Probes for Imaging Actin Filaments
Actin is a highly conserved family of proteins that form microfilaments. Due to its intracellular abundance in eukaryotic cells, well within micromolar concentrations, actin participates in the most protein-protein interactions than any other known protein. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in fixed cells and tissues, as well as, for visualizing individual actin filaments in vitro.Fluorescent phalloidin derivatives have been used as an important tool in the study of actin networks at high resolution.
Actin possesses a unique ability to dynamically polymerize into filamentous actin (F-actin) from its monomeric state. Interactions between F-actin and regulatory proteins such as actin-binding proteins rapidly assemble and disassemble actin filaments organizing them into actin bundles and cytoskeletal networks. These higher-order structures provide the mechanical and structural support essential for a multitude of cellular processes including intracellular transport, cytokinesis, cell motility, polarity and cell shape, gene regulation and signal transduction. Because actin is essential in so many biological processes, tools (actin-specific antibody and phalloidin derived stains) for the visualization of these actin structures are essential in research.
Detecting Filamentous Actin (F-Actin)
F-Actin stains are commonly used to characterize the stucture and functionality of the cytoskeleton. Because the actin cytoskeleton is a very dynamic and labile structure in living cells it typically requires either cold methanol or paraformaldehyde fixation prior to labeling actin. In fixed cells, actin structures can be visualized by actin antibodies, fluorescent phalloidin, or electron microscopy. Antibodies recognize both monomer and polymer (filamentous or F-actin) actin and hence tend to have a high background compared to probes that bind only F-actin. Correctly designed fluorescent phalloidin only binding to the native quaternary structure of F-actin and therefore have a low background.
Phalloidin, the main representative of the phallotoxin family, is a bicyclic heptapeptide isolated from the poisonous death cap mushroom, Amanita phalloides. It possesses a high binding affinity for the grooves between F-actin subunits over monomeric G-actin. Compared to actin antibodies, non-specific binding of phalloidin is negligible, thus providing minimal background and high-contrast during cellular imaging. Once bound to F-actin, phalloidin shifts the equilibrium of monomers and filaments toward the filaments position, and inhibits ATP-hydrolysis. The interaction stabilizes actin filaments by preventing subunit dissociation, and it promotes actin polymerization by lowering the critical concentration. As a consequence, these characteristics have made phalloidin derivatives useful stains in visualizing F-actin and cytoskeletal networks.