Cellular responses to external threats probed by super-resolution microscopy

Fluorescence microscopy has been an essential tool in biology. However, its imaging resolution has been limited around >200 nm in lateral dimensions, and >500 nm in axial dimension, leaving many biological structures too small to study. However the recent developments of several super-resolution techniques, this limit has been overcome. Here we present our approach to this matter, using our three-dimensional (3D) multicolor super-resolved single fluorophore microscopy. Especially we report imaging of a near-IR dye for imaging thick and dense structures for multicolor colocalization studies. Then we present three applications of the super-resolution imaging technique to the following three biological systems. Viral infection in mammalian cells triggers the immune response, a cascade of antiviral signaling proteins. For double stranded RNA (dsRNA) virus, the innate immune response starts from retinoic acid-inducible gene-I (RIG-I) protein which is a dsRNA sensor. For certain types of viruses, RIG-I localizes in cytoplasmic granular aggregates called as antiviral granules (AVGs). We infected/transfected cells by influenza virus lacking NS1 (IAVΔNS1) or polyinosinic:polycytidylic acid (poly I:C) to induce AVGs. We found in AVGs, RIG-I forms clusters of around 110nm in diameter. Then we treated intact cells with various stress conditions to from stress granules (SGs) and could also observe RIG-I clusters in SGs. RIG-I was also clustered in intact cells, but the clustering percentage and diameter were small than in AVGs or SGs, so we conclude that the intrinsically clustered RIG-I relocalizes into granular structures upon external stimuli, and its clustering is enhanced. To verify RIG-I clustering, we imaged TIAR, a marker for SGs, which showed much less clustering. Also we conducted various tests on our clustering algorithm, as well as structural illumination microscopy (SIM) imaging that all support the idea of the clustering of RIG-I. In bacterial cells, upon vast global DNA damage, an error-prone DNA repair response called SOS response occurs. SOS response is initiated by RecA, a protein essential for maintenance of DNA. It was reported that RecA forms a bundle to connect uncut and cut locus of in the event of double strand break, supporting the idea that RecA mediates homology search. We report that RecA also forms bundles in the SOS response, and these bundles are ribbon-like structures, i.e. flat at one side and wide at the other side, hypothetically wrapping around DNA damaged sites. The conventional approach of applying broad-spectrum antibiotics to treat bacterial infections contributes to the emerging of antibiotic resistance. To cope with this, species specific and narrow-spectrum antibiotics draw attention. Plantazolicin (PZN) is a natural antibiotic that is highly specific against B. anthracis which is the agent of anthrax and a category A priority pathogen, but the mechanism of how PZN kills B. anthracis has been unknown. Recent investigation showed PZN depolarizes B. anthracis membrane, and it was supported by the super-resolution imaging that PZN foci localize on the membrane of cells