illustration of Innexin distribution in a cell
illustration of Innexin distribution in a cell
Figure legend: Daigram of two coupled pupal salivary gland cells illustrating the three regions where innexin protein is consistently observed: 1/ the most intense innexin staining is always in the large vesicles (green, presumably annular junctions) just below the basal (non-lumen) surface of the cell. 2/ gap junction plaques (red) are detected in the lateral plasma membrane with decreasing intensity going from the basal-to-apical direction (Image: Inx2 protein detection patterns vary with depth through polarised cells). Plaques are very uncommon in the plasma membrane region located roughly between the apical end of the nucleus and the apical cell surface (near the lumen) - as illustrated in the 3D reconstructions presented below. 3/ the small vesicles around the nucleus (black) reflect innexins being incorporated into membrane in the golgi complex or early stage transport vesicles budding from the golgi complex prior to transport of innexin to the plasma membrane and incorporation into plaques.
The Innexin distribution pattern described above is based on separate immunocytochemical experiments to detect endogenous Inx2, Inx3 and Ogre protein. All three of these innexins are found in these locations in pupal salivary gland cells. Note that even within this single cell type the subcellular location of plaques (Image: Inx cellular distribution changes with age) and quantity of innexin protein incorporated into the cell membrane (see webpage Inx protein levels change over time) is dynamic and changes according to the developmental stage of the cells. Descriptions of innexin antibody staining in the published literature generally do not distinguish between different subcellular locations - understandably, given the small size of the cells involved. One notable exception describes the distribution of four innexins in cells of the follicle. In that study, Bohrmann and Zimmermann (2008) also observed that innexin distribution is dynamic eg. Inx2 is found as a cytoplasmic cloud after stage 10 in nurse cells but is transported into the oocyte chamber as nurse cells regress (Image: Distribution of Ogre, Inx2, Inx3 and Zpg in the follicle). Comparison of their descriptions to the distributions observed in the pupal salivary gland indicate that subcellular innexin distribution differs significantly between cell types. All of the innexin-immunopositive objects, and their general subcellular locations, are suprisingly similar to previously described connexin subcellular distribution patterns: plaques (Falk, 2000), annular junctions (Leithe et al. 2006) and transport vesicles (Gaietta et al. 2002).
The model above is based on anti-innexin antibody-stained tissues and 3D rendering of image-arrays generated by scanning confocal microscopy as shown below
Figure legend: Generation of 3D reconstructions from series of 2D optical sections in order to better visualise innexin distribution and plaque structure. A, Diagram illustrating a pair of pupal salivary gland cells showing the location of the series of 2D optical sections (grey box extending from basal to apical ends of cells) used to generate the 3D reconstruction. B, 3D reconstruction of the interface between two neighbouring wild type salivary gland cells (similar to the area of the grey box shown in diagram A). Inx2-containing plaques (p) were detected using polyclonal rabbit anti-Inx2 antibody and a monoclonal anti-rabbit Cy5-conjugated secondary antibody (red). The Na+ / K+ ATPase α subunit (green) of the sodium pump (s) was detected as fluorescence from the gfp-tagged endogenous gene (line ATPαG00109) and is most strongly fluorescent at the apical pole of the cells (this is easier to see in the rotating 3D structures shown in C and D). Small sections of the cell nuclei (n) from each of the neighbouring cells (blue, DAPI) can be seen in the 3D reconstruction. The image processing involved in making 3D reconstructions removes weaker staining, thus, innexin-immunopositive transport vesicles near the nucleus cannot be seen. C+D, Rotation of the reconstructed 3D volume shows the position of the nuclei (blue) approximately half-way between the cell poles. Inx2 is present in plaques in the baso-lateral cell membrane and annular junctions at the basal end of the cell. Na+ / K+ ATPase α (green) is present throughout the plasma membrane but is most intense at the apical end of the cells - opposite to Inx2. Four hundred optical 2D Z-sections were recorded to include the whole length of the cells and 3D reconstruction and animation was performed using ImageJ software