Single-Molecule Studies in Bacterial Cells
My research group focuses on bacterial cell division, transcription and gene regulation. We combine sensitive, high resolution single-molecule methods with well established genetic and biochemical methods to address challenges in bacterial cell biology.
Dynamics and structure of the division complex
Prokaryotic cell division is a conserved process that requires the formation of a multi-protein complex (termed divisome) to carry out constriction. Although many of the molecular constituents have been identified, the structural organization of the divisome remains elusive. The central component of the divisome is FtsZ, a highly conserved prokaryotic tubulin homolog that polymerizes at midcell to form a ring-like structure (termed the Z-ring). The FtsZ-ring is not only required for the assembly of all other division proteins, but may also generate a constrictive force during cytokinesis. We are interested in understanding how FtsZ and other components of the divisome are orchestrated to function and coordinate with other essential cellular events. Specifically, we focus on the following questions in E. coli cells:
- What is the structural organization of the divisome?
- How does the divisome reorganize during cell wall construction?
- What's the mechanism for force generation during cell wall constriction?
these goals we established single-molecule based superresolution imaging methods to illustrate the structure and dynamics of the FtsZ-ring with its associated proteins. Our recent publications are:
Buss, J., C. Coltharp, T. Huang, C. Pohlmeyer, S.C. Wang, C. Hatem, and J. Xiao, In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy. Mol Microbiol, 2013. 89(6): p. 1099-120. PubMed
Coltharp, C., R.P. Kessler, and J. Xiao, Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements. PLoS One, 2012. 7(12): p. e51725. PubMed
Coltharp, C. and J. Xiao, Superresolution microscopy for microbiology. Cell Microbiol, 2012. 14(12): p. 1808-18. PubMed
Fu, G., T. Huang, J. Buss, C. Coltharp, Z. Hensel, and J. Xiao, In vivo structure of the E. coli FtsZ-ring revealed by photoactivated localization microscopy (PALM). PLoS One, 2010. 5(9): p. e12682. PubMed
Spacial organization and dynamics of transcription Prokaryotic transcription has been extensively studied in the past half a century. However, there often exists a gap between the structural, mechanistic description of transcription obtained from in vitro biochemical studies, and the cellular, phenomenological observations from in vivo genetic studies. It is now accepted that a living bacterial cell is a complex entity; the heterogeneous cellular environment is drastically different from the homogenous, well-mixed situation in vitro. Where molecules are inside a cell may be important for their functions. Hence, examining the in-vivo spatial organization and dynamics of transcription in live cells is of particular important in finding new insight, possibly bridging the gap. Here is how we think of this problem:
Weng, X. and J. Xiao, Spatial organization of transcription in bacterial cells. Trends in Genetics, 2014. in press.
Hensel, Z., X. Weng, A.C. Lagda, and J. Xiao, Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells. PLoS Biol, 2013. 11(6): p. e1001591. Article
Noise control mechanisms in gene regulatory networksGene 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. How do cells function with amazing precisions 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 including transcription factors in real time. The publications listed here are a few examples about how we address this problem:
Hensel, Z. and J. Xiao, Single-molecule methods for studying gene regulation in vivo. Pflügers Archiv-European Journal of Physiology, 2013. 465(3): p. 383-95.
Hensel, Z., H. Feng, B. Han, C. Hatem, J. Wang, and J. Xiao, Stochastic expression dynamics of a transcription factor revealed by single-molecule noise analysis. Nat Struct Mol Biol, 2012. 19(8): p. 797-802.
Hensel, Z. and J. Xiao, A mechanism for stochastic decision making by bacteria. Chembiochem, 2009. 10(6): p. 974-6.
Yu, J., J. Xiao, X. Ren, K. Lao, and X.S. Xie, Probing gene expression in live cells, one protein molecule at a time. Science., 2006. 311(5767): p. 1600-3.
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.