Dr. Ling Chin Hwang
Tel: 0114 222 2847
We use multidisciplinary techniques such as single-molecule imaging, synthetic biology, biochemistry and microfluidics to study the molecular mechanisms of spatial organization in bacteria using cell-free systems. We are interested in understanding how bacteria, despite their small size and lack of obvious cytoskeleton, are able to spatially organize genomic DNA and proteins to specific locations in the cell. These bacterial positioning systems form distinctive patterns within the cells, and are involved in important cellular functions such as DNA segregation, cell division and motility.
Chromosome and plasmid segregation
How do you ensure that each daughter cell inherits a copy of genomic DNA for its survival? Most bacterial chromosomes and low copy number plasmids encode an active segregation machinery called ParABS to partition replicated DNA prior to cell division. It was observed that low copy number plasmids are precisely localized to opposite cell halves upon replication (top left panel) but the mechanism underlying this positioning is not well understood. We reconstituted the plasmid partition system in vitro and coated a flow cell with a ‘DNA carpet’ to act as a biomimetic of the bacterial nucleoid (bottom panel). Purified proteins and plasmids that were fluorescently-labeled were flowed in and we directly watched their dynamics using Total Internal Reflection Fluorescence Microscopy (TIRFM). We found that plasmid dynamics are driven by transient protein gradients or patterns that coat the DNA carpet (top right panel). Future studies will be carried out on understanding how similar protein patterns drive chromosome segregation in bacteria.
Cell division dynamics
In E. coli, the cell division machinery MinDE oscillates from pole-to-pole to position the cell division septum in the middle (top panel). We are interested in the spatial patterning on a molecular level and have reconstituted the Min oscillations on a supported lipid bilayer that mimics the E. coli cell membrane (bottom panel). These traveling waves and spirals are reminiscent of patterns found in nature (Turing patterns) such as zebra stripes and leopard spots, which are based on similar reaction-diffusion mechanism. These in vitro Min patterns will allow us to explore the characteristics of self-organization in the cell.
Single-molecule fluorescence microscopy allows us to watch the movement and interactions of individual DNA and protein molecules, which is inaccessible with conventional biochemical approaches. Our lab uses TIRF microscopy to image surface-immobilized molecules and surface-mediated processes to directly observe single-molecule to mesoscale dynamics. We are interested in developing and applying single-molecule and super-resolution microscopy to answer biological questions as part of the Imagine-Imaging Life program.
Biophysics, biochemistry, single-molecule fluorescence microscopy, bacterial chromosome segregation and cell division
Level 3 Modules
- Brooks AC & Hwang LC (2017) Reconstitutions of plasmid partition systems and their mechanisms. Plasmid, 91, 37-41. View this article in WRRO
- Duchi D, Bauer DLV, Fernandez L, Evans G, Robb N, Hwang LC, Gryte K, Tomescu A, Zawadzki P, Morichaud Z , Brodolin K et al (2016) RNA Polymerase Pausing during Initial Transcription. Molecular Cell, 63(6), 939-950. View this article in WRRO
- Vecchiarelli AG, Li M, Mizuuchi M, Hwang LC, Seol Y, Neuman KC & Mizuuchi K (2016) Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD. Proceedings of the National Academy of Sciences, 113(11), E1479-E1488.
- Vecchiarelli AG, Hwang LC & Mizuuchi K (2013) Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism. Proceedings of the National Academy of Sciences, 110(15), E1390-E1397.
- Robb NC, Cordes T, Hwang LC, Gryte K, Duchi D, Craggs TD, Santoso Y, Weiss S, Ebright RH & Kapanidis AN (2013) The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection. Journal of Molecular Biology, 425(5), 875-885.
- Cordes T, Santoso Y, Tomescu AI, Gryte K, Hwang LC, Camará B, Wigneshweraraj S & Kapanidis AN (2010) Sensing DNA Opening in Transcription Using Quenchable Förster Resonance Energy Transfer. Biochemistry, 49(43), 9171-9180.
- Heilemann M, Hwang LC, Lymperopoulos K & Kapanidis AN (2009) Single-Molecule FRET Analysis of Protein-DNA Complexes, 503-521.
- Santoso Y, Hwang LC, Le Reste L & Kapanidis AN (2008) Red light, green light: probing single molecules using alternating-laser excitation. Biochemical Society Transactions, 36(4), 738-744.
- Hwang LC & Wohland T (2007) Recent Advances in Fluorescence Cross-correlation Spectroscopy. Cell Biochemistry and Biophysics, 49(1), 1-13.
- Liu P, Sudhaharan T, Koh RML, Hwang LC, Ahmed S, Maruyama IN & Wohland T (2007) Investigation of the Dimerization of Proteins from the Epidermal Growth Factor Receptor Family by Single Wavelength Fluorescence Cross-Correlation Spectroscopy. Biophysical Journal, 93(2), 684-698.
- Hwang LC, Leutenegger M, Gösch M, Lasser T, Rigler P, Meier W & Wohland T (2006) Prism-based multicolor fluorescence correlation spectrometer. Optics Letters, 31(9), 1310-1310.
- Hwang LC & Wohland T (2005) Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies. The Journal of Chemical Physics, 122(11), 114708-114708.
- Hwang LC & Wohland T (2004) Dual-Color Fluorescence Cross-Correlation Spectroscopy Using Single Laser Wavelength Excitation. ChemPhysChem, 5(4), 549-551.
- Hwang LC, Vecchiarelli AG, Han Y-W, Mizuuchi M, Harada Y, Funnell BE & Mizuuchi K () ParA-mediated plasmid partition driven by protein pattern self-organization. The EMBO Journal, 32(9), 1238-1249.