Elucidating the functional mechanisms of cells, and their dysfunction in diseases, requires tools to dissect the intricate molecular processes present in cells. In particular, we need imaging tools with nanometer-scale resolution and dynamic imaging capability to allow direct visualization of these molecular processes. My lab develops imaging methods with single-molecule sensitivity and nanometer-scale resolution to meet these challenges and applies these methods to problems of biomedical interest. Our current research effort focuses on three main directions: (1) developing superresolution fluorescence microscopy techniques to allow imaging of cells, tissues, and animals with molecular-scale resolution; (2) applying superresolution imaging to study problems in cell biology and neurobiology; and (3) using single-molecule approaches to investigate how proteins and nucleic acids interact, with emphasis on chromatin remodeling.
Superresolution Fluorescence Microscopy—Technological Development
Numerous breakthroughs in biology have been enabled by fluorescence microscopy. The live-cell compatibility and molecular specificity make fluorescence microscopy a particularly powerful tool for biological research. 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, preventing a detailed characterization of most subcellular structures. A major challenge in bioimaging is to visualize cells and tissues with molecule-specific contrast, molecular-scale resolution, and real-time dynamics.
To meet this challenge, we invented a superresolution light microscopy method, stochastic optical reconstruction microscopy (STORM). In this method, we introduced the use of photoswitchable fluorescent probes to temporally separate the spatially overlapping images of individual molecules, allowing the construction of superresolution images. During STORM imaging, only a sparse subset of fluorophores are switched on and imaged at any given instance, allowing the positions of these fluorophores to be determined with high precision. Over time, the positions of numerous fluorophores are determined and used to construct an image with sub-diffraction-limit resolution. Using STORM, we originally achieved fluorescence imaging of molecular complexes and cells with ~20-nm resolution (Figure 1). Subsequently, we extended STORM to three-dimensional (3D) imaging by using astigmatism and achieved 3D superresolution imaging with ~20-nm lateral resolution and ~50-nm axial resolution (Figure 2).
Our recent experiments have improved the spatiotemporal resolution of STORM. Using a dual-objective scheme, we obtained ~10-nm lateral and ~20-nm axial resolution (Figure 3). With a nondiffracting Airy beam, we further improved the axial resolution, such that it is as high as the lateral resolution. We also developed ultrabright photoactivatable dyes, which allowed us to push the resolution to below 10 nm. With fast-switchable dyes, we achieved live-cell STORM imaging with 1-sec time resolution (Figure 4). We further improved the time resolution by developing imaging analysis algorithms that allow a higher density of fluorescent molecules to be localized in each camera frame.
Photoswitchable probes are essential for STORM imaging. Over the years, we have discovered many photoswitchable dyes with different colors and a variety of photoswitching mechanisms. These probes allowed multiple-color STORM imaging. More recently, we developed a novel photochemical mechanism to create photoactivatable dyes with 105 to 106 detectable photons per activation event, 2–3 orders of magnitude greater than the previous record. We discovered cell-permeable photoswitchable membrane dyes that allow STORM imaging of membrane organelles in living cells. We developed photoactivatable fluorescent proteins with undetectable dimerization tendency and high signaling efficiency for superresolution imaging of proteins in live cells free of dimerization artifact.
We continue to advance superresolution imaging capabilities with the goal of enabling real-time, in vivo imaging at the true molecular-scale resolution.
Superresolution Fluorescence Microscopy—Applications to Cell Biology and Neurobiology
STORM 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 and neurobiology. These studies have led to the discoveries of previously unknown subcellular structures and answered some long-standing questions.
A novel membrane skeleton structure in axons. Using STORM imaging, we recently discovered a strikingly regular and periodic membrane skeleton structure in axons (Figure 5). In this membrane skeleton, actin filaments form a ring-like structure that wraps around the circumference of the axons. The actin rings are evenly spaced along the axon shafts with a periodicity of 180–190 nm. Adjacent actin rings are connected by spectrin tetramers. This submembrane lattice structure influences the molecular organization of the axon membrane by ordering important membrane proteins, such as sodium channels, along the axon. This periodic membrane skeleton may also play an important role in axon stability by providing elastic and stable mechanical support for the axonal membrane. We are further investigating the molecular organization of this novel axonal structure, its functional roles, and its implications in neurological diseases.
Molecular architecture of synapses. In collaboration with Catherine Dulac (HHMI, Harvard University), we have established STORM as an effective tool to analyze the molecular architecture of synapses with nanometer precision in brain tissues (Figure 6). We have determined the spatial organization of many protein components of the presynaptic active zone and the postsynaptic density. We have observed a highly oriented organization of presynaptic scaffolding proteins, a laminar distribution of postsynaptic proteins, variations in the neurotransmitter receptor distribution among synapses, and a surprisingly large population of immature synapses with activity-dependent plasticity in the adult accessory olfactory bulb. We are continuing our investigations of the spatial organization and dynamics of synaptic proteins.
Telomere structure. Telomeres are essential elements at the ends of linear eukaryotic chromosomes that stabilize the chromosome and prevent DNA damage responses. We used STORM to image the structure of functional and dysfunctional mouse telomeres in collaboration with Titia de Lange (The Rockefeller University). We observed that functional telomeres frequently adopt a loop configuration (Figure 7). The TRF2 protein in the shelterin complex is required for the formation and/or maintenance of the loop structure, whereas other shelterin proteins are not required. Packaging of telomeres into the t-loop structure can explain why nonhomologous end joining and ATM signaling are inhibited by TRF2. We are extending our studies to the chromatin structures in other regions of the chromosome.
We have also investigated other cellular structures, for example, the calcium signaling domain in the sperm tail in collaboration with David Clapham (HHMI, Harvard Medical School), the clathrin-dependent endocytic structure in collaboration with Pietro de Camilli (HHMI, Yale University), and the molecular architecture of bacteria in collaboration with Sunney Xie (Harvard University).
Single-Molecule Studies of Chromatin Remodeling
The highly sensitive distance dependence of fluorescence resonance energy transfer (FRET) allows it to be used as a spectroscopic ruler for detecting structural changes at the single-molecule level. We have used single-molecule FRET to investigate RNA folding, ribonucleoprotein assembly, retroviral reverse transcription, and chromatin remodeling. Our current focus is on chromatin remodeling.
The packaging of DNA into chromatin represses essential nucleic acid transactions, such as transcription, replication, repair, and recombination. The structure of chromatin is in part regulated by ATP-dependent chromatin-remodeling enzymes, which can be classified into several distinct families. Among these, the ISWI family remodelers promote heterochromatin formation and transcriptional silencing by mobilizing nucleosomes and generating regularly spaced nucleosome arrays. We used single-molecule FRET to monitor the remodeling of individual nucleosomes in real time and investigated how ISWI family enzymes, such as human ACF and yeast ISW2, translocate and space nucleosome.
We have found that several ISWI family members translocate nucleosomes with a similar stepping pattern and a relatively small step size of only a few base pairs (Figure 8). DNA movements at different sites of the nucleosomes are coordinated. Surprisingly, movement of DNA into the entry side of the nucleosome occurs only after several base pairs of DNA have been pumped to the exit side. Each entry-side step draws in a few base pairs of DNA at a time, allowing the DNA to be extruded to the exit side one base pair at a time.
The nucleosome-spacing activity of ISWI family enzymes arises from regulation of nucleosome translocation by the length of extranucleosomal linker DNA. We have investigated the mechanism underlying linker DNA length sensing using human ACF, a prototypcial ISWI enzyme and found that ACF senses linker DNA length through an interplay between its accessory subunit Acf1 and catalytic subunit Snf2h. Acf1 senses and allosterically transmits the linker length information to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing an autoinhibitory domain (AutoN) of Snf2h to inhibit its ATPase activity. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to displace the AutoN from the ATPase and thereby activate ACF (Figure 8).
We are extending our study to other families of remodeling enzymes.
These projects are supported in part by the National Institutes of Health.
As of July 21, 2014