Zinquin identifies subcellular compartmentalization of zinc in cortical neurons. Relation to the trafficking of zinc and the mitochondrial compartment

Abstract

Zinquin (Zn 2+ selective fluorophore), when used to visualize intracellular Zn 2+, typically shows brightly fluorescent perinuclear endosome-like structures, presumably identifying Zn 2+ containing organelles. In this study, zinquin identified numerous and widespread sites of Zn 2+ compartmentalization in primary cultures of embryonic rat cortical neurons. Nuclear fluorescence, however, was absent. We labeled neuronal mitochondria with MitoTracker Green in the presence of zinquin and show that the fluorescent patterns of MitoTracker Green and zinquin were distinct and clearly different in both the perinuclear region and in processes. The mitochondrial compartment was much larger than the sum of the areas of zinquin fluorescence, as indicated by the small amount (<10% MitoTracker Green over zinquin) of overlap of MitoTracker Green on zinquin. Zinquin fluorescence was unaffected by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) treatment. The zinquin fluorescent objects were generally spherical in shape with a average diameter of about 0.6 μm. Most fluorescent objects, nearly two thirds on average, appeared to be docked, but both anterograde and retrograde movements were observed by time lapse image analysis. Although some fluorescent objects moved as much as 1 μm in 5 min, typical movements were smaller, usually 0.5 μm or less. Colchicine treatment caused striking aggregation of MitoTracker Green most noticeable in the perinuclear region. Zinquin fluorescence similarly showed reduced distribution throughout the cytoplasm, suggesting that zinquin fluorescent structures were associated with microtubules. Treatment with cytochalasin D had little noticeable effect on either the pattern of zinquin and MitoTracker Green fluorescence or their coincidence. Thus, numerous Zn 2+ sequestering organelles/structures are present in perinuclear regions and processes of cultured neurons and are sometimes found coincident with mitochondria. We demonstrated real time trafficking of sequestered Zn 2+, using zinquin fluorescence, apparently associated with an endosome-like compartment or protein complexes in the cytosol.