Cell Structure and Function

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors.

Publication Date: 2012 Jan 24 PMID: 22277578
Authors: Kamioka, Y. - Sumiyama, K. - Mizuno, R. - Sakai, Y. - Hirata, E. - Kiyokawa, E. - Matsuda, M.
Journal: Cell Struct Funct

Genetically-encoded biosensors based on the principle of Forster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

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Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus.

Publication Date: 2012 Jan 17 PMID: 22251795
Authors: Yamamoto-Hino, M. - Abe, M. - Shibano, T. - Setoguchi, Y. - Awano, W. - Ueda, R. - Okano, H. - Goto, S.
Journal: Cell Struct Funct

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial- and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120kDa and Syntaxin16 (Syx16); however the anti-120kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.

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Ultraviolet A induces endoplasmic reticulum stress response in human dermal fibroblasts.

Publication Date: 2012 Jan 17 PMID: 22251794
Authors: Komori, R. - Taniguchi, M. - Ichikawa, Y. - Uemura, A. - Oku, M. - Wakabayashi, S. - Higuchi, K. - Yoshida, H.
Journal: Cell Struct Funct

The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism against the accumulation of unfolded proteins in the ER (ER stress) that consists of three response pathways (the ATF6, IRE1 and PERK pathways) in mammals. These pathways regulate the transcription of ER-related genes through specific cis-acting elements, ERSE, UPRE and AARE, respectively. Because the mammalian ER stress response is markedly activated in professional secretory cells, its main function was thought to be to upregulate the capacity of protein folding in the ER in accordance with the increased synthesis of secretory proteins. Here, we found that ultraviolet A (UVA) irradiation induced the conversion of an ER-localized sensor pATF6alpha(P) to an active transcription factor pATF6alpha(N) in normal human dermal fibroblasts (NHDFs). UVA also induced IRE1-mediated splicing of XBP1 mRNA as well as PERK-mediated phosphorylation of an alpha subunit of eukaryotic initiation factor 2. Consistent with these observations, we found that UVA increased transcription from ERSE, UPRE and AARE elements. From these results, we concluded that UVA irradiation activates all branches of the mammalian ER stress response in NHDFs. This suggests that the mammalian ER stress response is activated by not only intrinsic stress but also environmental stress.

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Proteomic Screening for Rho-kinase Substrates by Combining Kinase and Phosphatase Inhibitors with 14-3-3zeta Affinity Chromatography.

Publication Date: 2012 Jan 17 PMID: 22251793
Authors: Nishioka, T. - Nakayama, M. - Amano, M. - Kaibuchi, K.
Journal: Cell Struct Funct

The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.

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Mechanical constraint converts planar waves into helices on tunicate and sea urchin sperm flagella.

Publication Date: 2012 Jan 13 PMID: 22130451
Authors: Ishijima, S.
Journal: Cell Struct Funct

The change in the flagellar waves of spermatozoa from a tunicate and sea urchins was examined using high-speed video microscopy to clarify the regulation of localized sliding between doublet microtubules in the axoneme. When the tunicate Ciona spermatozoa attached to a coverslip surface by their heads in seawater or they moved in seawater with increased viscosity, the planar waves of the sperm flagella were converted into left-handed helical waves. On the other hand, conversion of the planar waves into helical waves in the sea urchin Hemicentrotus spermatozoa was not seen in seawater with an increased viscosity as well as in ordinary seawater. However, the sea urchin Clypeaster spermatozoa showed the conversion, albeit infrequently, when they thrust their heads into seawater with an increased viscosity. The chirality of the helical waves of the Clypeaster spermatozoa was right-handed. When Ciona spermatozoa swam freely near a glass surface, they moved in relatively large circular paths (yawing motion). There was no difference in the proportion of spermatozoa yawing in either a clockwise or counterclockwise direction when viewed from above, which was also different from that of the sea urchin spermatozoa. These observations suggest that the planar waves generally observed on the sperm flagella are mechanically regulated, although their stability must depend on the Ca(2+) concentration in the cell. Furthermore, the chirality of the helical waves may be determined by the intracellular Ca(2+) concentration and changed by transmitting the localized active sliding between the doublet microtubules around the axoneme in an alternative direction.

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