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Single-Molecule Imaging of Biomolecular and Cellular Processes
Summary: Xiaowei Zhuang develops and applies optical imaging techniques to monitor the behavior of individual biological molecules and complexes in vitro and in live cells. Her current research focuses on (1) developing super-resolution optical microscopy that allows cell and tissue imaging with molecular-scale resolution and applying this technology to cell biology and neurobiology; (2) studying how biomolecules function, especially how proteins and nucleic acids interact, through single-molecule approaches; (3) developing live-cell imaging techniques and using them to investigate virus-cell interactions.
Super-resolution Optical Imaging
Optical microscopy is one of the most widely used imaging methods in biological research. Several advantages make light microscopy a particularly powerful tool. First, it allows noninvasive imaging of biological samples. Second, the availability of fluorescent probes in many colors and the ability to label specific gene products efficiently with these probes confer great molecular specificity in fluorescence imaging. Third, the relatively fast time resolution of optical microscopy allows us to probe dynamic processes in cells and tissues. However, the spatial resolution of optical microscopy, classically limited by the diffraction of light to ~300 nm, is substantially larger than typical molecular-length scales in cells, leaving many biological problems beyond the reach of light microscopy. Today, an important challenge in bioimaging is to visualize cells and tissues with both molecular specificity and molecular-scale spatial resolution.
To meet this challenge, we invented a new form of super-resolution light microscopy, stochastic optical reconstruction microscopy (STORM). In this method, we introduced the use of photoswitchable fluorescent probes to temporally separate the otherwise spatially overlapping images of individual molecules, allowing the construction of super-resolution images. The STORM imaging process includes multiple imaging cycles. In each cycle, only a fraction of the fluorophores are switched on, such that each of the active fluorophores is optically resolvable from the rest. This allows the position of these fluorophores to be determined with nanometer accuracy. Over the course of many activation cycles, the positions of numerous fluorophores are determined and used to construct a high-resolution image.
Using STORM, we have achieved fluorescence imaging of molecular complexes and cells with ~20-nm lateral resolution (Figure 1). Furthermore, we discovered a family of photoswitchable probes. Each of the probes consists of a reporter fluorophore that can be switched between fluorescent and dark states and an activator dye that facilitates photoactivation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Using this design, we demonstrated multicolor super-resolution imaging of cells (Figure 2). More recently, we extended the STORM technology into the third dimension. By introducing astigmatism with a cylindrical lens in the imaging path, we achieved three-dimensional (3D) STORM imaging by determining the axial positions of individual fluorophores from the ellipticity of their images without sample scanning. With 3D STORM, we have obtained spatial resolution of ~20 nm in the lateral dimensions and ~50 nm in the axial dimension, ~10 times smaller than the diffraction limit in all three dimensions (Figure 3). We hope to advance STORM capabilities to ultimately enable real-time imaging of live cells and tissues with resolution at the true molecular-length scale. This new form of fluorescence microscopy allows molecular interactions in cells and cell-cell interactions in tissues to be imaged at the nanometer scale. We are applying the STORM technology to cell biology and neurobiology.
Cellular Entry of Viruses
Viruses must deliver their genome into cell to initiate infection. This process—from viral attachment to the cell surface to genome delivery—is referred to as viral entry, a subject of fundamental importance as well as a therapeutic target for viral disease treatment. However, understanding viral entry mechanisms is challenging because of the involvement of multiple entry pathways and multiple steps on the pathway, each featuring dynamic interactions of the viruses with different cellular structures. A great way to study virus trafficking is thus to take a ride with the virus particle on its journey into the cell. To realize this goal, we have developed real-time imaging methods to track the behavior of individual virus particles and viral components in live cells. This approach allows us to follow the fate of individual viruses, dissect the entry pathways into microscopic steps, and determine the molecular mechanism of each step.
Using this approach, we have observed internalization, transport, and fusion of individual influenza viruses in live cells. We have observed distinct active-transport stages that occur before viral fusion. We have directly visualized multiple entry pathways for influenza virus—both clathrin-mediated and clathrin- and caveolin-independent pathways lead to viral fusion. We have discovered distinct populations of early endosomes and found that different cargoes are differentially targeted to these endosomal populations. Influenza viruses are preferentially delivered, via microtubule-dependent transport, to a dynamic, rapidly maturing population of early endosomes.
More recently, we have extended our research to nonenveloped viruses, studying the entry mechanism of poliovirus. We combine the single-virus tracking assay with an infectivity-based virological assay to characterize the early events in poliovirus infection. We observed that genome release by poliovirus is highly efficient and rapid and thus does not limit overall infectivity or the infection rate. We showed that poliovirus enters the cell by a clathrin- and caveolin-independent but tyrosine kinase– and actin-dependent endocytic mechanism and releases its genome from vesicles located within 100 to 200 nm of the plasma membrane (Figure 4). These results settle a long-standing debate about whether poliovirus directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell.
Nucleic Acid–Protein Interaction
A variety of essential cellular reactions, such as DNA telomere extension, messenger RNA editing, and protein synthesis, are catalyzed by ribonucleoprotein (RNP) complexes. Abnormal assembly of RNPs causes human disease. It is thus of fundamental and medical importance to decipher cellular mechanisms for RNP biogenesis. To obtain such knowledge, we developed single-molecule methods to visualize the folding process of individual biomolecules and the assembly of individual molecular complexes.
We use FRET (fluorescence resonance energy transfer) to image single RNA molecules in real time. These experiments have revealed transient states and multiple kinetic paths that are difficult to detect by classical ensemble experiments. We have observed complex, heterogeneous structure dynamics of RNA between different conformations and multiple folding pathways toward native structures. These behaviors indicate that RNA folds across a highly rugged energy landscape. In cells, the rugged-landscape problem is mitigated by the association of RNA with protein cofactors. We directly observed the structural dynamics of RNA enzymes as they assembled with their protein cofactors and detected complex, multiphase folding dynamics.
More recently, we have been investigating the assembly mechanisms of telomerase RNPs, essential cellular enzymes that solve the end replication problem and maintain chromosome stability by adding telomeric DNA to the termini of linear chromosomes. We have demonstrated a single-molecule approach to dissecting the individual assembly steps of telomerase in real time and established a hierarchical RNP assembly mechanism directed by multiple steps of protein-mediated RNA folding (Figure 5). This hierarchical assembly process is facilitated by an evolutionarily conserved structural motif within the telomerase RNA. These results have not only identified the RNA-folding pathway during telomerase biogenesis, but also defined the mechanism of action for an essential telomerase holoenzyme protein.
We are investigating the relation between the structural dynamics and various functional properties of the telomerase. Our single-molecule studies also extend to the investigation of other protein–nucleic acid complexes. In particular, we are studying the dynamic interactions between HIV reverse transcriptase and the various nucleic acid substrates it encounters during HIV infection.
These projects are supported in part by the National Institutes of Health, National Science Foundation, John D. and Catherine T. MacArthur Foundation, and David and Lucile Packard Foundation.
Last updated: January 8, 2009
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