Zhuang Research Lab

The Zhuang research lab works on the forefront of single-molecule biology and bioimaging, developing and applying advanced optical imaging techniques to study the behavior of individual biological molecules and molecular assemblies in vitro and in live cells. Students and postdoctoral fellows in the Zhuang lab apply their diverse backgrounds in chemistry, physics, biology, and engineering to develop new imaging probes and methods and applying these tools to study a variety of interesting biological systems. Our current research is focused on three major directions: (1) Developing super-resolution optical microscopy that allows cell and tissue imaging with nanoscopic scale resolution and applying this technology to cell biology and neurobiology, (2) investigating how biomolecules function, especially how proteins and nucleic acids interact, using single-molecule fluorescence imaging and spectroscopy; (3) Investigating how viruses and cells interact using imaging techniques with high spatiotemporal resolution.

(1) Super-resolution fluorescence microscopy

Fluorescence microscopy is one of the most widely used imaging methods in biomedical research. It's molecular/chemical specificity and live-cell compatibility have made fluorescence microscopy a particularly powerful tool for biological research, and numerous breakthroughs in biology have been enabled by this imaging modality. However, the spatial resolution of light microscopy, classically limited by the diffraction of light to several hundred nanometers, is substantially larger than the typical length scales of molecules in cells, preventing a detailed characterization of most subcellular structures.

To overcome this limit, we have developed a super-resolution 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 positions of these molecules to be precisely determined and super-resolution images to be reconstructed from these molecular coordinates. Using STORM, we have achieved three-dimensional, multicolor fluorescence imaging of cells and tissues with sub-diffraction-limit resolution. Through innovations in optics and molecular probes, we reached a spatial resolution of ~10 nm and, in some cases, sub-10 nm. We have demonstrated live-cell STORM imaging with sub-second time resolution. We are currently working on advancing super-resolution imaging by further increasing the spatial and temporal resolution and enhancing deep-tissue, live-animal imaging capabilities.

We have applied STORM to a variety of problems in cell biology and neurobiology. These studies have led to the discoveries of previously unknown cellular structures and elucidated the high-resolution structures of many molecular assemblies in cells. For example, we discovered a periodic membrane-bound cytoskeletal structure in neurons made of actin, spectrin, and associated proteins, and determined the molecular mechanism underlying the preferential formation of this periodic membrane skeleton structure in axons over dendrites. Our studies have also provided novel insights into chromatin structures in the nucleus, the molecular architecture of synapses, the spatial distributions and molecular identities of synapse on neurons, and other cellular structures. Currently, we are focusing on the following biological areas: sub-cellular structures in the neurons, neural circuits in the brain, and the three-dimensional structure and dynamics of chromatin and chromosomes in the nucleus.

STORM Image Gallery

Click here for a more detailed description of recent research in the lab.

STORM Workshop (August 2010, April 2012)

Workshop Information

Selected publications:

Mapping Synaptic Input Fields of Neurons with Super-Resolution Imaging
Y.M. Sigal, C.M. Speer, H.P. Babcock, X. Zhuang
493-505 (2015)

Developmental mechanism of the periodic membrane skeleton in axons
G. Zhong, J. He, R. Zhou, D. Lorenzo, H.P. Babcock, V. Bennett, X. Zhuang
DOI:10.7554/eLife.04581 (2014)

Structurally distinct Ca2+ signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility
J. Chung, S. Shim, R. Everley, S. Gygi, X. Zhuang, D. Clapham
808-822 (2014)

Isotropic three-dimensional super-resolution imaging with a self-bending point spread function
S. Jia, J. Vaughan, X. Zhuang
Nature Photonics
302-306 (2014)

Super-resolution fluorescence imaging reveals TRF2-dependent t-loop formation
Y. Doksani, J. Wu, T. de Lange, X. Zhuang
345-356 (2013)

Ultra-bright Photoactivatable Fluorophores Created by Reductive Caging
J. Vaughan, S. Jia, X. Zhuang
Nature Methods
1181-1184 (2012)

Super-resolution Fluorescence Imaging of Organelles in Live Cells with Photoswitchable Membrane Probes
S-H. Shim, C. Xia, G. Zhong, H.P. Babcock, J.C. Vaughan, B. Huang, X. Wang, C. Xu, G-Q. Bi, X. Zhuang
Proc. Natl. Acad. Sci.
13978-13983 (2012)

Fast, three-dimensional super-resolution imaging of live cells
S. Jones, S-H. Shim, J. He, X. Zhuang
Nature Methods
499-505 (2011)

Super-resolution imaging of chemical synapses in the brain
A. Dani, B. Huang, J. Bergan, C. Dulac, X. Zhuang
843-856 (2010)

Three-dimensional Super-resolution Imaging by Stochastic Optical Reconstruction Microscopy
B. Huang, W. Wang, M. Bates, X. Zhuang
810-813 (2008)

Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes
M. Bates, B. Huang, G. T. Dempsey, X. Zhuang
1749-1753 (2007)

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
M. J. Rust, M. Bates, X. Zhuang
Nature Methods
793-795 (2006)

Short-range spectroscopic ruler based on a single-molecule optical switch
M. Bates, T. R. Blosser, X. Zhuang
Phys. Rev. Lett.
108101 (2005)

(2) Single-Molecule biology

We have developed and applied single-molecule fluorescence imaging and spectroscopy techniques to study complex biomolecular systems in vitro. In particular, we use the highly sensitive distance dependence of fluorescence resonance energy transfer (FRET) to detect structural changes at the single-molecule level. We are also developing novel single-molecule methods to detect dynamics within biomolecular complexes.

An area of particular interest to us is the interactions of proteins with nucleic acids. Many essential cellular reactions, such as DNA replication, transcription, RNA processing, and protein synthesis, involve DNA-protein or RNA-protein complexes. Understanding nucleic acid-protein interactions is thus crucial for deciphering the molecular mechanisms underlying many important biological processes. Using single-molecule methods, we directly visualize the assembly and function of these molecular complexes in real time. These experiments allow us to observe transient states that are difficult to detect by classical ensemble experiments, to probe the dynamic interactions between DNA, RNA and proteins, and to determine the relationship between the structural dynamics and function for these molecular complexes. Using this approach, we have studied the assembly process, catalytic cycle, and structure-function relationship of several nucleic acid-interacting enzymes, including ribozymes, telomerase, HIV reverse transcriptase and chromatin-remodeling enzymes.

Our current focus is on ATP-dependent chromatin remodeling enzymes. We are studying how these enzymes remodel nucleosome structures and how the remodeling process is regulated.

Click here for a more detailed description of recent research in the lab.

Selected publications:

Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA
W.L. Hwang, S. Deindl, B.T. Harada, X. Zhuang
213-217 (2014)

ISWI remodelers slide nucleosomes with coordinated multi-base-pair entry steps and single-base-pair exit steps
S. Deindl, W.L. Hwang, S.K. Hota, T.R. Blosser, P. Prasad, B. Bartholomew, X. Zhuang
442-452 (2013)

Functional importance of telomerase pseudoknot revealed by single-molecule analysis
M. Mihalusova, J.Y. Wu, X. Zhuang
Proc. Natl. Acad. Sci.
20339-20344 (2011)

Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription
S. Liu, B.T. Harada, J.T. Miller, S.F.J. Le Grice, X. Zhuang
Nature Struct. Mol. Biol.
1453-1460 (2010)

Dynamics of Nucleosome Remodeling by Individual ACF Complexes
T. Blosser, J. Yang, M. Stone, G. Narlikar, X. Zhuang
1022-1027 (2009)

Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates
S. Liu, E. Abbondanzieri, J. W. Rausch, S. F. J. Le Grice, X. Zhuang
1092-1097 (2008)

Dynamic binding orientations direct activity of HIV reverse transcriptase
E. Abbondanzieri, G. Bokinsky, J. W. Rausch, J. X. Zhang, S. F. J. Le Grice, X. Zhuang
184-189 (2008)

Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein
M. D. Stone, M. Mihalusova, C. M. O'Connor, R. Prathapam, K. Collins, X. Zhuang
458-461 (2007)

(3) Single-virus tracking

Our research in this direction focuses on virus-cell interactions and related cellular trafficking pathways. Viruses must deliver their genome into cells to initiate infection. This entry process 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 in the pathway, each featuring interactions of the viruses with different cellular structures. What could be a better way to study viral trafficking than 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 individual virus particles in live cells. This approach allows us to follow the fate of individual viruses, to dissect the infection pathways into microscopic steps, and to determine the molecular mechanism of each step. By combining this approach with other biochemical methods, we have studied the entry mechanisms of influenza virus, poliovirus, dengue virus and non-viral gene delivery vectors. Our research also extends to the post entry trafficking, assembly and budding mechanisms of viruses.

Click here for a more detailed description of recent research in the lab.

Selected publications:

Dual function of CD81 in influenza uncoating and budding
J. He, E. Sun, M.B. Bujny, D. Kim, M.W. Davidson, X. Zhuang
PLOS Pathogens
e1003701 (2013)

Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells
H.M. van der Schaar, M.J. Rust, C. Chen, H. van der Ende-Metselaar, J. Wilschut, X. Zhuang, J.M. Smit
PLOS Pathogens
e1000244 (2008)

Epsin1 is a cargo specific adaptor for the clathrin-mediated endocytosis of influenza virus
C. Chen, X. Zhuang
Proc. Natl. Acad. Sci. USA
11790-11795 (2008)

Virus trafficking - learning from single-virus tracking
B. Brandenburg, X. Zhuang
Nat. Rev. Microbiology
197-208 (2007)

Imaging poliovirus entry in live cells
B. Brandenburg, L. Y. Lee, M. Lakadamyali, M. J. Rust, X. Zhuang, J. M. Hogle
PLoS Biol.
1543-1555 (2007)

Assembly of endocytic machinery around individual influenza viruses during viral entry
M. J. Rust, M. Lakadamyali, F. Zhang, X. Zhuang
Nature Struct. Mol. Biol.
567-573 (2004)

Visualizing infection of individual influenza viruses
M. Lakadamyali, M. J. Rust, H P. Babcock, X. Zhuang
Proc. Natl. Acad. Sci. USA
9280-9285 (2003)