Gap junction protein tissue distribution and abundance in the adult brain in drosophila

Abstract

The distribution of gap junction (GJ) protein in Drosophila tissues and developmental stages was determined by probing immuno-blots with an anti-Drosophila GJ protein antibody (R2AP18). All tissues and developmental stages examined contained 18, 24 or 72 kD GJ protein. GJ protein was notably abundant in immuno-blots of homogenates of adult brain tissue. This was confirmed by the direct visualization of GJs in thin sections of adult brain by electron microscopy. GJs were particularly large and numerous between glial cells in the optic lobes and peripheral glial sheath. R2AP18 reactivity was used to identify GJ protein in immunoblots of cell fractions from isolated adult heads. The final GJ-enriched pellets, derived by extracting crude membrane fractions with urea and N-lauroyl sarcosine, contained GJs with reduced profile widths (13–15 nm vs 16–18 nm for native GJs) and which, unlike native GJs in the crude membrane fractions, were immuno-labelled by R2AP18. Immuno-blots of the ureasarcosine extracted GJ pellets and supernatant contained higher molecular weight R2AP18 immuno-reactive proteins in addition to the 18 kD form which was present in the tissue homogenate and crude membrane fractions. The results confirm previous observations that urea-sarcosine causes alterations in GJ structure and suggest that urea-sarcosine treatment exposes antigenic determinant(s) which are unavailable for R2AP18 binding in non-extracted native GJs. The abundance of GJs in the adult brain and the relatively simple R2AP18 staining patterns in immuno-blots of GJ-enriched fractions from isolated adult heads suggest that this tissue will be useful for further biochemical and molecular studies of GJs in Drosophila.