Our results are thus consistent with findings in vertebrates that suggest a close link between space junction and adherens junction biogenesis

Our results are thus consistent with findings in vertebrates that suggest a close link between space junction and adherens junction biogenesis. Our experiments further indicate that Coracle, a homologue of the vertebrate membrane-skeleton Protein 4.1, may be another Innexin 2 conversation. study, to our knowledge, of cellular distribution and proteinCprotein interactions of an Innexin space junctional channel protein in the developing epithelia of mutants have been as yet recognized in and has demonstrated an essential role for these space junctional channel proteins in electrical signal transmission. Mutations in the and genes result in loss-of-coordination phenotypes (Starich mutants display feeding defects that result from impaired of the electrical coupling between pharynx muscle tissue (Starich and genes interfere with transmission at electrical synapses of the giant fiber system (Phelan gene was recently shown to play a role in the survival of early germ cells during gametogenesis (Tazuke is required to control foregut morphogenesis in response to the Wingless/WNT signaling cascade (Bauer genes have been extensively analyzed by in situ hybridization during oogenesis and embryogenesis, virtually no studies exist on protein distribution, trafficking, assembly, turnover, or interactions with other cellular proteins. Here, we present the first study of space junction protein distribution in epithelial cells. We demonstrate an essential function for Innexin 2 for epithelial morphogenesis, and we provide evidence of conversation between Innexin 2 and other junctional core proteins in polarized epithelial cells. MATERIALS AND METHODS Cloning and Transgene Production An fragment corresponding to the full-length fusion construct a polymerase chain reaction (PCR) fragment coding for full-length was cloned via fusion fragment was excised Reversine using P16 that was used in this study is usually a transcript-null allele (Bauer 5 was a gift from A. Fehon, Duke University or college, Durham, Rabbit Polyclonal to PPP1R7 NC. Homozygous mutant balancer. High levels of ectopic Innexin 2 were induced by crossing the 2 2 lines with the ubiquitously driving maternal V32-Gal4 strain (gift from D. St. Johnston) or the effector strain (Xiao was obtained from the Bloomington Stock Center, Indiana University or college, Bloomington, IN. To express p35 together with innexin 2, w; virgin females. The effect of overexpression was analyzed in the progeny by performing anti-Innexin 2 staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Germ collection clones of P16 mutants were induced as explained by Chou and Perrimon (1996 ) by using BL#1844 as FRT collection and BL#1813 as ovoD1 collection (stocks were obtained from the Bloomington Stock Center). Immunohistochemistry For antibody staining, embryos were fixed in 4% formaldehyde, 100 mM phosphate buffer. Normally, antibody staining was performed as explained previously (Bauer embryos (0C24 h) were collected, rinsed with water, dechorionated with 50% bleach for 3 min, and rinsed with 0.1% Triton X-100. For the following immunoprecipitation, the embryos were homogenized either in radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris [pH 8.0]) or immunoprecipitation (IP) buffer (CHAPS-buffer; Laing PCR product was Reversine generated using the LD 11658 clone as template together with the following primers: 5-ggg atc ccg Reversine aag tcc tgg gaa ggc gga-3 (5 primer), and 5-cga gct cgt cgg aag gcg tag aaa tt-3 (3 primer). Subsequently, the Innexin 2 PCR amplification product was cloned in frame with hSOS into the bait vector via the 5-cDNA library (catalog no. 200444; Stratagene) together with 100 g of the bait construct were transformed into the yeast temperature-sensitive mutant strain cdc25H. This strain contains a point mutation at amino acid residue 1328 of the defect and to activate the yeast Ras signaling pathway (for details, observe Broder embryo, we generated an anti-Innexin 2 antibody against a peptide made up of the C-terminal amino acids 297C310 of Innexin 2 (observe MATERIALS AND METHODS; Physique 1A). This antibody acknowledged the 43-kDa protein band of in vitro-translated Innexin 2 protein and a band of the same size in embryonic extracts of wild-type embryos (Physique 1B). To test the Reversine specificity of the antibody, we overexpressed an Innexin 2-GFP construct in a pair rule pattern in every other segment of the epidermis by using transgenic flies and performed immunostaining by using the Innexin 2 antibody. As shown in Physique 1, CCE, the antibody detects the stripe pattern in the ectopic expression experiment. In contrast, anti-Innexin 2 antibody staining was not detected in the epidermis (Physique 1, FCH) nor in other epithelial organs such as the salivary glands (Physique 1, ICK) of late zygotic mutants, whereas other proteins such as the -catenin homolog Armadillo are expressed. Consistent with these in vivo results, protein extracts of zygotic mutants, which are transcript null.