A Model for Integrin Inside-Out Activation and Clustering

<p>Cellular stimulation induces a conformational change in talin that exposes its talin head domain. The talin head domain binds to the β cytoplasmic tail, which displaces the α tail from its complex with the β tail, which in turn leads to an unclasping and a membrane-associated structural change of the cytoplasmic face (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Vinogradova1" target="_blank">Vinogradova et al. 2002</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Vinogradova2" target="_blank">2004</a>). Notice the proposed shifted membrane interface for both membrane-proximal helices before and after unclasping (green bars), which suggests a “fanning-out” unclasping process (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Vinogradova2" target="_blank">Vinogradova et al. 2004</a>). The unclasping initiates the opening of the integrin C-terminal stalks—including the transmembrane domains (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Luo1" target="_blank">Luo et al. 2004</a>)—which is necessary for the switchblade shift of the extracellular headpiece from the bent to the extended form for high-affinity ligand binding (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Takagi2" target="_blank">Takagi et al. 2002</a>). The α subunit is in blue and the β subunit is in red. The ligated integrins cluster, possibly via oligomerization of transmembrane domains (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Li1" target="_blank">Li et al. 2003</a>). The model was generated based on the crystal structure of α_vβ₃ extracellular domain (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Xiong1" target="_blank">Xiong et al. 2001</a>) and the nuclear magnetic resonance structure of the cytoplasmic domain (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Vinogradova1" target="_blank">Vinogradova et al. 2002</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020169#pbio-0020169-Vinogradova2" target="_blank">2004</a>) with the helices extending to the transmembrane domain.</p

COUP-TFII is not Essential for Maintenance of Leydig Cell Function.

<p>A) Histological examination of testis and epididymis. Spermatazoa (arrow) were observed in testes and epididymis of 4-month-old control and Cre/+ F/F mice, which were treated with tamoxifen at the age of two months. B) Immunohistochemistry for Leydig cell markers, 3β-HSD, P450Scc and EST. C) Immunoblotting for COUP-TFII, 3β-HSD, P450Scc and EST. Tam OD indicates the day before tamoxifen injection when mice are two month old. Tam 60D means 60 days after tamoxifen injection.</p

Inducible Ablation of <i>COUP-TFII</i> at Pre-puberty Stage Leads to Infertility and Hypogonadism.

<p>A) Scheme of inducible ablation of <i>COUP-TFII</i> at Pre-puberty stage. Tamoxifen or oil was intraperitonealy injected into P14 animals to induce the deletion of <i>COUP-TFII</i> gene, and mice were sacrificed at P90. B) Immunoblotting of COUP-TFII was performed to examine the deletion efficiency in mutant mice, and tissues were collected from the comparison littermates. F/F, Tam: <i>COUP-TF^flox/flox</i> treated with tamoxifen: Cre/+ F/F, Oil: <i>Cre-ER^{TM (+/−)} COUP-TFII^flox/flox</i> treated with oil and Cre/+ F/F, Tam: <i>Cre-ER^{TM (+/−)} COUP-TFII^flox/flox</i> treated with tamoxifen. C) The photograph depicts the appearance of male reproduction organs from 3-month-old littermate. D) Relative weight of reproduction organs normalized with body weight. Results are expressed as the mean (±SD) of the ratios for each genotype. Statistical comparison was done with a student test. * P<0.05, ** P<0.01</p

COUP-TFII Plays Roles in Testis Organogenesis and Progenitor Leydig cell Formation.

<p>A) Immunohistochemistry for COUP-TFII at embryonic 18.5 (E18.5) and P7. Tamoxifen was injected to pregnant mothers at E18.5. B) Immunohistochemistry for Leydig cell markers, 3β-HSD at E18.5, P14 and P21. Arrow indicated progenitor Leydig cells, and arrowhead was fetal Leydig cells. C) Quantitative results of progenitor Leydig cell number peri-seminiferous tubule. Data in (C) indicate mean±SD. * P<0.05; ** P<0.01. D) H& E staining of the testes and epididymis from P60 littermate of control and mutant mice. Spermatazoa was indicated by arrow. Immunohistochemistry result for Leydig cell markers, 3β-HSD and EST. E) The photograph depicts the appearance of testes from control and mutant mice at E18.5 and P14.</p

Arrest of Spermatogenesis at the Round Spermatid Stage in the Null Mice.

<p>H&E staining of paraffin-embedded testes (A, A') and epididymis (B). A' is the large magnification of the box area in the A. (C) Quantitative realtime RT-PCR analysis of the germ cell differentiation markers. RNA was isolated from 3-months-old littermates. Expression levels of each gene were normalized to the levels of the 18sRNA (n = 6). Data in (C) indicate the mean±SD. * P<0.05; ** P<0.01</p

<i>COUP-TFII</i> Null Mice Display Leydig Cell Hypoplasia.

<p>(A) Immunohistological detection of COUP-TFII expression in the testes from adult wild type mice. Green, COUP-TFII; Blue: DAP1 (B) <i>COUP-TFII</i> deletion efficiency was examined by qRT-PCR. Testes were collected from the littermates. N = 6; ** P<0.01 (C) Serum testosterone, LH, and FSH levels in 3-month-old males: F/F; Tam, Cre/+ F/F; Oil and Cre/+ F/F; Tam. (n = 8, 10, and 7, respectively). (D-I) H&E staining of paraffin-embedded testes (D). Immunohistochemistry results of Leydig cell marker P450Scc (E), EST (F), 3β-HSD (G) and CYP19 (H) indicated that mutant mice display Leydig cell hypoplasia. However, Sertoli cells in the null mice are normal (I).</p

Indispensable Roles of COUP-TFII in Progenitor Leydig Cell Differentiation.

<p>A) Evaluation of Leydig cell differentiation in the mutant testes, indicated by Leydig cell marker 3β-HSD immunostaining. The samples were collected at P14, P21, P28, P60 and P90, which correspond to 0, 7, 14, 46 and 76 days after tamoxifen injection, respectively. B) Testosterone treatment could not rescue the hypoplasia of Leydig cells. There were no increases in the intensity of P450Scc (C) and EST (D) signals or localization changes of 3β-HSD (B) signals in the rescued animal. E) qRT-PCR analysis of the expression of Leydig cell marker. RNA was isolated from 3-months-old littermates implanted with control or testosterone pellet (n = 6). Expression levels of each gene were normalized to the levels of the 18sRNA. Data in (E) indicate mean±SD. * P<0.05; ** P<0.01</p

Hypogonadism and Spermatogenesis Defects were Rescued by Testosterone Replacement.

<p>Gross appearance of testes and accessory glands after testosterone treatment. The size of testes, epididymis, seminal vesicles (A) and prostate (B) grew markedly in Cre/+ F/F mice implanted with testosterone pellet compared with the null mice implanted with control pellet. H& E staining demonstrates the resumption of spermatogenesis in the null mine treated with testosterone. The elongated spermatid or spermatozoa could be observed in the mutant testes (C, D) and epididymis (E). In addition, loss of secretary protein in seminal vesicles could be observed in the rescued mice (F; arrow). A, anterior prostate; V ventral prostate; DL, dorsal-lateral prostate.</p

Arylations of Substituted Enamides by Aryl Iodides: Regio- and Stereoselective Synthesis of (<i>Z</i>)‑β-Amido-β-Arylacrylates

Arylations of substituted enamides by aryl iodides were achieved for the first time via an unusual PdCl₂(COD)/Ag₃PO₄ catalytic system. A broad range of (<i>Z</i>)-β-amido-β-arylacrylates were prepared regio- and stereoselectively in a highly efficient manner

Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane

<div><p>RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of <i>PAH1</i> (<u>p</u>hosphatidic <u>a</u>cid phospho<u>h</u>ydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in <i>pah1Δ</i> yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in <i>pah1</i>Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the <i>Arabidopsis</i> Pah2p in <i>Nicotiana benthamiana</i> decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.</p></div