Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the Drosophila stomach

Abstract

Copper cells were originally identified in Drosophila midgut epithelium by their striking orange fluorescence in copper-fed larvae. Here, we examined copper cell fluorescence in light of the previous observations that (1) a similar fluorescent signal in yeast is produced by a complex between copper and metallothionein, and (2) metallothionein is expressed constitutively in the copper cell region and inducibly in other regions of the Drosophila midgut. Pulse-feeding experiments with 1 mM CuCl2 revealed that fluorescence appeared rapidly in copper cells (<5 min) and slowly in other cells of the midgut (days), suggesting a constitutive cofactor in the former and an inducible cofactor in the latter. Fluorescence was also detected in Drosophila S2 tissue culture cells after induction of metallothionein synthesis by addition of CuCl2 to the growth medium. Thus, fluorescence coincided spatially and temporally with the expression of metallothionein. Fluorescence was also linked to the acid-secreting activity of copper cells. Fluorescence was not observed when acid secretion was inhibited by a mutation in the alpha spectrin gene and acidification was blocked in copper-fed wild-type larvae. However, acidification was restored after a 1-day chase period in which the fluorescent signal became sequestered within a vesicular compartment. We therefore conclude that copper cell fluorescence is most probably attributable to a cytoplasmic copper-metallothionein complex, suggesting an unanticipated role for metallothionein in acid-secreting cells.

Homeostatic Mechanisms for Iron Storage Revealed by Genetic Manipulations and Live Imaging of Drosophila Ferritin

“…Our finding that ferritin expression in the iron region is constitutive (and remains so even under iron-deficient conditions), but is inducible in the anterior midgut, has a parallel with the expression of metallothioneins, which are the copper storage proteins (Egli et al 2006). Interestingly, cuprophilic cells that are present anterior to the iron region show constitutive metallothionein expression (Mcnulty et al 2001), whereas cells in both the anterior and posterior midgut induce metallothionein expression and copper/metallothionein fluorescence at higher copper concentrations (Poulson and Bowen 1952;Mcnulty et al 2001) (Figure 5J). Results presented in this article, combined with previous reports, lead to the following fundamental conclusion with respect to metal metabolism in the midgut of Drosophila: the insect midgut contains two sets of specialized cells, one of which constitutively expresses metallothionein and a second constitutively expresses ferritin.…”

“…(I) Cumetallothionein fluorescence (orange, asterisks) is prominent in the posterior midgut. (J) Cu-metallothionein fluorescence is also found in a cluster of cells anterior to the iron region, where constitutive metallothionein expression has been reported (Mcnulty et al 2001). (K and L) Time course of iron-dependent ferritin induction in (K) anterior midgut and (L) middle midgut dissected from third instar larvae.…”

“…Copper-containing (cuprophilic) cells function in the acidification of the midgut and are present at different sites than the iron region (Poulson and Bowen 1952;Hoppler and Bienz 1994;Dubreuil et al 2001). Copper-metallothionein complexes in these cells fluoresce orange-red upon ultraviolet illumination (Mcnulty et al 2001). We imaged simultaneously the copper-metallothionein fluorescence and GFP-ferritin in midguts from larvae fed a diet containing 1 mm Cu…”

The orphan pentameric ligand-gated ion channelpHCl-2is gated by pH and regulates fluid secretion inDrosophilaMalpighian tubules

“…Copper cells are the principal site of acid secretion in the midgut, and, like the Malpighian tubules, are thought to transport protons via apically localized V-type H + -ATPases (Dubreuil, 2004;Shanbhag and Tripathi, 2005). While a clear relationship between H + -ATPase output and copper uptake has yet to be elucidated, it has been shown that copper uptake is impaired in flies whose copper cells are deficient in acid secretion, and that the copper cell midgut region is less acidic following copper feeding (Dubreuil, 2004;McNulty et al, 2001). Similar to our proposed model in the Malpighian tubules, pHCl-2 may influence ATPase output in copper cells by regulating chloride counter-ion availability, which, in turn, could affect the rate of copper uptake.…”

The Digestive Tract of Drosophila melanogaster

“…The middle midgut, notably the copper-cell region, is devoted to the absorption of these metals (63,118). This region is stained by Prussian blue because of the presence of iron and becomes bright luminescent orange upon www.annualreviews.org • The Digestive Tract of Drosophila melanogastercopper ingestion as a result of the fixation of copper by metallothionein (58,111).…”