Transferrin receptor recycling in the absence of perinuclear recycling endosomes

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.     

Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed,using the Arabidopsis genome sequence and radish genetic mapping

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by H. C. BeckerS. Giancola and S. Marhadour contributed equally to this work     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.     

Wild type and mutant p53 differentially regulate the gene expression of human collagenase-3 (hMMP-13)

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions including arthritis and cancer metastasis. Recently we have shown for the first time that the human MMP-1 gene is a p53 target gene subject to repression by wild type p53 (Sun, Y., Sun, Y. I., Wenger, L., Rutter, J. L., Brinckerhoff, C. E., and Cheung, H. S. (1999) J. Biol. Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down-regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter- and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis.     

The Arabidopsis AtEPR1 extensin-like gene is specifically expressed in endosperm during seed germination

Screening of 10 000 Arabidopsis transgenic lines carrying a gene-trap (GUS) construct has been undertaken to identify markers of seed germination. One of these lines showed GUS activity restricted to the endosperm, at the micropylar end of the germinating seed. The genomic DNA flanking the T-DNA insert was cloned by walking PCR and the insertion was shown to be located 70 bp upstream of a 2285 bp open reading frame (AtEPR1) sharing strong similarities with extensins. The AtEPR1 open reading frame consists of 40 proline-rich repeats and is expressed in both wild-type and mutant lines. The expression of the AtEPR1 gene appears to be under positive control of gibberellic acid, but is not downregulated by abscisic acid during seed germination. No expression was detected in organs other than endosperm during seed germination. The putative role of AtEPR1 is discussed in the light of its specific expression in relation to seed germination.