15 research outputs found

    The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells

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    <div><p>Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (<i>Xbp1</i>) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional <i>Xbp1</i> mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated <i>Ire1α</i> deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, <i>Ire1α</i> deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon <i>Ire1α</i> deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, <i>Ire1α</i> deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific <i>Ire1α</i> deletion. The results demonstrate that glucose activates IRE1α-mediated <i>Xbp1</i> splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.</p></div

    Data collection

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    a<p>This counts split data (i.e. far and near side) as different data sets (see text for details).</p>b<p>All statistics are calculated over the respective number of data sets.</p>c<p>The phase residual is a measure for the homogeneity and quality of the data. Values below 45° are considered excellent; values below 55° are acceptable.</p

    Proximity of residue Arg<sup>406</sup> (Arg<sup>403</sup> in cardiac myosin) to the actin interface.

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    <p>Arg<sup>406</sup> (red spheres) is immediately adjacent to residues of the cardiomyopathy loop that were previously implicated in actin binding by docking studies (407–414; green). While the conformation of the Arg<sup>406</sup> in the smooth muscle myosin crystal structure points away from the interface (A), it can easily reach actin by simple, stereochemically permitted bond angle rotations (B). The resulting conformation does not generate serious clashes with other myosin residues. Myosin is shown in blue, the interacting actin filament subunits in grey. Residue Pro<sup>333</sup> of actin, the closest to myosin Arg<sup>406</sup>, is shown as spheres.</p

    Mechanism of Filament Nucleation and Branch Stability Revealed by the Structure of the Arp2/3 Complex at Actin Branch Junctions

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    <div><p>Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.</p> </div

    <i>KO</i> islets exhibit ER stress.

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    <p>(A) qRT-PCR of UPR genes in islets isolated 6 wk post-Tam and incubated in 11 mM glucose 16 h ([<i>n</i> = 5], [<i>p</i> ≤ 0.05]). (B) Immunofluorescence microscopy of pancreas sections stained for KDEL (BIP and GRP94) (green), the plasma membrane protein GLUT2 (red), and nuclei DAPI (blue). Overlap of red/green channels represents defective compartmentalization that was found to be increased in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> as shown in yellow. Scale bars, 400x = 50 μm, 1,000x = 10 μm, 5,180x = 2 μm and 10,500x = 1 μM. Additional examples are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s007" target="_blank">S3B Fig</a>. (C) EM of adult mouse (16 wk old) islets and their β cells from mice 2 wk post-Tam. Scale bars, both panels, 1 μm. Distended mitochondria are outlined with yellow dashes. (D) Conventional PCR flanking the 26 nt intron in <i>Xbp1</i> mRNA spliced by IRE1α from the islet complementary DNAs (cDNAs) used for mRNA-Seq analysis, 6 mM versus 18 mM glucose. Results representative of <i>n</i> = 5 per genotype. (E) Global heatmap for the ~22,000 mRNAs detected by mRNA-Seq for 18 mM <i>KO</i><sup><i>Fe/-; Cre</i></sup> & <i>WT</i><sup><i>Fe/+</i></sup> samples; green and red indicate increased and decreased expression. The blue box indicates genes with inverse expression dependent on IRE1α and high glucose.</p

    Difference and structural variability maps.

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    <p>4-nm wide slices perpendicular to the helix axis of several maps are shown on the left. Only peaks significant at a confidence level of 99.99% are shown. A: Wild-type smooth muscle actomyosin in the absence of nucleotide. The motor domain (MD) and light-chain (LC) regions are labeled. A faint ghost image of this map is also overlaid on C and E to aid visualization. B: A difference map generated by subtracting the R403Q mutant smooth muscle actomyosin apo state reconstruction from the R403Q mutant smooth muscle actomyosin ADP state reconstruction. A clear difference peak can be identified in the light-chain region. A faint ghost image of the wild-type ADP state reconstruction is overlaid to aid visualization. This image is also overlaid on D and F. C: Difference map between two independently generated R403Q mutant apo state reconstructions. Only occasional, randomly distributed, isolated pixels can be seen, no coherent difference peaks exist. D: Difference map between two independently generated R403Q mutant ADP state reconstructions. E: Structural variability (AVID map) of R403Q mutant apo state reconstruction. Only randomly distributed peaks can be seen, there is no consistent structural variability in any confined region. F: Structural variability (AVID map) of R403Q mutant ADP state reconstruction. Bar:10 nm. G: Surface representation of the difference map shown in B. The cyan density represents additional density in the R403Q mutant ADP state reconstruction if compared to the R403Q mutant apo state reconstruction. The apo state (pink) and ADP state (blue wireframe) wild-type reconstructions are also shown. The difference between the mutant reconstructions is located in the light-chain region and correlates with the changes observed in wild-type smooth muscle actomyosin.</p

    mRNA sequencing identifies IRE1α- and glucose-dependent mRNAs in islets.

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    <p>(A) mRNA-Seq data on β cell-specific mRNAs. The results show no significant change to INS1 or INS2 in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> samples, while MAFA, GCG, and PC5 are increased by deletion ([<i>n</i> = 5], [18 mM <i>KO</i><sup><i>Fe/-; Cre</i></sup>, <i>p</i>-values ≤ 0.05]). mRNA-Seq expression fold changes were normalized relative to the 6 mM <i>WT</i><sup><i>Fe/+</i></sup> islet context. (B) Four-way Venn diagrams of <i>WT</i><sup><i>Fe/+</i></sup> versus <i>KO</i><sup><i>Fe/-; Cre</i></sup> islets during 6 mM versus 1 8mM glucose exposur<i>e</i> for 72 h. <i>Ire1α</i>-dependent mRNAs are in bold italics, while those also dependent on high glucose are in bold, italicized, and underlined font. At the center, bar graphs representing the <i>Ire1α</i>- and glucose-dependent trends of interest are labeled “Induction” and “Repression.” (C) Combined <b>DAVID</b> (the Database for Annotation, Visualization and Integrated Discovery) and “ConceptGen” GO analysis of <i>Ire1α-</i> and glucose-dependent mRNAs. Categories shown are specifically found in the genotype, while the shared categories have been omitted for simplicity, although no single mRNA was common between the groups. (D) Mass spectrometry of murine islets infected with <i>Ad-IREα-K907A (Ad-ΔR)</i> versus <i>Ad-β-Galactosidase</i> (<i>β-Gal</i>). Proteins with ≥5 unique peptides detected per protein increased or decreased upon infection in triplicate were analyzed for GO using ConceptGen and DAVID web resources (<i>n</i> = 3). The proteins shown (Fig 3D) exhibit the same expression dependence for IRE1α as measured by mRNA-Seq (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s002" target="_blank">S2 Data</a>).</p

    Localization of the Labels Attached to Arp2, Arp3, Arc40/ARPC1, and Arc18/ARPC3 at the Actin Branch Junction

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    <div><p>Color codes used: Arp2 (pink), Arp3 (orange), Arc40/ARPC1 (green), and Arc18/ARPC3 (red).</p> <p>(A) 2D average projection maps of the branches obtained with Arp2-GFP (row 1), Arp3-GFP (row 2), Arc40/ARPC1-YFP (row 3), and Arc18/ARPC3-GFP (row 4).</p> <p>(B) Difference maps calculated between maps obtained with labeled and unlabeled complexes.</p> <p>(C) Difference maps superimposed with the projection maps. The position of the difference peaks was cross-validated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#s3" target="_blank">Materials and Methods</a>).</p> <p>(D) The average projection map obtained with the unlabeled complex.</p> <p>(E) The main difference peaks are superimposed with the unlabeled projection map.</p> <p>(F and G) Circles of 3.9-nm radius centered on the difference peaks indicate the possible locations of the C-termini of each labeled subunit. The GFP/YFP label was attached to the C-terminus of the relevant subunit with an eight-amino-acid flexible linker that in fully extended conformation can reach a length of up to approximately 3.2 nm. The distance of the N-terminus of GFP or YFP from the center of mass of its beta-barrel (14 × 8 × 8 nm) is approximately 2.5 nm. The centers of the peaks determined from the difference maps probably coincide with the center of mass.</p> <p>Bar = 10 nm.</p></div

    Structure Models of the Arp2/3 Complex at Actin Branch Junction

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    <div><p>Color codes used: Arp2 (light pink), Arp3 (orange), Arc40/ARPC1 (green), Arc35/ARPC2 (cyan), Arc19/ARPC4 (blue), Arc18/ARPC3 (dark pink), and Arc15/ARPC5 (yellow). Gray arrows indicate the mother and daughter filaments.</p> <p>(A) Orientation of the Arp2/3 complex relative to the mother and daughter filaments as determined using the labeling constraints.</p> <p>(B) Model rotated vertically anticlockwise by 90° from view in (A) and tilted so that the mother filament coincides with the vertical axis. The gray arrow is positioned to pass through the center of the complex.</p> <p>(C) Model rotated vertically by 180° from the view in (A).</p> <p>(D) Label positions and their corresponding C-termini localization. GFP or YFP, shown as ribbon diagram with the same color coding as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-g003" target="_blank">Figure 3</a>, were superimposed on the respective difference peak (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-g003" target="_blank">Figure 3</a>) with their orientation matching the peak shape.</p> <p>(E) Model superimposed on the projection density map (white corresponds to high density).</p> <p>(F) Model and ribbon diagram of a daughter filament (white) as it would grow after small relative rotations of Arp2 and Arp3 (see text) superimposed on the projection density map.</p> <p>(G and H) Model proposed by Beltzner and Pollard [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-b06" target="_blank">6</a>] (G) and by Aguda et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-b05" target="_blank">5</a>] (H) shown for comparison. Note that in (G), the daughter filament will be oriented out of the paper plane toward the reader.</p> <p>(I)Arp2/3 crystal structure in the same orientation as originally presented in Robinson et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-b12" target="_blank">12</a>].</p></div

    Surface representations of smooth muscle actomyosin constructs.

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    <p>The pointed end of the filaments is towards the top of the figure. A: Wild-type smooth muscle actomyosin in the presence of ADP. B: Wild-type smooth muscle actomyosin in the apo state. The contour level for A and B is chosen to represent the correct molecular mass. Note the well defined density and angle of the light-chain region (LC) C: R403Q mutant smooth muscle actomyosin in the presence of ADP. D: R403Q mutant smooth muscle actomyosin in the apo state. The contour level for C and D is chosen to show as much of the light chain domains as possible without completely obscuring the shape of the motor domain. E: Overlay of the maps in A–D. Color code and contouring as in A–D. F, G: Watershed segmentation of the maps in C (F) and D (G). These results reconfirm the orientation of the light-chain domains that correspond to those of the wild-type reconstructions (sketches) and the better definition of boundaries in the center of the filaments: the actin subunits are well segmented while there is no sub-segmentation of myosin domains as can be obtained for wild-type reconstructions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001123#pone.0001123-Volkmann4" target="_blank">[33]</a>. The sketches show central lines extracted from the density of the wild-type (grey) and the segmentation of the R403Q (black). Only the line segments extracted for the corresponding light-chain regions (LC) are shown, line segments corresponding to the motor domain region (MD) overlap almost completely for all maps.</p
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