While on the Molecular Biophysics Training Grant, I began my research developing a new multiphoton FRET microscopy technique using ultrafast laser pulse shaping. This technique will permit selective excitation of multiple fluorophores in a biological system, as well as provide quantitative information about the concentrations of donor and acceptor molecules and their temporal dynamics in cellular processes. While on the training grant, I implemented the ultrafast pulse shaping and used that to achieve selective excitation of fluorophores in solution. I have subsequently applied this pulse shaping technique to live-cell imaging, obtaining fluorescence images of live Cos-7 cells expressing fluorescent proteins. I have achieved selective excitation of the fluorophores in the cells and observed FRET in the cells. In addition, I have learned how to perform fluorescence lifetime measurements and used those results to determine the FRET efficiency of my system.
I have developed MATLAB code to design pulse shapes as well as perform image processing and analysis, and have also extended existing FRET theory to quantify my results. I am currently performing calculations on my live-cell image data. In addition, I am exploring other applications of ultrafast pulse shaping to live-cell imaging, such as selective excitation in different systems and imaging systems containing multiple spectrally-overlapped fluorophores. I have been trained in the use of ultrafast lasers, basic wet lab techniques, the use of a two-photon laser scanning microscope, and tissue culture techniques to maintain, transfect, and image living cells. I have also taken classes introducing me to a variety of nonlinear optical and biophysical techniques, their theoretical bases, and recent advances in the field.
“Pulse-shaping-based Two-photon FRET Microscopy,” Meredith H. Brenner, Dawen Cai, Samuel W. Straight, Joel A. Swanson, and Jennifer P. Ogilvie. Proceedings of Ultrafast Phenomena 2012.
“Pulse Shaping Multiphoton FRET Microscopy.” Meredith H. Brenner, Dawen Cai, Sarah R. Nichols, Samuel W. Straight, Adam D. Hoppe, Joel A. Swanson, and Jennifer P. Ogilvie. Proceedings of SPIE 8226, 82260R, 2012.
I began my research in the Kopelman lab working to refine techniques used for external calibration of organically modified silane (ORMOSIL) nanoparticles utilized for intracellular electric field sensing in cancer cells. In the last few years, my research has primarily been focused on nanoparticle-mediated photothermal therapy (PTT) of cancer cells. My research has successfully elucidated that covalently linked Coomassie Blue dye polyacrylamide (PAA) nanoparticles can successfully be used for effective PTT of cancer cells, enabling another step in a multimodal approach to cancer detection and treatment using the same nanoplatform that was used for optical and photoacoustic imaging. Finally, my most recent research includes the efficient delivery of femtosecond laser ablation generated gold nanoparticles into cancer cells nuclei, aimed at enabling more effective PTT, as well as other forms of therapies. Upon completion of my Ph.D program in Physics, I would like to expand my research to include the use of nanoplatforms in the detection, imaging, and treatment of malignant cell lines.
Antonuk LE, El-Mohri Y, Du H, Behravan M, Curry T, Zhao Q, Koniczek M, Street R, and Lu JP: Exploration of the Potential Performance of Polycrystalline Silicon-based Active Matrix Flat Panel Imagers Incorporating Active Pixel Sensor Architectures. SPIE Medical Imaging 2008 - Physics of Medical Imaging, San Diego, CA, February 16-21, 2008. (Abstract and oral presentation.)
Behravan M, Antonuk LE, El-Mohri Y, Zhao Q, Yeakey M, Martelli C, McDonald J, Curry T and Koniczek M: Noise Characterization of Polycrystalline Silicon Thin Film Transistors for X-ray Imagers Based on Active Pixel Sensor
Architectures. Materials Research Society Spring 2008 Meeting: Symposium A – Amorphous and Polycrystalline Thin-Film Silicon Science and Technology, San Francisco, CA, March 24 - 28, 2008. (Abstract and oral presentation.)
Taeyjuana Curry, Tamir Epstein, Ron Smith, & Raoul Kopelman. Photothermal Therapy of Cancer Cells mediated by Blue Hydrogel Nanoparticles. Under review.
Taeyjuana Curry, Wei Qian, & Raoul Kopelman. Surface Optimization of Gold Nanoparticles for Controlled Delivery Into Cancer Cell Nuclei. Submitted.
At the outset of my studies, little was known of the functions and importance of CH O hydrogen bonding in biology. To address the dearth of information on this subject, I originated a collaboration between the Al-Hashimi and Trievel labs to bring multiple biophysical and biochemical techniques to bear, as well as developing techniques specifically to examine CH O hydrogen bonding. I focused my research on the role of CH O hydrogen bonds in AdoMet-dependent methylation, as it is of prime importance to many cellular functions. Previous studies had suggested that the catalytic methyl group might form CH O hydrogen bonds within the methyltransferase active site. Using multiple techniques, I was able to show that not only are CH O hydrogen bonds present in all seven structural classes of AdoMet-dependent methyltransferases, but that they are required for both cofactor binding and catalysis. Additionally, using NMR spectroscopy and X-ray crystallography, I have experimentally determined the atomic-scale structure and motions of the catalytic methyl group and its hydrogen bonding partners, and found that these interactions aid in catalysis both by stabilizing the positively charged transition state, as well as restricting methyl group motion. Of note, as these interactions are the only known conserved element in the many different structural classes of AdoMet-dependent methyltransferases, we believe that these interactions were a critical element in methyltransferase convergent evolution, and are fundamental to the methyl transfer reaction.
Rafiee P, Theriot ME, Nelson VM, Heidemann J, Kanaa Y, Horowitz SA, Rogaczewski A, Johnson CP, Ali I, Shaker R, Binion DG. Human Esophageal Microvascular Endothelial Cells Respond to Acidic pH Stress by PI3K/AKT and p38 MAPK-regulated Induction of Hsp70 and Hsp27 Am J Physiol Cell Physiol, 291(5) C931-945 (2006)
Horowitz S, Binion DG, Nelson VM, Kanaa Y, Javadi P, Lazarova Z, Andrekopoulos C, Kalyanaraman B, Otterson MF, Rafiee P. Increased Arginase Activity and Endothelial Dysfunction in Human Inflammatory Bowel Disease Am. J. Physiol Gastrointest Liver Physiol, 292(5)G1323-G1336 (2007)
Krishnan S*, Horowitz S*, Trievel RC. Structure and Function of Histone H3 Lysine 9 Methyltransferases and Demethylases Chembiochem, 12(2):254-263 (2011)
Horowitz S, Yesselman JD, Al-Hashimi HM, Trievel RC. Direct Evidence for Methyl Group Coordination by Carbon-Oxygen Hydrogen Bonds in the Lysine Methyltransferase SET7/9, J. Biol. Chem, 286(21):18658-63 (2011)
Horowitz S, Dirk LMA, Yesselman JD, Nimtz J, Del Rizzo PA, Mehl RA, Houtz RL, Al-Hashimi HM, Trievel RC. Methyl CH O Hydrogen Bonds Orchestrate AdoMet-Dependent Methylation Under review
Frank AT, Horowitz S, Andricioaei I, Al-Hashimi HM. Use of 1H NMR Chemical Shifts in Determining RNA Structure and Dynamics In preparation
Horowitz S, Trievel RC. Carbon-Oxygen Hydrogen Bonds in Biology, J. Biol. Chem, invited review article, submitted
Yesselman JD*, Horowitz S*, Trievel RC, Brooks CL. Systematic Analysis of Methyl CH O Hydrogen Bonding in Proteins In preparation
Horowitz S*, Fick RJ*, Trievel RC. Mechanistic Aspects of AdoMet-Dependent Methylation invited review article, to be published May 2013
*denotes equal contribution
The past decade has seen a rapid expansion of interest in the construction of nanoscale materials and devices from DNA owing to its predictable self-assembly properties and the ease with which it can be functionalized to position and interact with other functional materials. The maturation of this field requires a detailed understanding of the impact of design parameters on assembly yield and performance. To this end, I am using single-particle fluorescence techniques to study the kinetic and chemical properties of DNA nanomaterials.
In particular, I have developed two techniques: nanoscale chemical fingerprinting and single-origami kinetic assays. The first technique combines single-particle localization with reversible binding of fluorescent probes to specific features of a target structure – in this case, a DNA origami tile with patterned surface features – to generate a quantitative nanoscale map of the interactions between the structure and components in solution. Using a two-color approach, I can distinguish different patterns of surface features and monitor enzymatic modifications of the surface patterns. I have also discovered stable spatial distributions of binding, or “fingerprints” of interactions, that are unique to each DNA tile and not predictable based on the design. This technique empowers us to characterize subtle, but important chemical properties of single nanostructures with unprecedented spatial and temporal detail.
The second technique, single-origami kinetic assays, involves monitoring the cumulative fluorescence or FRET (Förster resonance energy transfer) signal from individual nanoscale DNA origami tiles as they bind fluorescently labeled components from solution.. Using this technique, I find that interactions between adjacent binding sites have a significant impact on DNA hybridization kinetics and overall yield. These findings have important implications for the growing number of applications in which components are assembled on DNA scaffolds with controlled stoichiometry and spacing.
Johnson-Buck, Alexander E.; Blanco, Mario R.; Walter, Nils G. (2012). Single-molecule fluorescence resonance energy transfer. In Encyclopedia of Biophysics. 1 (G. Roberts, Ed.), Springer, in press.
Blanco, Mario R.; Johnson-Buck, Alexander E.; Walter, Nils G. (2012). Hidden Markov modelling. In Encyclopedia of Biophysics. 1 (G. Roberts, Ed.), Springer, in press.
Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.; Walter, Nils G. (2011) Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. DOI: 10.1002/wnan.170.
Marek, Matt S.; Johnson-Buck, Alexander E.; Walter, Nils G. (2011). The shape-shifting quasispecies of RNA: One sequence, many functional folds. Physical Chemistry Chemical Physics. 13, p. 11524-11537.
Johnson-Buck, Alexander E.; McDowell, Sarah E.; Walter, Nils G. (2011). Metal ions: Supporting actors in the playbook of small ribozymes. In Metal Ions Life Sci. 9 (A. Sigel, H. Sigel, R.K.O. Sigel, Eds.), The Royal Society of Chemistry, Cambridge, UK, in press.
Lund, Kyle A.; Manzo, Anthony J.; Dabby, Nadine; Michelotti, Nicole; Johnson-Buck, Alexander; Nangreave, Jeanette; Taylor, Steven; Pei, Renjun; Stojanovic, Milan N.; Walter, Nils G.; Winfree, Erik; Yan, Hao. (2010). Molecular robots guided by prescriptive landscapes. Nature 465, 206-210.
Michelotti, Nicole, de Silva, Chamaree, Johnson-Buck, Alexander E., Manzo, Anthony J., Walter, Nils G. (2010). A bird's eye view: tracking slow nanometer-scale movements of single molecular nano-assemblies. Methods in Enzymology 475, 121-148.
Johnson-Buck, Alex; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Kopelman, Raoul. Fabrication, Characterization, and Spectral Properties of Indigo Blue Nanocrystals. (2009). Mol. Cryst. Liq. Cryst. 501, 138-144.
Vibrational spectroscopy has proven to be a unique tool in determining chemical behaviors of a variety of biological systems. These techniques offer intrinsic selectivity, as these techniques are able to probe different chemical signatures. Such methods have been particularly applicable for use in non-invasive imaging and surface characterization.
The health, growth, and preservation of human oocytes have been the targets of many advanced screening methods which determine viability as well as likelihood of eventual fertilization. The most accurate and widely used assays for determining oocyte health are based on morphological scoring, which are prone to the most error. Part of my thesis research has focused on developing coherent anti-Stokes Raman scattering (CARS) microscopy into a powerful non-invasive analytical tool in screening for cryopreservation candidate selection and providing a method to permit modification cryoprotectant exposure regimens to improve oocyte cryotolerance. To date, I have successfully developed a method to calculate total lipid content which has been shown to directly correlate with certain developmental stages in oocyte maturation. My eventual goal is to modify oocytes with established cryoprotectant exposure protocols and determine whether a chemical difference can be determined which then can be used to refine previous protocols.
Another facet of my thesis research also focuses on the biological interactions of peptides at an abiotic surface. Although peptides are often associated with an aqueous environment, it is important to be able to build a surface which contains active proteins in the absence of bulk water. This has applications in textiles, medical devices as well as the military where environments are not necessarily conducive to certain proteins. Currently I am using sum frequency generation (SFG) vibrational spectroscopy to characterize surfaces with chemically immobilized anti-microbial peptides (AMPs). SFG is a surface-sensitive technique which can be used to monitor protein secondary structure. The next goal of this research is to modify this abiotic surface in order to facilitate these peptides to keep their proper secondary structure and AMP activity.
Khamaladze A, Matz RS, Epstein T, Jasensky J, Banaszak-Holl MM, and Chen Z. Cell volume changes during apoptosis monitored in real time using digital holographic microscopy J. Struct. Biol. 2012, 178(3):270-278.
Ye SJ, Li HC, Wei F, Jasensky J, Boughton AP, Yang P, and Chen Z. Observing a Model Ion Channel Gating Action in Model Cell Membranes in Real Time in Situ: Membrane Potential Change Induced Alamethicin Orientation Change J. Am. Chem. Soc. 2012, 14(134):6237-6243.
Liu Y, Jasensky J, and Chen Z. Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy Langmuir 2012, 28(4):2113-2121.
Khamaladze A, Jasensky J, Zhang C, Han XF, Ding J, Seeley E, Liu XR, Smith GD, and Chen Z. Hyperspectral microscopic imaging by multiplex coherent anti-Stokes Raman scattering (CARS) Proc. SPIE 2011, 8158, 815805.
Zhang C, Wang J, Khmaladze A, Liu Y, Ding B, Jasensky J, and Chen Z. Examining Surface and Bulk Structures Using Combined Nonlinear Vibrational Spectroscopies Opt. Letts. 2011, 36(12):2272-2274.
Stephanie Le Clair
While on the training grant, Sum Frequency Generation (SFG) was utilized to monitor the real-time membrane interaction of truncated forms of human and rat islet amyloid polypeptide (IAPP). IAPP is the main component in amyloid plaques found on the pancreatic islets of Langerhans in type II diabetes patients. Truncated human IAPP(1-19) has been shown to be cytotoxic and does not form fibers. Whereas truncated rat IAPP(1-19) only differs from human IAPP(1-19) at residue 18, it is significantly less cytotoxic. The goal of the research was to ascertain the difference in cytotoxicity between these two peptides. Using SFG, the amide I band of each peptide was deconvoluted to determine the secondary structure and membrane orientation. This was done for membranes of diverse compositions and under several buffer conditions. FTIR-ATR was also used to cross-validate the orientations obtained with SFG. Differences in the SFG limits of detection were shown when comparing rat IAPP(1-19) and human IAPP(1-19).
When the training grant ended, I continued research under the NSF graduate research fellowship and changed projects. The goal of my current project is to characterize the structure and dynamics of the full-length cytochrome b5-cytochrome P450 (cyt b5-cyt P450) complex in a membrane environment. These two proteins have an important role in the metabolism of a variety of endogenous and exogenous compounds but little is known regarding how the full-length proteins interact with one another. Solution NMR was used to probe the complex formation between these two proteins in isotropic bicelles, both in the presence and absence of a cyt P450 substrate. The interaction interface of cyt b5 for cyt P450 was mapped out and this information, as well as double mutant cycle analysis data, were used to deduce the cyt b5-cyt P450 complex structure with HADDOCK. Through a series of NMR experiments, we have also begun to address the role of electrostatic interactions in the encounter complex formation between cyt b5 and cyt P450 and have determined the key hydrophobic “hot spot” residues of cyt b5 which are essential in binding to cyt P450. We are also unraveling the kind of role that cyt P450 ligands play in the interaction of cyt P450 with cyt b5. Specific regions of cyt b5 and cyt P450 are also being studied to understand their role in complex formation: the linker region of cyt b5 and the transmembrane domain of cyt P450.
Le Clair, S. V.; Zhang, M.; Im, S.-C.; Waskell, L.; Ramamoorthy A. “Anatomy of the cyt b5 interface in its stereospecific complex and encounter complexes with cytochrome P450.” In preparation.
Ahuja, S.; Jahr, N.; Im, S.-C., Vivekanandan, S.; Popovych, N.; Xu, J.; Soong, R.; Le Clair, S. V.; Nanga, R. P.; Yamamoto, K.; Bridges, A.; Waskell, L.; and Ramamoorthy, A. “A dynamic structure of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data.” Submitted.
Nguyen, K.; Le Clair, S. V.; Ye, S.; and Chen, Z. “Orientation Determination of Protein Helical Secondary Structure Using Linear and Nonlinear Vibrational Spectroscopy.” J. Phys. Chem. B. 2009, 113, 12169-12180.
Nguyen, K.; Le Clair, S. V.; Ye, S.; and Chen, Z. “Molecular Interaction between Magainin 2 and Model Membranes in Situ.” J. Phys. Chem. B. 2009, 113, 12358-12363.
Ye, S.; Nguyen, K. T.; Le Clair, S. V.; and Chen, Z. “In Situ Molecular Level Studies on Membrane Related Peptides and Proteins in Real Time Using Sum Frequency Generation Vibrational Spectroscopy.” J. Struct. Biol. 2009, 168, 61-77.
Le Clair, S. V.; Nguyen, K.; and Chen, Z. "Sum Frequency Generation Studies on Bioadhesion: Elucidating the Molecular Structure of Proteins at Interfaces.” J. Adhesion. 2009, 85, 484-511.
Esmonde-White, K. A.; Le Clair, S. V.; Roessler, B. J.; Morris, M. D. “Effect of Conformation and Drop Properties on Surface-Enhanced Raman Spectroscopy of Dried Biopolymer Drops.” Appl. Spectrosc. 2008, 62, 503-511.
The cell is an extremely complex system that is highly regulated both spatially and temporally. Subcellular cargoes including organelles, endosomes, lysosomes, and mRNA granules are often directed to specific locations in the cell in a time-dependent fashion. This targeted intracellular trafficking is usually accomplished by molecular motor proteins such as kinesin and dynein, which drive cargo motion along cytoskeletal tracks. Collections of multiple motors working together are thought to be required to generate sufficient force and distance for transport of a large cargo through the dense cytoplasm. Over the past two decades, highly precise single-molecule experiments have shown how a single kinesin or dynein motor generates force in vitro. Considerations of multiple-motor cargo transport in live cells are muddled by the complexity of the cell, and multiple-motor in vitro experiments are highly artificial and do not allow ideal experimental precision.
We propose to reconcile these two types of experiments by developing a synthetic biology method to create well-characterized multiple motor complexes. We use a protein-based method to specifically attach known numbers of motor proteins to scaffolds of known length and flexibility. We express these custom-designed constructs in mammalian COS-7 cells and assemble multiple-motor complexes in live cells. The motility properties of these complexes can then be studied in live cells or in vitro via TIRF (Total Internal Reflection Fluorescence) microscopy. This method allows us to investigate many outstanding questions in the motor field with a high degree of accuracy and precision. Although these synthetic biology methods are being applied to molecular motors here, one could conceivably use these tools to create more general multi-protein complexes in live cells where genetic fusion does not suffice.
(In preparation – Biophysical Journal): J.A. Cribb, P.A. Vasquez, P. Moore, S. Norris, S. Shah, M.G. Forest, and R. Superfine. Nonlinear signatures of entangled polymer solutions in active microbead rheology.
I am exploring how B lymphocyte’s antigen receptor (BCR) signaling utilizes lipid organization to regulate signaling. Although lipid phase separation in model membranes has been shown extensively, there is little direct evidence that lipid organization exists and has a function in live cells. However, many theories explaining how BCR antigen binding is communicated across the plasma membrane assert that lipid organization either drives or regulates this process by controlling the partitioning of membrane proteins to the BCR proximal. I aim to clarify the role of lipid-mediated heterogeneity in BCR signal regulation through the use of two-color super-resolution microscopy. I have applied highly accurate image registration to our lab’s pre-existing super-resolution imaging techniques to allow for accurate two-color single molecule microcopy. This has facilitated observations of lipid reorganization which are dependent upon BCR antigen binding. Branched and saturated lipids respond to BCR clustering differently, which is consistent with lipid phase behavior seen in model membranes. An understanding of how lipid organization affects signaling through by the BCR may allow for novel drugs targeting lipids to treat B cell based autoimmune diseases and cancers. In addition, I am characterizing the fluorescence of indocarbocyanine dyes for use in quantitative two-color super-resolution and developing new analysis methods based on correlation functions, both of which will allow for highly accurate measurements of spatio-temporal correlation between separately labeled biomolecules. I hope to share these developments through publications and conferences in order to facilitate further research in these areas by other research groups.
Chen, Ran, Tatsiana A Ratnikova, Matthew B Stone, Sijie Lin, Mercy Lard, George Huang, JoAn S Hudson, and Pu Chun Ke. 2010. “Differential uptake of carbon nanoparticles by plant and Mammalian cells.” Small (Weinheim an Der Bergstrasse, Germany) 6 (5) (March 8): 612-617.