Our research goal is to understand cellular processes at high spatial and temporal resolution using novel single-molecule manipulation and imaging methods, including Holographic Optical Tweezers, smFRET, and super-resolution PALM/STORM microscopy. We aim for interdisciplinary research that integrates state-of-the-art instrumentation, critical biological problems, and quantitative data analysis and modeling guided by statistical physics.
Advanced Optical Microscopy and Trapping
We are endeavoring to innovate optical microscopy. We recently developed a one-of-a-kind optical microscope (integrating wide-field and focused-light microscopies) capable of simultaneous epi-fluorescence, total internal reflection fluorescence microscopy, selective plane illumination, and holographic optical tweezers using six lasers. Using this highly versatile microscope, we are performing a variety of advanced microscopy assays (e.g., live-cell imaging, multi-color single-molecule fluorescence, smFRET, PALM/STORM, FRAP, force spectroscopy) on biological systems. We continue to develop the instrument to enable multimodal single-molecule assays inside living cells.
Single-molecule Localization Microscopy (SMLM)
The resolution of all conventional optical microscopies is fundamentally limited to about half the wavelength of light due to diffraction. Single-molecule localization microscopy, also known as PALM and STORM, circumvents this limit by temporally separating stochastically blinking fluorescent molecules one at a time, followed by single-molecule localization using Point Spread Function (PSF) fitting. Our lab retains expertise in this novel microscopy from prior work on bacterial and eukaryotic cell imaging, including E. coli FliM, B. subtilis SpoIIIE, mitochondrial double membranes, and the mitochondrial fission protein Drp1. We are currently applying SMLM to many other systems in collaboration with Rutgers biologists to reveal previously unresolved intracellular structures at super-resolution.
Mitochondrial DNA Packaging and Transcription
Human mitochondria have their own DNA, called mtDNA. Although it encodes only a small number of genes (13 proteins, 2 rRNAs, 22 tRNAs), they are all critical for oxidative phosphorylation. Mitochondrial transcription involves three primary proteins: TFAM (Mitochondrial Transcription Factor A), POLRMT (Mitochondrial RNA Polymerase), and TFB2M (Mitochondrial Transcription Factor B2). TFAM is particularly interesting because it bends mtDNA by almost 180 degrees upon binding. The binding of TFAM to two specific sites on mtDNA (called LSP and HSP promoters) initiates the recruitment of POLRMT and transcription. Remarkably, TFAM can also bind to mtDNA nonspecifically, and the subsequent DNA bending tightly packs mtDNA, similar to how histone proteins condense nuclear DNA into nucleosomes. However, the nonspecific interaction does not incur transcription. We are trying to understand the molecular details of specific vs nonspecific interactions between TFAM and mtDNA using single-molecule methods. This work is in progress in collaboration with the Smita Patel laboratory at Rutgers.
Innate Immune Response
We are interested in the molecular mechanisms of the innate immune response in human cells. Using innovative experimental methods, we are trying to understand how cells detect viral RNA with high specificity and amplify the signal. We are particularly interested in the two top upstream signaling pathways mediated by RIG-I and MAVS. This work is in progress in collaboration with the Smita Patel laboratory at Rutgers.
Plant Cell Wall Synthesis
Cellular production of complex polysaccharides from simple carbohydrate precursors plays critical roles in a variety of metabolic processes essential to the survival of every living organism. In the context of photosynthetic organisms like plants and algae, cell wall polysaccharides synthesis is particularly of interest for emerging biomass and bioenergy-related applications. In this DOE-funded project, we aim to develop an innovative multimodal single-molecule manipulation/imaging optical instrument through integration of holographic optical tweezers, super-resolution fluorescence microscopy, single-particle tracking, and surface-enhanced Raman spectroscopy (SERS) to investigate in vivo cell wall regeneration of protoplasts from Arabidopsis and Poplar, along with collaborators at Rutgers (Shishir Chundawat, Eric Lam, and Laura Fabris), Vanderbilt University, and Oak Ridge National Laboratory.
- DOE grant: https://www.energy.gov/articles/department-energy-announces-40-million-bio-based-research
- Read about the $1.5M DOE grant at the Daily Targum
- Read the story at the SAS website
- Rutgers Access newsletter cover story
We have finally succeeded in capturing the dynamics of how cellulose synthesizes and self-assembles into a dense fibril network on a plant cell surface, for the first time since Robert Hooke’s first microscopic observation of cell walls in 1667. We discovered that cellulose fibrillar networks on plant protoplast cell surfaces develop in multiple stages, and our observations provide fresh insights into how cell walls are made in plants, which has important implications for developing transgenic crops that are optimized for cellulosic bioenergy and agricultural food/feed production.
