Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models

Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed "trio-FPs" consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.