Single-Molecule Studies in Live Cells
My research group is interested in taking single-molecule approaches into live cells to study the dynamics of cellular processes as they occur in real time. In particular, we are interested in unraveling the noise-control mechanisms of gene regulatory networks and the assembly mechanism of the E. coli division complex using single-molecule fluorescence detection and live-cell imaging.
In recent years, due to contributions from numerous research groups, single-molecule approaches have changed the ways many biological problems are addressed. Single-molecule approaches offer superior sensitivity for studying molecular properties of individual molecules, resolving and analyzing heterogeneities in subpopulations, and most importantly, following dynamic motions and interactions of macromolecular machineries in real time. Many single-molecule studies are carried out in vitro and have generated new insights on important and compelling scientific issues. Today, the new challenge is to probe the dynamics of individual protein molecules and record heir "molecular movies" under their physiological social context in live cells. The following is our efforts toward achieving this goal.
Dynamics and structure of the E. coli division complex
Cell division is essential for the survival and development of all organisms. In E. coli, at least ten essential proteins assemble at the midcell to form a ring-like division complex, the divisome, in an ordered fashion to carry out cytokinesis. We are interested in knowing how this complex is orchestrated to function and regulated to coordinate with other essential cellular events. Specifically, we focus on the following questions:
We will address these questions for ten essential division proteins employing highly sensitive single-molecule fluorescence detection and live-cell imaging.
- When does each division protein localize to the midcell during the cell cycle?
- How does each division protein move to the midcell to assemble into the divisome?
- Where does each division protein locate within the divisome?
Noise control mechanisms in gene regulatory networks
Gene expression is stochastic in nature as the components involved exist in small copy numbers. Such stochasticity inevitably leads to output noise. However, "Noisy gene expression" is intuitively at odds with the reliable formation of precise gene expression patterns cells and organisms exhibit during development and growth. We wonder: How do cells function with amazing precisions and robustness when the underlying molecular events are inherently stochastic?
To answer this question, we have developed single-molecule gene expression fluorescence reporters that allow us to directly monitor the production of single protein molecules in real time. We are currently characterizing the noise properties of four basic gene regulation modules using synthetic constructs and a natural gene regulatory network using the genetic switch of ? phage as a model system.
Development of Better fluorescent Reporters
We are also interested in developing better fluorescent reporters and single-pair fluorescent resonance energy transfer (spFRET) reporters to allow the probing of fast kinetics of cellular processes and interactions among protein complexes.
The use of single-molecule fluorescence microscopy methods, in combination with molecular biological methods, will not only complement traditional population studies, but also shed new lights on the mechanisms of these cellular processes at an unprecedented level. The methodology developed in the research will open a new dimension in characterizing biological systems in live cells.
Xiao J., Elf, J., Li, G., Yu, J., Xie X. S., Imaging gene expression in living cells at the single molecule level. Single Molecules: A Laboratory Manual, edited by Selvin P., and Ta H. 2007, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Singleton SF., Roca AI., Lee AM., Xiao J., Probing the structure of RecA-DNA filaments. Advantages of a fluorescent guanine analog. Tetrahedron, 2007, 63(17):3553-3566
Yu J., Xiao J., (equal contribution), Ren X., Lao K., Xie X.S., Probing gene expression in live E. coli cells: one molecule at a time. Science, 2006, 311(5767):1600-3
Xiao J., Lee, A., Singleton SF. Direct evaluation of a kinetic model for RecA-mediated DNA-strand exchange: the importance of nucleic acid dynamics and entropy during homologous genetic recombination. Chembiochem. 2006, 7(8):1265-78
Xiao J., Lee, A., Singleton SF. Construction and evaluation of a kinetic scheme of RecA mediated DNA strand exchange. Biopolymers. 2006, 81(6):473-96
Lee A., Xiao J.,Singleton SF. Origins of sequence selectivity in homologous genetic recombination: insights from rapid kinetic probing of RecA-mediated DNA strand exchange. J Mol Biol. 2006, 360(2):343-59
Xiao J., Singleton SF., Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA∙triple-stranded DNA complex using fluorescence resonance energy transfer. J. Mol. Biol, 2002 Jul 12;320(3):529-58
Singleton SF., Xiao J., The Stretched DNA geometry of recombination and repair nucleoprotein filaments. Biopolymers, 2001-2002;61(3):145-58
Martin SR, Lu AQ, Xiao J, Kleinjung J, Beckingham K, Bayley PM., Conformational and metal-binding properties of androcam, a testis-specific calmodulin-related protein from Drosophila. Protein Science, 1999, Nov 8(11):2444-54.