Visualising innexin distribution and gap junctions in large cells
Figure legend: Image of an early pupal-stage salivary gland cell revealing the position of the gap junction protein subunit Innexin 2 (green). Blue = nuclei. Red = F-actin. The image is a projection of 100 Z-series optical sections through the cell. Two distinct populations of innexin staining are observed: plaques at the plasma membrane and large internal objects. The projected image gives the impression that there is a continuous large gap junction plaque around the cell, however, single sections (see Inx plaque morphology) reveal instead that there are lots of disconnected little plaques in the cell membrane.
One of the advantages of studying connexin-based vertebrate gap junctions is that the cells in which they are expressed are generally quite large (particularly in cultured cell in vitro experiments) making it easier to visualise connexin distribution and gap junction morphology. Although innexins are expressed in most Drosophila cells (Stebbings et al. 2002) these cells are generally too small to get a good idea of the distribution of innexin proteins within the cell or the overall morphology of innexin-based gap junction plaques. Many of the images on this website are pictures of innexins in pupal salivary gland cells because:
- - salivary gland cells are very large (approx. 50μm in diameter).
- - salivary gland cells express the endogenous innexins Ogre (Inx1)(Ogre review), innexin 2 (Inx2 review) and innexin 3 (Inx3 review) which can be visualised using existing innexin-specific antibodies.
- - glands are easy to dissect out of the developing animal for subsequent antibody staining.
A brief description of larval/pupal salivary glands is appropriate here in order to follow some of the observations described elsewhere on the website. The salivary gland is essentially a sack of cells, the center of the sack being an empty space (the lumen) that forms a tube leading off towards the mouth. Immediately prior to pupation, glue is secreted from salivary gland cells into the lumen and out through the mouth, enabling the pupa to attach itself to a surface. During pupation the salivary gland cells initiate a process of programmed cell death (apoptosis) and disintegrate. Salivary gland cells are polarized; they have a top (apical- the lumen end) and a bottom (basal- the end exposed to surrounding fly tissues). A detailed description of salivary glands can be found on The Interactive Fly website.
Examination of the distribution of gap junction proteins in these large cells reveals that they are found at a number of distinct subcellular locations:
- - Innexins are found at the plasma membrane…as expected for gap junction forming proteins. In the projected image above, Inx2 protein outlines the cell. Inx3 and Ogre (Ogre in pupal salivary gland cells) are also located at the plasma membrane. Interestingly, one innexin , Inx7 has been reported at the cell membrane, but also in the nucleus of certain cell types (Ostrowski et al, 2008).
- - Innexins also appear in large internal vesicles (green spots within the cell in the image above) - these are putative annular junctions (Close-up images of Drosophila annular junctions) - double membrane vesicles associated with the internalisation of gap junctions.
- - A population of innexin-containing small vesicles is also detected within the cell but cannot be seen in the Z-series projection image above. But they can be seen in single optical slices here.
- - A diagram representing the location of the main subcellular locations occupied by innexin proteins is presented here. Note that different cell types can express multiple innexin family members and the different family members do not always occupy similar positions in the cell. For example, in follicle cell plasma membranes Ogre is predominantly baso-lateral whereas co-expressed endogenous Inx2 is mostly located at the basal pole/baso-laterally (see Distribution of Ogre, Inx2, Inx3 and Zpg in the follicle).