Development of a novel AAK1 inhibitor via Kinobeads-based screening.

A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and ß/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.

Aromatic oil from lavender as an atopic dermatitis suppressant.

In atopic dermatitis (AD), nerves are abnormally stretched near the surface of the skin, making it sensitive to itching. Expression of neurotrophic factor Artemin (ARTN) involved in such nerve stretching is induced by the xenobiotic response (XRE) to air pollutants and UV radiation products. Therefore, AD can be monitored by the XRE response. Previously, we established a human keratinocyte cell line stably expressing a NanoLuc reporter gene downstream of XRE. We found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan metabolite and known inducer of the XRE, increased reporter and Artemin mRNA expression, indicating that FICZ-treated cells could be a model for AD. Lavender essential oil has been used in folk medicine to treat AD, but the scientific basis for its use is unclear. In the present study, we investigated the efficacy of lavender essential oil and its major components, linalyl acetate and linalool, to suppress AD and sensitize skin using the established AD model cell line, and keratinocyte and dendritic cell activation assays. Our results indicated that lavender essential oil from L. angustifolia and linalyl acetate exerted a strong AD inhibitory effect and almost no skin sensitization. Our model is useful in that it can circumvent the practice of using animal studies to evaluate AD medicines.

Cancer stem cells as the source of tumor associated myoepithelial cells in the tumor microenvironment developing ductal carcinoma in situ.

The heterogeneous cell population in the stromal microenvironment is considered to be attributed to the multiple sources from which the cells originate. Tumor associated myoepithelial cells (TAMEs) are one of the most important populations in the tumor microenvironment (TME) especially in breast cancer. On the other hand, cancer stem cells (CSCs) have previously been described to be the origin of tumor-associated cellular components in the TME. We prepared a cancer stem cell model converting mouse-induced pluripotent stem cells (miPSCs) in the presence of conditioned medium of breast cancer cell line MDA-MB-231 cells. The converted cells developed tumors progressing into invasive carcinoma with ductal carcinoma in situ (DCIS) like structure when transplanted into mouse mammary fat pads. The primary cultured cells from the tumor further exhibited markers of CSC such as Sox2, Oct3/4, - CD133 and EpCAM, and mammary gland-related TAME markers such as α-smooth muscle actin, cytokeratin 8, whey acidic protein, prolactin receptor and progesterone receptor as well. These results indicated that the CSCs could be an origin of TAMEs contributing to mammary gland epithelial cell differentiation and the progression to invasive carcinoma during tumor development. The gene expression profiles confirmed the enhanced signaling pathways of PI3K/AKT and MAPK, which have been demonstrated to be enriched in the CSC models, together with the estrogen receptor signaling which was peculiar to mammary gland-derived character.

Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response.

GlycoMaple is a new tool to predict glycan structures based on the expression levels of 950 genes encoding glycan biosynthesis-related enzymes and proteins using RNA-seq data. The antioxidant response, protecting cells from oxidative stress, has been focused on because its activation may relieve pathological conditions, such as neurodegenerative diseases. Genes involved in the antioxidant response are defined within the GO:0006979 category, including 441 human genes. Fifteen genes overlap between the glycan biosynthesis-related genes defined by GlycoMaple and the antioxidant response genes defined by GO:0006979, one of which is FUT8. 5-Hydroxy-4-phenyl-butenolide (5H4PB) extracted from Chinese aromatic vinegar induces the expression of a series of antioxidant response genes that protect cells from oxidative stress via activation of the nuclear factor erythroid 2-related factor 2-antioxidant response element pathway. Here, we show that FUT8 is upregulated in both our RNA-seq data set of 5H4PB-treated cells and publicly available RNA-seq data set of cells treated with another antioxidant, sulforaphane. Applying our RNA-seq data set to GlycoMaple led to a prediction of an increase in the core fucose of N-glycan that was confirmed by flow cytometry using a fucose-binding lectin. These results suggest that FUT8 and core fucose expression may increase upon the antioxidant response.

An Electron Tomographic Analysis of Giantin-Deficient Golgi Proposes a New Function of the Golgin Protein Family.

The Golgi apparatus is an organelle that mediates modifications, sorting, and transport of proteins and lipids. Golgins are a group of proteins with coiled-coil structures that localize to the Golgi and are thought to function as tethers to facilitate the docking of vesicles, Rab GTPases, and cytoskeleton components to the Golgi stack. Giantin is the longest golgin and has been thought to function as a tether for COPI vesicles along with other golgins, such as p115 and GM130. Contrary to our expectation that the loss of the tether will result in an increase in untethered COPI vesicles in the cytoplasm, our electron microscopy observations showed that the fenestrae normally present in Golgi cisternae were reduced upon Giantin knockdown. We also found that this structural change is accompanied by altered secretion of cargo proteins and cell surface glycosylation. These results indicate that there exists a correlation between Golgi structural changes caused by the loss of Giantin and Golgi function. Here, we describe electron tomography methods for the detection of structural changes in the Golgi.

A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi Apparatus After Experimental Manipulation.

The visual classification of cell images according to differences in the spatial patterns of subcellular structure is an important methodology in cell and developmental biology. Experimental perturbation of cell function can induce changes in the spatial distribution of organelles and their associated markers or labels. Here, we demonstrate how to achieve accurate, unbiased, high-throughput image classification using an artificial intelligence (AI) algorithm. We show that a convolutional neural network (CNN) algorithm can classify distinct patterns of Golgi images after drug or siRNA treatments, and we review our methods from cell preparation to image acquisition and CNN analysis.

The microbiota catabolites of quercetin glycosides concertedly enhance the resistance against acetaldehyde-induced oxidative stress.

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (OPAC) are the predominant catabolites of quercetin glycosides, such as quercetin 4'-O-ß-glucoside from the onion, produced by intestinal microbiota. Although each catabolite has been reported to protect the cells from acetaldehyde-induced cytotoxicity, the effect of their combination remains to be clarified. The purpose of this study was to determine whether the combination of DOPAC and OPAC enhances the resistance against the acetaldehyde-induced oxidative stress in the cultured hepatocytes. The pretreatment of the combination of DOPAC (5 µM) and OPAC (5 µM) showed significant protection against the acetaldehyde- and hydrogen peroxide-induced cytotoxicity, even though each compound at the same concentration did not. This combination also significantly inhibited the intracellular dichlorofluorescin diacetate-detectable reactive oxygen species (ROS) level, whereas the solo treatment did slightly, suggesting that reducing mechanisms of ROS or compounds that enhance ROS production are involved in the cytoprotective effect. The combinatory treatment significantly enhanced the gene expression of not only the aldehyde dehydrogenases (ALDHs), but also glutamate-cysteine ligase, catalytic subunit, the first rate-limiting enzyme of glutathione (GSH) synthesis. Accordingly, both the intracellular GSH level and the total ALDH activity were enhanced by DOPAC plus OPAC. Involvement of GSH in the cytoprotection as well as ALDH up-regulation by the combination was confirmed by the experiments using a GSH biosynthesis inhibitor, buthionine sulfoximine. Taken together, the present results suggested that the quercetin microbiota catabolites concertedly protect the cells from acetaldehyde through a pre-enhanced resistance against oxidative stress by the GSH-dependent up-regulation of ALDHs.

Immune State Conversion of the Mesenteric Lymph Node in a Mouse Breast Cancer Model.

Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-γ production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-γ expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103+CD11b+ dendritic cells (DCs) into the mLN, along with increased (1→3)-ß-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103+CD11b+ DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

Design and Synthesis of Glycosylated Cholera Toxin B Subunit as a Tracer of Glycoprotein Trafficking in Organelles of Living Cells.

Glycosylation of proteins is known to be essential for changing biological activity and stability of glycoproteins on the cell surfaces and in body fluids. Delivering of homogeneous glycoproteins into the endoplasmic reticulum (ER) and the Golgi apparatus would enable us to investigate the function of asparagine-linked (N-) glycans in the organelles. In this work, we designed and synthesized an intentionally glycosylated cholera toxin B-subunit (CTB) to be transported to the organelles of mammalian cells. The heptasaccharide, the intermediate structure of various complex-type N-glycans, was introduced to the CTB. The synthesized monomeric glycosyl-CTB successfully entered mammalian cells and was transported to the Golgi and the ER, suggesting the potential use of synthetic CTB to deliver and investigate the functions of homogeneous N-glycans in specific organelles of living cells.

A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity.

Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases.

Evaluation of skin sensitization based on interleukin-2 promoter activation in Jurkat cells.

Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T-cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T-cell activation to supplement data on skin sensitization related to KE4. To evaluate T-cell activation, the Jurkat T-cell line stably expressing luciferase downstream of the pro-inflammatory cytokine interleukin-2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T-cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno-suppressiveness of chemical substances and supplements KE4 T-cell activation data, and may thus contribute to reducing the use of animal experiments.

Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm.

Eukaryotic cells are composed of organelles, and each organelle contains proteins that play a role in its function. Therefore, the localization of a protein, especially to organelles, is a clue to infer the function of that protein. In this study, we attempted to identify novel mitochondrially localized proteins in the budding yeast Saccharomyces cerevisiae using a fluorescent protein (GFPdeg) that is rapidly degraded in the cytoplasm. Of the budding yeast proteins predicted to localize to mitochondria by the prediction tool Deeploc-1.0, those with known mitochondrial localization or functional relevance were eliminated, and 95 proteins of unknown function were selected as candidates for analysis. By forced expression of GFPdeg fusion proteins with these proteins and observation of their localization, we identified 35 uncharacterized proteins potentially localized to mitochondria (UPMs) including 8 previously identified proteins that localize to mitochondria. Most of these had no N-terminal mitochondrial localization signal and were evolutionarily young "emerging genes" that exist only in S. cerevisiae. Some of these genes were found to be upregulated during the postdiauxic shift phase when mitochondria are being developed, suggesting that they are actually involved in some mitochondrial function.

Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

Glycoprotein Semisynthesis by Chemical Insertion of Glycosyl Asparagine Using a Bifunctional Thioacid-Mediated Strategy.

Glycosylation is a major modification of secreted and cell surface proteins, and the resultant glycans show considerable heterogeneity in their structures. To understand the biological processes arising from each glycoform, the preparation of homogeneous glycoproteins is essential for extensive biological experiments. To establish a more robust and rapid synthetic route for the synthesis of homogeneous glycoproteins, we studied several key reactions based on amino thioacids. We found that diacyl disulfide coupling (DDC) formed with glycosyl asparagine thioacid and peptide thioacid yielded glycopeptides. This efficient coupling reaction enabled us to develop a new glycoprotein synthesis method, such as the bifunctional thioacid-mediated strategy, which can couple two peptides with the N- and C-termini of glycosyl asparagine thioacid. Previous glycoprotein synthesis methods required valuable glycosyl asparagine in the early stage and subsequent multiple glycoprotein synthesis routes, whereas the developed concept can generate glycoproteins within a few steps from peptide and glycosyl asparagine thioacid. Herein, we report the characterization of the DDC of amino thioacids and the efficient ability of glycosyl asparagine thioacid to be used for robust glycoprotein semisynthesis.

Synthesis and characterization of conductive flexible cellulose carbon nanohorn sheets for human tissue applications.

BACKGROUND: Conductive sheets of cellulose and carbon nanomaterials and its human skin applications are an interesting research aspect as they have potential for applications for skin compatibility. Hence it is needed to explore the effects and shed light on these applications. METHOD: To fabricate wearable, portable, flexible, lightweight, inexpensive, and biocompatible composite materials, carbon nanohorns (CNHs) and hydroxyethylcellulose (HEC) were used as precursors to prepare CNH-HEC (Cnh-cel) composite sheets. Cnh-cel sheets were prepared with different loading concentrations of CNHs (10, 20 50,100 mg) in 200 mg cellulose. To fabricate the bio-compatible sheets, a pristine composite of CNHs and HEC was prepared without any pretreatment of the materials. RESULTS: The obtained sheets possess a conductivity of 1.83 × 10- 10 S/m and bio-compatible with human skin. Analysis for skin-compatibility was performed for Cnh-cel sheets by h-CLAT in vitro skin sensitization tests to evaluate the activation of THP-1 cells. It was found that THP-1 cells were not activated by Cnh-cel; hence Cnh-cel is a safe biomaterial for human skin. It was also found that the composite allowed only a maximum loading of 100 mg to retain the consistent geometry of free-standing sheets of < 100 µm thickness. Since CNHs have a unique arrangement of aggregates (dahlia structure), the composite is homogeneous, as verified by transmission electron microscopy (TEM) and, scanning electron microscopy (SEM), and other functional properties investigated by Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), conductivity measurement, tensile strength measurement, and skin sensitization. CONCLUSION: It can be concluded that cellulose and CNHs sheets are conductive and compatible to human skin applications.

Horseradish peroxidase interacts with the cell wall peptidoglycans on oral bacteria.

Salivary peroxidase and myeloperoxidase are known to display antibacterial activity against oral microbes, and previous indications have pointed to the possibility that horseradish peroxidase (HRP) adsorbs onto the membrane of the major oral streptococci, Streptococcus mutans and Streptococcus sanguinis (S. sanguinis). However, the mechanism of interaction between HRP and the bacterial cell wall component is unclear. Dental plaques containing salivary glycoproteins and extracellular microbial products are visualized with 'dental plaque disclosing agent', and are controlled within dental therapy. However, current 'dental plaque disclosing agents' are difficult to evaluate with just dental plaques, since they stain and disclose not only dental plaques but also pellicle formed with salivary glycoproteins on a tooth surface. In this present study, we have demonstrated that HRP interacted with the cell wall component of the major gram-positive bacterial peptidoglycan, but not the major cell wall component of gram-negative bacteria lipopolysaccharide. Furthermore, we observed that the adsorbed HRP labeled with fluorescence was detected on the major oral gram-positive strains S. sanguinis and Streptococcus salivarius (S. salivarius), but not on a gram-negative strain, Escherichia coli (E. coli). Furthermore, we have demonstrated that the combination of HRP and chromogenic substrate clearly disclosed the dental plaques and the biofilm developed by S. sanguinis, S. salivarius and the major gram-postive bacteria Lactobacillus casei on tooth surfaces, and slightly disclosed the biofilm by E. coli. The combination of HRP and chromogenic substrate did not stain either the dental pellicle with the salivary glycoprotein mucin, or naked tooth surfaces. These results have suggested the possibility that the adsorption activity of HRP not only contributes to the evaluation of dental plaque, but that enzymatic activity of HRP may also contribute to improve dental hygiene.

Highly reliable, targeted photothermal cancer therapy combined with thermal dosimetry using a near-infrared absorbent.

Photothermal therapy (PTT) using a photo-absorbent in the near-infrared (NIR) region is an effective methodology for local cancer treatment. Before PTT using a NIR absorbent is executed, the operator generally determines the two parameters of fluence rate and irradiation time. However, even if the irradiation parameters are unchanged, the therapeutic effect of PTT is often different for individual tumors. Hence, we examined the therapeutic effect of PTT using a NIR absorbent (ICG lactosome) while changing two parameters (fluence rate and irradiation time) in various combinations. As a result, there was no robust correlation between those parameters and the therapeutic effect. Compared to those parameters, we found that a more reliable determinant was maintenance of the tumor temperature above 43 °C during NIR irradiation. To reconfirm the significance of the determinant, we developed a new system that can regulate the temperature at the NIR irradiation site at a constant level. By using the new system, we verified the treatment outcomes for tumors in which the NIR absorbent had accumulated. All of the tumors that had been kept at 43 °C during NIR irradiation were cured, while none of the tumors that had been kept at a temperature below 41 °C were cured. In conclusion, PTT using a NIR absorbent with thermal dosimetry is a highly reliable treatment for cancer.

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles.

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

Development of an experimental method of systematically estimating protein expression limits in HEK293 cells.

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.

The Golgin Protein Giantin Regulates Interconnections Between Golgi Stacks.

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi.