| Many biological processes are dynamic and asynchronous. Rather than probing a fixed end result that is averaged over an ensemble of transient states, real-time fluorescence microscopy enables direct visualization of biological targets in live cells with high spatial- and temporal-resolution. Here I present applications of this technique to the studies of influenza viral infection and polymer-mediated gene delivery.;The majority of influenza viruses infect cells by hijacking clathrin-mediated endocytosis, an important process used by cells to maintain many cellular functions. A detailed characterization of this viral entry pathway not only is medically important but also can yield insights into our understanding of the complex network of ligand-host interactions during clathrin-mediated endocytosis. By labeling influenza viral membrane with fluorescent dye molecules, individual viruses can be visualized and tracked with high precision during their internalization into cells. This, combined with simultaneous imaging of fluorescently-tagged cellular proteins, has allowed the identification of a specific adaptor protein that is critical for influenza viral entry through the clathrin-mediated pathway.;The influenza viral genome consists of eight strands of ribonuclearproteins (RNPs). Their intracellular trafficking is tightly regulated to ensure a successful infection. By tracking fluorescently-labeled RNPs microinjected into cells, RNPs are found to move by diffusion in both the cytoplasm and the nucleus. They interact with nuclear pore complex (NPC) before they are imported into the nucleus. The viral protein M1, which regulates RNPs nuclear transport, is shown not to affect the diffusion of RNPs but blocks the RNP-NPC interaction.;Finally, imaging of polymer-gene complexes along with cell surface proteoglycans, early and late endosomes, in combination with bulk biochemical assays and drug treatment, has revealed a common entry pathway used by various proteoglycan-bind ligands including cationic lipids and polypeptides. Being clathrin- and caveolin-independent, this pathway displays many distinct features: it requires flotillin-1 and dynamin, and leads to the rapid accumulation of cationic ligands in late endosomes without early endosomal sorting and microtubule-dependent transport. |
