Our understanding of living organisms has greatly benefited from various imaging and visualization tools. In particular, understanding the inner workings of a cell requires imaging techniques with molecular-scale resolution and dynamic imaging capability such that molecular interactions and processes inside the cell can be directly visualized. We are developing imaging methods with single-molecule sensitivity and nanometer-scale resolution to meet these challenges. Our current research effort focuses on three main directions: (1) developing super-resolution fluorescence microscopy techniques to allow imaging of cells and tissues with molecular-scale resolution and applying these methods to cell biology and neurobiology; (2) using single-molecule approaches to investigate how proteins and nucleic acids interact, with emphasis on chromatin remodeling, telomere regulation, and retroviral reverse transcription; (3) using high-resolution live-cell imaging approaches to study virus-cell interactions, in particular how viruses enter cells.
Super-resolution Fluorescence Microscopy
Fluorescence microscopy is one of the most widely used imaging methods in biological research. The noninvasive live-cell compatibility and molecular specificity make fluorescence microscopy a particularly powerful tool. However, the spatial resolution of light microscopy, classically limited by the diffraction of light to several hundred nanometers, is substantially larger than the typical molecular-length scales in cells, leaving many biological problems beyond reach. Today, a major 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 invention, 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 imaging process includes multiple cycles. In each cycle, only a fraction of 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 high precision. Over the course of many activation cycles, the positions of numerous fluorophores are determined and used to construct an image with sub-diffraction-limit resolution.
Using STORM, we have achieved fluorescence imaging of molecular complexes and cells with ~20-nm resolution (Figure 1). Furthermore, we have discovered a family of photoswitchable probes with many different colors and demonstrated multicolor super-resolution imaging (Figure 2). More recently, we have extended the STORM technology into the third dimension. By introducing astigmatism with a cylindrical lens in the imaging path, we have achieved three-dimensional (3D) super-resolution imaging by determining the axial positions of individual fluorophores from the ellipticity of their images. We have obtained spatial resolution that is 10 times better than the diffraction limit in both lateral and axial dimensions (Figure 3). We are advancing 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 has the potential to transform biomedical research, by allowing molecular interactions in cells and cell-cell interactions in tissues to be imaged at the nanometer scale.
We have applied STORM to various problems in cell biology. As an example, we have studied the spatial relationship between clathrin and various curvature-generating or stabilization proteins during clathrin-mediated endocytosis (collaboration with Pietro De Camilli [HHMI, Yale University]; Figure 3E).
Recently, we have extended our interest into neurobiology. We have studied the molecular architecture of chemical synapses in the brain in collaboration with Catherine Dulac [HHMI, Harvard University]. Multicolor, 3D STORM allows the distributions of synaptic proteins to be measured with nanometer precision (Figure 4). We have determined the organization of 10 protein components of the presynaptic active zone and the postsynaptic density. We have observed a highly oriented organization of presynaptic scaffolding proteins, a differential compartmental distribution of PSD components, and variations in the neurotransmitter receptor composition and distribution among synapses and across different brain regions (Figure 4).
Nucleic AcidProtein Interaction
A variety of essential cellular reactions related to gene expression and regulation involve nucleoprotein assemblies. It is of critical importance to understand the biogenesis of these molecular assemblies and the interactions between enzymes and their nucleic acid substrates during these reactions. To obtain such knowledge, we have developed single-molecule methods to visualize the assembly and function of individual molecular complexes. These experiments have revealed transient states and multiple kinetic paths that are difficult to detect by classical ensemble experiments, allowing better dissection of these processes. In the following, we describe three examples in telomerase assembly, retroviral reverse transcription, and chromatin remodeling.
Telomerase is an essential cellular ribonucleoprotein that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes. Genetic mutations that abrogate normal assembly of telomerase cause human disease. It is thus important to decipher cellular strategies for telomerase biogenesis. We have demonstrated a single-molecule approach to dissect 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). These results have not only identified the RNA-folding pathway during tetrahymena telomerase biogenesis but also defined the mechanism of action for an essential telomerase holoenzyme protein (collaboration with Kathleen Collins [University of California, Berkeley]).
The reverse transcriptase (RT) of human immunodeficiency virus (HIV) catalyzes a series of reactions to convert the single-stranded viral RNA into double-stranded DNA for host-cell integration. This task requires RT to discriminate various nucleic acid substrates for different catalytic functions, including DNA polymerization, RNA cleavage, and strand displacement synthesis. Using single-molecule fluorescence imaging, we have studied the interactions between RT and a variety of nucleic acid substrates in real time (collaboration with Stuart Le Grice [National Cancer Institute]). We observed highly dynamic enzyme-substrate interactions (Figure 6). The enzyme exhibits large-scale orientational and translation dynamics that facilitate multiple stages of the reverse transcription pathway, including the initiation, DNA polymerization, and strand displacement synthesis. These dynamics are regulated by non-nucleoside RT inhibitors, suggesting novel functional mechanisms of anti-HIV drugs.
The packaging of DNA into chromatin represses essential nucleic acid transactions, such as transcription, replication, repair, and recombination. This repression is in part regulated by chromatin-remodeling enzymes. One of these enzymes, ACF, functions to generate regularly spaced nucleosomes required for heritable gene silencing. Using single-molecule FRET, we have monitored the remodeling of individual nucleosomes by ACF and revealed novel remodeling intermediates and dynamics (collaboration with Geeta Narlikar [University of California, San Francisco]). ACF-catalyzed nucleosome translocation occurs in well-defined steps, with the initial step being ~7 base pairs and the subsequent steps being 34 base pairs (Figure 7). The ACF homodimer is a processive and bidirectional nucleosome translocase that can move the nucleosome back and forth many times before dissociation. We are conducting experiments to elucidate the nucleosome translocation mechanism by ACF and other chromatin remodelers.
Cellular Entry of Viruses
Viruses must deliver their genome into specific sites in the cell to initiate infection. This process, referred to as viral entry, is 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 between the virus and different cellular structures. To overcome this challenge, 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, to dissect the entry pathways into microscopic steps, and to determine the molecular mechanism of each step. Our major focus is the influenza virus.
Using this approach, we have observed internalization, transport, and fusion of individual influenza viruses in live cells. The virus trajectories reveal several active-transport stages prior to viral fusion, each with distinct molecular mechanisms. We have directly visualized multiple entry pathways used by influenza virus. The majority of virus particles enter cells through clathrin-mediated endocytosis. These virions exploit epsin1 as a cargo-specific adaptor and enter cells by triggering the de novo formation of clathrin-coated pits. The remaining particle enters through a clathrin- and caveolin-independent pathway. Both pathways lead to viral fusion with similar efficiencies. After viral fusion, the released viral genetic materials are transported to the nucleus by diffusion. 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 (Figure 8A, B).
We have also extended this approach to study other viruses. For example, in collaboration with Jolanda Smit (University of Groningen), we have found that dengue virus enters cells exclusively via clathrin-mediated endocytosis and is trafficked through Rab5-positive early endosomes to Rab7-positive late endosomes, where viral fusion takes place. In collaboration with James Hogle (Harvard Medical School), we have shown that poliovirus enters the cell by a clathrin- and caveolin-independent but actin-dependent endocytic mechanism. Viral genome release by poliovirus is highly efficient, occurring immediately after the internalization of the virus particle in vesicles near the plasma membrane (Figure 8C). These results settle a long-lasting debate of whether poliovirus directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell.
These projects are supported in part by the National Institutes of Health, National Science Foundation, and David and Lucile Packard Foundation.
As of May 30, 2012