The anti-oxidative property of mesoporous silica nanoparticles (MSNs) has been proposed previously, which prompted us to investigate the potential protective effect of MSNs on human embryonic stem cells (hESCs) against oxidative stress. To this purpose, the cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Apoptosis was analyzed by Annexin V/propidium iodide double-staining method. The intracellular glutathione, superoxide dismutase and malondialdehyde were measured with commercial assay kits. The reactive oxygen species was detected by staining with fluorescent dye DCFH-DA. The relative levels of Nkx2.5, Mef2c, Tbx5, dHand and α-MHC transcripts were measured by real-time polymerase chain reaction. The protein levels of Connexin 43, Troponin C1 and GAPDH were determined by immunoblotting. The beating behavior of embryoid bodies (EBs) was visually examined. Our results demonstrated that MSNs reversed hydrogen peroxide (H2O2)-inhibited cell viability and ameliorated H2O2-induced cell apoptosis in vitro. The H2O2-elicited intracellular oxidative stress was significantly relieved in the presence of MSNs. Furthermore, MSNs improved H2O2-suppressed differentiation of hESC-derived EBs and the maturation of the cardiomyocytes. In addition, MSNs treatment enhanced the beating properties of EBs. MSNs effectively conferred protection on hESCs against oxidative stress with respect to cardiac differentiation.
Key words: Mesoporous silica nanoparticles, hydrogen peroxide, human embryonic stem cells, differentiation
The Golgi apparatus is a central station for protein trafficking in eukaryotic cells. A widely accepted model of protein transport within the Golgi apparatus is cisternal maturation. Each cisterna has specific resident proteins, which are thought to be maintained by COPI-mediated transport. However, the mechanisms underlying specific sorting of these Golgi-resident proteins remain elusive. To obtain a clue to understand the selective sorting of vesicles between the Golgi cisterenae, we investigated the molecular arrangements of the conserved oligomeric Golgi (COG) subunits in yeast cells. Mutations in COG subunits cause defects in Golgi trafficking and glycosylation of proteins and are causative of Congenital Disorders of Glycosylation (CDG) in humans. Interactions among COG subunits in cytosolic and membrane fractions were investigated by co-immunoprecipitation. Cytosolic COG subunits existed as octamers, whereas membrane-associated COG subunits formed a variety of subcomplexes. Relocation of individual COG subunits to mitochondria resulted in recruitment of only a limited number of other COG subunits to mitochondria. These results indicate that COG proteins function in the forms of a variety of subcomplexes and suggest that the COG complex does not comprise stable tethering without other interactors.
Key words: The Golgi apparatus, COG complex, yeast, membrane trafficking, multi-subunit tethering complex
For more than a century, hematoxylin and eosin (H&E) staining has been the de facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.
Key words: in vivo imaging, histology, machine learning, molecular activity
Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galβ1-3GlcNAcβ1-2Manα1-6[Galβ1-3GlcNAcβ1-2Manα1-3]Manβ1-4GlcNAcβ1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.
Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H
Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.
Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube
The Warburg effect is one of the hallmarks of cancer cells, characterized by enhanced aerobic glycolysis. Despite intense research efforts, its functional relevance or biological significance to facilitate tumor progression is still debatable. Hence the question persists when and how the Warburg effect contributes to carcinogenesis. Especially, the role of metabolic changes at a very early stage of tumorigenesis has received relatively little attention, and how aerobic glycolysis impacts tumor incidence remains largely unknown. Here we discuss a novel paradigm for the effect of the Warburg effect that provides a suppressive role in oncogenesis.
Key words: Warburg effect, aerobic glycolysis, cell competition, EDAC