Lipid Membrane Interaction of Peptide/DNA Complexes Designed for Gene Delivery

Among gene delivery systems, peptide-based gene carriers have received significant attention because of their selectivity, biocompatibility, and biodegradability. Since cellular membranes function as a barrier toward exogenous molecules, cell-penetrating peptides (CPPs), which are usually cationic and/or amphiphilic, can serve as efficient carriers to deliver cargo into the cytosol. Here, we examined the interactions of carrier peptides and their DNA complexes with lipid membranes using a quartz crystal microbalance (QCM) and high-speed atomic force microscopy (HS-AFM). The carrier peptides are a 12-residue partial presequence of yeast cytochrome c oxidase subunit IV (Cytcox) and BP100, which are a mitochondria-targeting signal peptide and a CPP, respectively. QCM data showed that BP100 has a higher binding affinity than Cytcox to both plasma membrane- and mitochondrial membrane-mimicking lipid bilayers. The DNA complexes with either Cytcox or BP100 exhibited the same tendency. Furthermore, HS-AFM data demonstrated that the DNA complexes of either peptide can disrupt the lipid membranes, forming larger pores in the case of Cytcox. Our results suggest that the binding affinity of the peptide/DNA complex to the plasma membrane is more critical than its membrane disruption ability in enhancing the cellular uptake of DNA

C‐Terminal Domain Controls Protein Quality and Secretion of Spider Silk in Tobacco Cells

The remarkable mechanical strength and extensibility of spider dragline silk spidroins are attributed to the major ampullate silk proteins (MaSp). Although fragmented MaSp molecules have been extensively produced in various heterologous expression platforms for biotechnological applications, complete MaSp molecules are required to achieve instinctive spinning of spidroin fibers from aqueous solutions. Here, a plant cell-based expression platform for extracellular production of the entire MaSp2 protein is developed, which exhibits remarkable self-assembly properties to form spider silk nanofibrils. The engineered transgenic Bright-yellow 2 (BY-2) cell lines overexpressing recombinant secretory MaSp2 proteins yield 0.6-1.3  µg L⁻¹ at 22 days post-inoculation, which is four times higher than those of cytosolic expressions. However, only 10-15% of these secretory MaSp2 proteins are discharged into the culture media. Surprisingly, expression of functional domain-truncated MaSp2 proteins lacking the C-terminal domain in transgenic BY-2 cells increases recombinant protein secretion incredibly, from 0.9 to 2.8 mg L⁻¹ per day within 7 days. These findings demonstrate significant improvement in the extracellular production of recombinant biopolymers such as spider silk spidroins using plant cells. In addition, the results reveal the regulatory roles of the C-terminal domain of MaSp2 proteins in controlling their protein quality and secretion

Bilirubin is produced nonenzymatically in plants to maintain chloroplast redox status

血液の分解産物ビリルビンが植物で作られることを発見 --植物の効率的な光合成に寄与している可能性--. 京都大学プレスリリース. 2023-06-08.Bilirubin, a potent antioxidant, is a product of heme catabolism in heterotrophs. Heterotrophs mitigate oxidative stress resulting from free heme by catabolism into bilirubin via biliverdin. Although plants also convert heme to biliverdin, they are generally thought to be incapable of producing bilirubin because they lack biliverdin reductase, the enzyme responsible for bilirubin biosynthesis in heterotrophs. Here, we demonstrate that bilirubin is produced in plant chloroplasts. Live-cell imaging using the bilirubin-dependent fluorescent protein UnaG revealed that bilirubin accumulated in chloroplasts. In vitro, bilirubin was produced nonenzymatically through a reaction between biliverdin and reduced form of nicotinamide adenine dinucleotide phosphate at concentrations comparable to those in chloroplasts. In addition, increased bilirubin production led to lower reactive oxygen species levels in chloroplasts. Our data refute the generally accepted pathway of heme degradation in plants and suggest that bilirubin contributes to the maintenance of redox status in chloroplasts

Organellar Glue: A Molecular Tool to Artificially Control Chloroplast–Chloroplast Interactions

細胞小器官を接着する新技術「オルガネラグルー」を開発 --オルガネラ間コミュニケーションの操作に期待--. 京都大学プレスリリース. 2022-09-30.Organelles can physically interact to facilitate various cellular processes such as metabolite exchange. Artificially regulating these interactions represents a promising approach for synthetic biology. Here, we artificially controlled chloroplast–chloroplast interactions in living plant cells with our organelle glue (ORGL) technique, which is based on reconstitution of a split fluorescent protein. We simultaneously targeted N-terminal and C-terminal fragments of a fluorescent protein to the chloroplast outer envelope membrane or cytosol, respectively, which induced chloroplast–chloroplast interactions. The cytosolic C-terminal fragment likely functions as a bridge between two N-terminal fragments, thereby bringing the chloroplasts in close proximity to interact. We modulated the frequency of chloroplast–chloroplast interactions by altering the ratio of N- and C-terminal fragments. We conclude that the ORGL technique can successfully control chloroplast–chloroplast interactions in plants, providing a proof of concept for the artificial regulation of organelle interactions in living cells

Deuterium- and Alkyne-Based Bioorthogonal Raman Probes for In Situ Quantitative Metabolic Imaging of Lipids within Plants

Plants can rapidly respond to different stresses by activating multiple signaling and defense pathways. The ability to directly visualize and quantify these pathways in real time using bioorthogonal probes would have practical applications, including characterizing plant responses to both abiotic and biotic stress. Fluorescence-based labels are widely used for tagging of small biomolecules but are relatively bulky and with potential effects on their endogenous localization and metabolism. This work describes the use of deuterium- and alkyne-derived fatty acid Raman probes to visualize and track the real-time response of plants to abiotic stress within the roots. Relative quantification of the respective signals could be used to track their localization and overall real-time responses in their fatty acid pools due to drought and heat stress without labor-intensive isolation procedures. Their overall usability and low toxicity suggest that Raman probes have great untapped potential in the field of plant bioengineering

Three-dimensional nanoscale analysis of light-dependent organelle changes in Arabidopsis mesophyll cells

一つの植物細胞を丸ごと3次元で再現 --光依存的なオルガネラの変化をナノスケールで探る--. 京都大学プレスリリース. 2022-10-19.Different organelles function coordinately in numerous intracellular processes. Photorespiration incidental to photosynthetic carbon fixation is organized across three subcellular compartments: chloroplasts, peroxisomes, and mitochondria. Under light conditions, these three organelles often form a ternary organellar complex in close proximity, suggesting a connection with metabolism during photorespiration. However, due to the heterogeneity of intercellular organelle localization and morphology, organelles' responses to changes in the external environment remain poorly understood. Here we used array tomography by field emission scanning electron microscopy to image organelles inside the whole plant cell at nanometer resolution, generating a three-dimensional (3D) spatial map of the light-dependent positioning of chloroplasts, peroxisomes, nuclei, and vacuoles. Our results show, in light-treated cells, the volume of peroxisomes increased, and mitochondria were simplified. In addition, the population of free organelles decreased, and the ternary complex centered on chloroplasts increased. Moreover, our results emphasized the expansion of the proximity area rather than the increase in the number of proximity sites inter-organelles. All of these phenomena were quantified for the first time on the basis of nanoscale spatial maps. In summary, we provide the first 3D reconstruction of Arabidopsis mesophyll cells, together with nanoscale quantified organelle morphology and their positioning via proximity areas, and then evidence of their light-dependent changes

Long-term persistence of X-ray-induced genomic instability in quiescent normal human diploid cells.

Ionizing radiation can induce genomic instability in the progeny of irradiated cells, as was demonstrated in various experimental systems. Most in vitro studies have utilized replicating cells, but it is not clear whether radiation-induced genomic instability persists in quiescent cells. Here we show the induction of X-ray-induced genomic instability in normal human diploid cells irradiated and maintained in a quiescent state for up to 24 months while cells were subcultured approximately once every 2-3 months. Every 12 months, a fraction of the irradiated cell population was stimulated to divide by culturing at a low density, and we found that these cells showed increased frequencies of phosphorylated ATM foci, decreased colony-forming ability, and increased frequency of chromosomal aberrations. No significant increases in ROS levels were detected in long-term cultured cells. These results suggest that there are ROS-independent mechanism(s) induced by radiation, which can generate persistent delayed effects in quiescent cells, and could ultimately contribute to carcinogenesis

Mitochondrial movement during its association with chloroplasts in Arabidopsis thaliana

葉緑体との相互作用におけるミトコンドリア運動を発見 --相互作用の制御による効率的な物質代謝の可能性に期待--. 京都大学プレスリリース. 2021-03-19.Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis

Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter) were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence

Passive Oral Immunization by Egg Yolk Immunoglobulin (IgY) to Vibrio cholerae Effectively Prevents Cholera

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera