditions. Mitochondria (green; GFP) and chloroplasts (red; chlorophyll autofluorescence) were visualized with confocal microscopy after dark adaptation (A), immediately after wBL (470 nm, 4 µmol m⁻²s⁻¹) illumination for 4 h (B), after dark treatment for 6 h (C) and 10 h (D) following the 4-h wBL illumination, respectively. Bar = 50 µm.To our knowledge, this may be the first report that directly demonstrates that wBL regulates mitochondria and chloroplast positioning in a reversible manner, though the nuclei in A. thaliana leaf cells were also found to reverse their positions when transferred from sBL to dark conditions.¹⁵ Reversible regulation of organelle positioning in leaf cells should play critical roles in adaptation of plants to highly fluctuating light conditions in the nature. Since distribution patterns of mitochondria under wBL and sBL are identical to those of chloroplasts, we can assume that phototropins, the BL receptors for chloroplast photo-relocation movement,¹⁶ may have some role in the redistribution of mitochondria. On the other hand, we also found that red light exhibited a significant effect on mitochondria positioning (Islam et al. 2009), suggesting an involvement of photosynthesis. These possibilities are now under investigation.     

Increased visceral fat mass and insulin signaling in colitis-related colon carcinogenesis model mice

Leptin, a pleiotropic hormone regulating food intake and metabolism, plays an important role in the regulation of inflammation and immunity. We previously demonstrated that serum leptin levels are profoundly increased in mice which received azoxymethane (AOM) and dextran sulfate sodium (DSS) as tumor-initiator and -promoter, respectively, in a colon carcinogenesis model. In this study, we attempted to address underlying mechanism whereby leptin is up-regulated in this rodent model. Five-week-old male ICR mice were given a single intraperitoneal injection of AOM (week 0), followed by 1% DSS in drinking water for 7 days. Thereafter, the weights of visceral fats and the serum concentration of leptin were determined at week 20. Of interest, the relative epididymal fat pad and mesenteric fat weights, together with serum leptin levels in the AOM and/or DSS-treated mice were markedly increased compared to that in untreated mice. In addition, leptin protein production in epididymal fat pad with AOM/DSS-treated mice was 4.7-fold higher than that of control. Further, insulin signaling molecules, such as protein kinase B (Akt), S6, mitogen-activate protein kinase/extracellular signaling-regulated kinase 1/2, and extracellular signaling-regulated kinase 1/2, were concomitantly activated in epididymal fat of AOM/DSS-treated mice. This treatment also increased the serum insulin and IGF-1 levels. Taken together, our results suggest that higher levels of serum insulin and IGF-1 promote the insulin signaling in epididymal fat and thereby increasing serum leptin, which may play an crucial role in, not only obesity-related, but also -independent colon carcinogenesis.     

Dietary flavonoids suppress azoxymethane-induced colonic preneoplastic lesions in male C57BL/KsJ-db/db mice

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68–91% and BCAC by 64–71%, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans.     

Receptor-binding cancer antigen expressed on SiSo cells induces apoptosis via ectodomain shedding

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a secreted antigen that induces apoptosis in putative receptor-expressing cells, including peripheral lymphocytes and natural killer (NK) cells. RCAS1 expression is associated with aggressive characteristics and poor overall survival for 15 different human malignancies. The putative RCAS1 receptor has not been isolated and the mechanism of RCAS1 apoptosis induction remains unclear. This study explores how RCAS1 is involved in apoptosis initiation. The cell lines SiSo and MCF-7, human uterine carcinoma and breast adenocarcinoma, respectively, both express RCAS1, but RCAS1 secretion is undetectable in MCF-7 cells. SiSo and MCF-7 cells were stimulated to induce RCAS1 ectodomain shedding followed by assessment of RCAS1 expression and secretion. Additionally, the RCAS1 putative receptor-expressing human chronic myelogenous leukemia cell line K562 was co-cultured with SiSo, MCF-7, or soluble RCAS1 to follow RCAS1 secretion in apoptosis initiation. RCAS1 secretion was strongly suppressed by inhibitors of metalloproteases, protein kinase C (PKC)-δ, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK), epidermal growth factor (EGF), and G-protein-coupled receptor (GPCR). K562 apoptosis could be induced only by co-culturing with SiSo or soluble RCAS1. RCAS1 is thus secreted by ectodomain shedding, which may represent a pivotal step in RCAS1-induced apoptosis initiation.     

Constriction of the ductus arteriosus by selective inhibition of cyclooxygenase-1 and -2 in near-term and preterm fetal rats

We studied the transplacental ductal constrictive effects of a selective cyclooxygenase (COX)-1 inhibitor (SC560), six selective COX-2 inhibitors including rofecoxib, and a non-selective COX inhibitor (indomethacin). Each drug was administered to the pregnant rats, and fetal ductus arteriosus (DA) was studied with a whole-body freezing method. The inner diameter ratio of the DA to the main pulmonary artery (DA/PA) was 1.02+/-0.03 (mean+/-S.E.M.) in controls. Every drug constricted the DA dose-dependently. In preterm rats on the 19th day of gestation, 10mg/kg of SC560, rofecoxib and indomethacin caused ductal constriction, with DA/PA reduced to 0.76+/-0.02, 0.80+/-0.03 and 0.75+/-0.02, respectively. In near-term on the 21st day, 10mg/kg of them caused ductal constriction, with DA/PA to 0.74+/-0.04, 0.26+/-0.02 and 0.33+/-0.05. In conclusion, both COX-1 and COX-2 selective inhibitors constrict fetal DA. They are not better alternatives for the fetus than non-selective COX inhibitors for tocolysis.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.)

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.