Effective alignment method using a diamond notch knife for correlative array tomography.

Correlative array tomography, combining light and electron microscopy via serial sections, plays a crucial role in the three-dimensional ultrastructural visualization and molecular distribution analysis in biological structures. To address the challenges of aligning fluorescence and electron microscopy images and aligning serial sections of irregularly shaped biological specimens, we developed a diamond notch knife, a new tool for puncturing holes using a diamond needle. The diamond needle featured a triangular and right-angled tip, enabling the drilling of deep holes upon insertion into the polished block face. This study describes the application of the diamond notch knife in correlative array tomography.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the ß-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport ß-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

Bld10p/Cep135 determines the number of triplets in the centriole independently of the cartwheel.

The conserved nine-fold structural symmetry of the centriole is thought to be generated by cooperation between two mechanisms, one dependent on and the other independent of the cartwheel, a sub-centriolar structure consisting of a hub and nine spokes. However, the molecular entity of the cartwheel-independent mechanism has not been elucidated. Here, using Chlamydomonas reinhardtii mutants, we show that Bld10p/Cep135, a conserved centriolar protein that connects cartwheel spokes and triplet microtubules, plays a central role in this mechanism. Using immunoelectron microscopy, we localized hemagglutinin epitopes attached to distinct regions of Bld10p along two lines that connect adjacent triplets. Consistently, conventional and cryo-electron microscopy identified crosslinking structures at the same positions. In centrioles formed in the absence of the cartwheel, truncated Bld10p was found to significantly reduce the inter-triplet distance and frequently form eight-microtubule centrioles. These results suggest that the newly identified crosslinks are comprised of part of Bld10p/Cep135. We propose that Bld10p determines the inter-triplet distance in the centriole and thereby regulates the number of triplets in a cartwheel-independent manner.

Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle-Complex-Mediated DNA Delivery to Specific Plant Organelles.

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants.

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants.

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants.

Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.

Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells.

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth.

A Promising Vector for TCR Gene Therapy: Differential Effect of siRNA, 2A Peptide, and Disulfide Bond on the Introduced TCR Expression.

Adoptive immunotherapy using TCR gene-modified T-lymphocytes is an attractive strategy for targeting malignancies. However, TCR mispairings between endogenous and introduced TCR chains are a major concern, as they may induce mixed TCRs with unknown specificities and may reduce the expression of therapeutic TCRs. To overcome these problems, we have recently established a novel retroviral siTCR vector encoding small-interfering RNAs (siRNAs) to knockdown endogenous TCR genes for the efficient expression of therapeutic TCRs. In this study, to improve the efficacy of siTCR vectors, we developed 2A peptide-based siTCR vectors that could increase the expression levels of transduced TCRs compared with internal promoter-based siTCR vectors. We also evaluated the efficacy of an siTCR strategy and the addition of a new interchain disulfide bond created by cysteine modification. We found that the effect of the cysteine modification depended on TCR variations, while the siTCR strategy improved the expression of all TCRs tested. Furthermore, the combined effect of the siTCR and cysteine modification strategies was highly significant for certain TCRs. Therefore, our novel siTCR technology, in isolation or in combination with another strategy, may open the door to effective immunotherapy for cancer patients.Molecular Therapy - Nucleic Acids (2012) 1, e63. doi:10.1038/mtna.2012.52; published online 18 December 2012.

Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation.

Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

A mobile secretory vesicle cluster involved in mass transport from the Golgi to the plant cell exterior.

Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.

Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots.

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene.

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.

Proliferative activation up-regulates expression of CD4 and HIV-1 co-receptors on NK cells and induces their infection with HIV-1.

NK cells play important roles in immune surveillance against malignancy and virus-infected cells. NK cell functions are affected in patients infected with HIV-1; however, whether there is direct interaction between NK cells and HIV-1 remains controversial. In this study the expression of CD4, an important receptor for HIV-1, was up-regulated on NK cells co-cultured with an NK cell-selective stimulating cell line, HFWT, and rIL-2. Although the level of CD4 was lower on NK cells than on CD4+ T cells, expression of the HIV-1 co-receptor CCR5 was clearly up-regulated on CD4+ NK cells. CD4+ NK cells expressed higher levels of HLA-DR and CD25 than CD4- NK cells, suggesting that they were highly activated. Cell-free HIV-1 could not infect the NK cells, but NK cells were infected when co-cultured with HIV-1-infected T cells. Using this co-culture system, we can better understand how HIV-1 infects NK cells and how NK cell functions are affected in AIDS.

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells.

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.

Small bowel bacterial overgrowth is not seen in healthy adults but is in disabled older adults.

BACKGROUND/AIMS: Small bowel bacterial overgrowth (SBBO) is defined as an abnormal increase in the number of bacteria in the small intestine, and may be occult in older adults. The aim of this study was to determine whether small bowel bacterial overgrowth (SBBO) is found in healthy older people as a concomitant of normal aging or is seen only in disabled or frail older people, excluding patients with intestinal disease and those who had undergone upper intestinal or gastric surgery. METHODOLOGY: Forty-one relatively healthy older people, aged 74.6 +/- 1.7 (mean +/- SE) years, who engage in regular exercise, and 42 variously disabled older people, average age 78.8 +/- 1.1 years, who commute to a day-care center participated in this study. SBBO was determined by a breath hydrogen (H2) test after ingestion of 50g of glucose solution dissolved in 200mL of water. Physical activity was judged from the number of steps walked per day as measured by a pedometer. Food intake was assessed by an interview. RESULTS: No SBBO-positive subject was seen among the healthy, while 11 (25.6%) of the disabled older adults were positive. The number of steps/day taken by the disabled subjects was extremely low, only 22.3% of that of the healthy (P<0.001). No significant difference was seen between the groups in food intake. The mean age of the SBBO-positive patients was relatively high, 81.5 +/- 2.2 years, and 5 (45.4%) of them were underweight, with a BMI<18.5. CONCLUSIONS: Our results and previous studies indicate that SBBO is seen only in patients with intestinal disease and disabled or frail older people.

Small bowel bacterial overgrowth may not affect bone mineral density in older people.

BACKGROUND & AIMS: Small bowel bacterial overgrowth (SBBO) may be associated with malnutrition, diarrhea, and weight loss. Recently, bone mineral density (BMD) in patients with SBBO was reported to be lower, and SBBO may be an important factor in the development of metabolic bone disease. However, the subjects in these studies were relatively young patients with intestinal diseases. There is no information on the effect of SBBO on BMD in older people. METHOD: Seventeen relatively active and 33 disabled older people participated in this study. SBBO was determined by a breath hydrogen (H2) test after ingestion of a glucose solution. BMD of the lumbar spine and femur were measured using a dual energy X-ray absorptiometry scan (DEXA). RESULTS: One healthy control and 11 disabled subjects were SBBO-positive. The Z-scores of the lumbar spine were not statistically different between groups, and a high incidence of disorders, >70%, was seen in all groups. On the other hand, there were significant differences in the femoral BMD between the healthy controls and the SBBO-negative (P<0.001) and SBBO-positive (P<0.05) groups. No significant difference was seen in femoral BMD between SBBO-positive and SBBO-negative institutionalized people. CONCLUSION: SBBO seems to have little effect on BMD in people approximately 80 years old.