Sequestration of microinjected molecular probes from the cytoplasm of Amoeba proteus

The intracellular fate of different molecular probes (TRITC, TRITC-BSA, TRITC-BSA-Au(16)) microinjected into Amoeba proteus was analyzed in living and fixed cells with the fluorescence and electron microscope, respectively. Immediately after microinjection all of the probes distribute randomly without impeding the normal movement behavior or cellular morphology. However, within 45 min numerous aggregates appear in the cytosol which measure 0.25-1 μm in diameter and consist of the condensed molecular probe as well as an unknown homogeneous material. Larger aggregates are then encircled by elements of the rough endoplasmic reticulum and autophagosomes are formed. Later, these autophagosomes fuse with pre-existing lysosomes and their content is finally released by exocytosis. About 4-6 days after microinjection the molecular probes are completely sequestered from the cytoplasm. Quantitative evaluations have shown that the mode and rate of autophagosome formation is clearly influenced by the biochemical properties, the intracellular concentration and the microinjected volume of the molecular probes. Uncoupled TRITC is more slowly and TRITC-BSA-Au(16) more rapidly sequestered from the cytoplasm than TRITC-BSA. High intracellular BSA-concentrations accelerate the rate of autophagosome formation, whereas a corresponding increase of the TRITC-concentration exhibits the contrary effect. Under constant fluorochrome and protein conditions the total number of vacuoles formed increases with the injected volume. The results of the present paper demonstrate that Amoeba proteus has developed a specific autophagosomal sequestration mechanism which is influenced by both, qualitative and quantitative differences of microinjected molecular probes.