Innexin Gap Junctions

Manipulating Innexin subunit protein levels

Image showing how transgenic expression of an innexin subunit can alter innexin protein detection levels in salivary gland cells

Figure legend: Transgenic expression of an innexin subunit successfully alters the amount of that protein within cells. A, Image of a pupal salivary gland that is expressing UAS-Ogre-myc (a wild type Ogre gene tagged with myc epitope) under the transcriptional control of C96-GAL4. The tissue has been stained with antibodies to detect myc epitope (green) and endogenous Inx3 protein (red). It is immediately obvious that individual cells express different quantities from the transgene. Cells 1+2 are expressing less UAS-Ogre-myc than their neighbours. Strong UAS-Ogre-myc expressing cells 3+4 are producing so much protein that it appears to build-up in the cytoplasm giving hese cells a green background tint compared to cells 1+2. B, Measurements of Ogre-myc and Inx3 fluorescence intensity in annular junction vesicles (Description of annular junctions) within individual cells confirms what is visually apparent in panel A: cells 3+4 express much more Ogre-myc than cells 1+2. The level of endogenous Inx3 appears to remain stable despite the expression of UAS-Ogre-myc. Error bars = 1 standard deviation. Y-axis = fluorescent intensity (0-255 units).

Image showing relative levels of an endogenous innexin subunit compared to a transgenically expressed subunit

Figure legend: The relative levels of Inx3 and Ogre-myc proteins differ within subcellular domains. C, close-up image of annular junction vesicles from a pupal salivary gland cell expressing endogenous Inx3 (red) and transgenic Ogre-myc (green). The merged image suggests that some vesicles possess an equivent level of Ogre-myc and Inx3 (*, based on the yellow colour), some vesicles possess less Ogre-myc than Inx3 (arrow, vesicle in more red than yellow) and some have more Ogre-myc than Inx3 (arrowhead, vesicle is more green than yellow). D, A plot of the ratio of Inx3 and Ogre-myc fluorescence intensity measured from a population of individual annular junctions within single cells indicates that the relative protein levels is quite variable in these subcellular objects. Cells 1-4 correspond to those in image A.

The transgenic expression of proteins is an important tool in the arsenal for analysing protein function. The experiments described here indicate that transgenically expressed innexin protein is incorporated into the subcellular structures/domains that normally contain endogenous innexins. In image A (above), myc-tagged Ogre is detected in annular junctions but it can also be found in plaques (not shown). The fact that transgenically supplied innexins can rescue plaque formation involving other innexin family subunits (Rescue of Inx3 plaques in an inx2null mutant, Lehmann et al, 2006) and can rescue loss-of-function phenotypes (Curtin et al, 2002) also confirms that transgenic innexin proteins are produced and can interact with their normal partners whether they be other innexin family members or proteins involved in transport, junction formation or degradation. This approach may consequently prove fruitful in experiments that attempt to alter the stoichiometry of subunits within channels or to incorporate modified innexins and observe their biological effect. Moreover, endogenous innexin protein levels do not appear to be affected by different levels of transgenic expression (graph B). The observations raise some issues:

  • - high levels of transgene expression can lead to a build-up of protein in the cytoplasm which could have unanticipated consequences.
  • - Transgenic expression of innexins, even in cells that normally express that particular innexin, can lead to developmental defects via unknown mechanisms. (Images: UAS-ogre induces leg joint defects, Endogenous Ogre in leg discs, Perils of Innexin transgene expression)
  • - even within cells of the same tissue, some cells express significantly different amounts of transgene (as shown in salivary cells, image A above, when using the GAL4/UAS system (Brand and Perrimon, 1993)). Phenotypic effects could prove highly variable. This lack of consistency could become annoying if the phenotype under observation is labour intensive and quantitative eg.requires dye-coupling or electrophysiological measurements taken from neighbouring pairs of cells.

There are a number of possible explanations why the ratio of Inx3 to Ogre-myc levels (graph B and red/green ratios in plot D) are so variable:

  • - polyclonal antibodies were used to detect endogenous Inx3 protein. This will naturally introduce some variability and could be responsible for the differences in Inx3 staining intensity between cells 1-4 in plot B. But the error bars suggest that the staining is pretty consistent.
  • - transgenic Ogre-myc may be incorporated inconsistently into membrane in the golgi apparatus (due to steric impedance caused by the myc epitope); some plaques may then receive more Ogre subunits and some less.
  • - Myc-tagged Ogre may be degraded at a different rate than Inx3 in annular junctions.
  • - The annular junctions within a single cell will contain an Ogre-myc protein contribution recieved from all of the gap junction-coupled neighbouring cells (Description of annular junctions). Annular junctions produced from neighbours that express transgene strongly will have a much lower ratio of Inx3 to Ogre-myc (red/green ratio) than annular junctions produced from weakly expressing neighbours.

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