A mitochondrion-specific dye for multicolour fluorescent imaging of Trypanosoma brucei.

An increasing number of antibody probes and markers for specific subcellular structures is becoming available to study the cell biology of Trypanosoma brucei. When investigating a novel gene, the subcellular localization of its product is often a first hint to function. With efficient heterologous tagging [1] and gene transfer methods, this approach is straight forward and simultaneous fluorescent imaging of one or several established markers for specific subcellular structures together with the protein at question provides the maximum of spatial information. Although multicolour fluorescence and digital image processing is now within the reach of most laboratories [2], the number of primary antibodies which can be simultaneously detected by indirect immunofluorescence is restricted. Thus, organelle specific dyes like 4,6-diamino-2-phenylindole (DAPI) for nuclear and kinetoplast DNA are invaluable tools. The mitochondrion of living trypanosomes has been visualized with the potential sensitive vital stain rhodamine 123 [3–5]. Since the distribution of rhodamine 123 in the cell cannot be fixed, this dye is not usable together with immunofluorescence and it is very difficult to capture images from stained cells [4]. Rhodamine 123 has been used to probe the mitochondrial membrane potential in different life cycle forms of T. brucei [3,4]. During differentiation of proliferating long slender bloodstream forms to non-dividing short stumpy forms and further to procyclic forms in the tsetse midgut, the single mitochondrion undergoes dramatic changes of metabolic function [6,7]. Correspondingly, different electrochemical potentials across the mitochondrial membranes produce a characteristic staining pattern for slender and stumpy bloodstream forms [3]. The aldehyde fixable dye MitotrackerTM green FM (Molecular Probes, Eugene, OR) has been used for staining mitochondria of yeast and mammalian cells [8– Abbre6iations: DAPI, 4,6-diamino-2-phenylindole; FITC, fluorescein isothiocyanate. * Corresponding author. Tel.: +49 89 85783972; fax: +49 89 85783810; e-mail: [email protected]