This project represents the continuation of a program in single molecule studies of polymer and biological physics that began in 1998 and that has resulted in a number of advances in single-molecule approaches to the study of biological systems. The core focus of the continuation consists of three parts. (i) Single molecule fluorescence combined with force measurements will be integrated with structural and biochemical data to develop a mechanistic understanding of translation by the ribosome. (ii) Studies of in vivo cell signaling in dorsal ganglia root (DRG) neurons induced by neural growth factor (NGF) using quantum dot-labeled NGF and the photo-activated green fluorescent protein labeled TrkA receptor will be undertaken. Studies of further downstream signaling activity with high spatial and temporal resolution are part of this effort. (iii) A research program studying the cell adhesion molecule E-cadherin with combined single molecule FRET and atomic force measurements constitutes the third project. A new, broad-area, non-fluorescing afm tip has been developed and will be used to study the formation and structure of the extracellular adhesive complex and the cellular controls of the strength of cadherin adhesion using live cells. The downstream cell signaling functions of cadherin will be investigated with micro-fluidic methods.
In terms of broader impact, methods, such as optical tweezers, acousto-optic feedback control of optical tweezers, novel flow cells for polymer dynamics studies, FRET with immobilized molecules in stopped-flow geometries, improved bio-compatible surfaces for single molecule enzymology, and the suppression of dye-photobleaching were developed in the course of this research work and are now widely used by other researchers. Students and postdoctoral fellows trained by this P.I. in biophysics are now professors at major research institutions. The present focus on technique development and the training of students and postdocs is expected to continue this trend.
Funding for this award is provided by the Division of Physics in the Directorate for Mathematical and Physical Sciences and by the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.
NSF grant PHY-0647161 The main goals of this grant were to develop technologies for studying fast dynamics of biological reactions and to image macromolecules in physiologically relevant in vitro conditions and in living cells. Over the duration of the award, we have invented various new super-resolution imaging approaches and we have applied them to in vivo and in vitro systems. Due to the length limit of this report, we will focus only on a selection of the most recent developments. Major research areas and findings: Three-dimensional super-resolution imaging of live bacterial cell communities Ultrahigh-resolution imaging of complexes in living cells Super-resolution fluorescence imaging of signaling nanodomains in mammalian cells Single-molecule studies of human RNA Polymerase transcription 1) Three-dimensional super-resolution imaging of living bacterial cell communities Almost all of the bacteria live as a community known as biofilm in their natural habitat. Initially, some of the planktonic bacteria bind to the surface and synthesize a very complex extra-cellular polymeric substances (EPS) composed of carbohydrate polymers, proteins and chromosomal DNA. This process in turn recruits other bacteria to form tissue-like structure called the mature biofilm. Biofilms are directly related to many aspects of our daily lives including chronic diseases, bioenergy and transportation. Using novel imaging technologies, we have been able to characterize how certain biofilms develop from single cells and how they are organized. Molecular architecture and assembly principles of Vibrio cholerae biofilms. Berk V, Fong JC, Dempsey GT, Develioglu ON, Zhuang X, Liphardt J, Yildiz FH, Chu S. Science. 2012 Jul 13;337(6091):236-9. doi: 10.1126/science.1222981. 2) Ultrahigh-resolution imaging of complexes in living cells Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. To address this question, we developed an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We then used this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ. Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ. Pertsinidis A, Mukherjee K, Sharma M, Pang ZP, Park SR, Zhang Y, Brunger AT, Südhof TC, Chu S. PNAS. 2013 Jul 23;110(30):E2812-20. doi: 10.1073/pnas.1310654110. Epub 2013 Jul 2. 3) Super-resolution fluorescence imaging of signaling nanodomains in mammalian cells We have used photoactivated localization microscopy (PALM) to directly visualize individual RAF nanoclusters on the cell membrane. RAF is a key signaling protein in human cancer. Using PALM, we were able to support the possibility that RAF can dimerize and potentially form higher-order multimers, show that RAF multimer formation can result from multiple mechanisms. We concluded that RAF dimerization is a critical but not sufficient step for RAF activation. Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling. Nan X, Collisson EA, Lewis S, Huang J, Tamgüney TM, Liphardt JT, McCormick F, Gray JW, Chu S. PNAS. 2013 Nov 12;110(46):18519-24. doi: 10.1073/pnas.1318188110. Epub 2013 Oct 24. 4) Single-molecule studies of human RNA Polymerase transcription Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes. Transcription initiation by human RNA polymerase II visualized at single-molecule resolution. Revyakin A, Zhang Z, Coleman RA, Li Y, Inouye C, Lucas JK, Park SR, Chu S, Tjian R. Genes Dev. 2012 Aug 1;26(15):1691-702. doi: 10.1101/gad.194936.112. Epub 2012 Jul 18.
