Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal Biological Diversity

It is well-known that photosynthetic cells of various microalgae species display distinct fluorescent properties. The efficiency of self-fluorescence excitation and emission at different wavelengths depends on the structure of photosynthetic system and particularly on the structure of antenna complex of specific strains. The peculiar structure of blue-green algae light-harvesting complex allows to discriminate and classify known and new cells up to species/strain level by means of microscopic spectroscopy. In this chapter, a novel fluorescent spectroscopic technique for microalgae species discrimination will be presented. This method is based on a special data processing of a set of fluorescent spectra, obtained from a single photosynthetic cell of microalgae, particularly from cyanobacterial cells. According to the presented technique, single-cell self-fluorescence spectra are recorded by means of confocal laser scanning microscopy (CLSM), and data processing is conducted via linear discriminant analysis (LDA) and artificial neural networks (ANN)

Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics

Oncogenic mutations in the mitogen activated protein kinase (MAPK) pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2). We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells