3RD MIDWEST SINGLE MOLECULE WORKSHOP UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN

AUGUST 4 - 5, 2014

University of Illinois – Physics Department – 320 Loomis Lab, 1110 W. Green Street – Urbana-Champaign, Illinois 61801 – 217-333-3393

CONTENTS ORGANIZERS Program – 2 Prof. Yann R. Chemla – University of Illinois Oral presentation abstracts – 5 Dr. Jaya Yodh – University of Illinois Poster list – 17 Poster presentation abstracts – 20 Management team: Participants – 45 Angala Meharry – University of Illinois Maps – 49 Sandra Patterson – University of Illinois

Contact: VENUE [email protected] Alice Campbell Alumni Center http://cplc.illinois.edu/workshops/MWSMW2014 601 South Lincoln Avenue

Urbana, IL 61801 http://www.uiaa.org/alumnicenter/contact.html

SPONSORED BY

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PROGRAM

MONDAY, AUGUST 4, 2014 ALICE CAMPBELL ALUMNI CENTER

8:00 a.m. - 8:45 a.m. Registration & refreshments Welcome 8:45 a.m. - 9:00 a.m. Yann Chemla – University of Illinois at Urbana-Champaign

Keynote lecture: Stephen Kowalczykowski – University of California, Davis 9:00 a.m. - 10:00 a.m. “Single-Molecule Visualization of Protein-DNA Complexes: Understanding the Physics and Chemistry of Biology, One Molecule at a Time”

10:00 a.m. - 10:20 a.m. Coffee break

SESSION I: “Single-Molecule Interactions”

Chair: Yann Chemla – University of Illinois at Urbana-Champaign

Talk 1: Sanjeevi Sivasankar – Iowa State University 10:20 a.m. - 10:40 a.m. “Conformational Switching in Single Prion Proteins Promotes Oligomerization”

Talk 2: Charles Schroeder – University of Illinois at Urbana-Champaign 10:40 a.m. - 11:00 a.m. “Direct Observations of TALE Protein Search Dynamics Along DNA”

Talk 3: Yi Luo – The Ohio State University 11:00 a.m. - 11:20 a.m. “Nucleosomes Accelerate Transcription Factor Dissociation”

Talk 4: Sujay Ray – Kent State University 11:20 a.m. - 11:40 a.m. “G-quadruplex Formation in Telomeres Enhances POT1/TPP1 Protection Against RPA Binding”

Talk 5: Krishna Sigdel – University of Missouri 11:40 a.m. - 12:00 p.m. “Mechanical Insight into Lipid-protein Interactions Using Bee Venom”

12:00 p.m. - 2:30 p.m. LUNCH & POSTER SESSION I (Posters I-1 to I-27)

SESSION II: “Molecular machines”

Chair: Wei Cheng – University of Michigan

Talk 6: Peter Cornish – University of Missouri 2:30 p.m. - 2:50 p.m. “Structured mRNA Induces the Ribosome into a Hyper-rotated State” Talk 7: Julia Widom – University of Michigan 2:50 p.m. - 3:10 p.m. “Dissecting the Functions of RNA Helicases in Splicing by Single-molecule FRET” Talk 8: Maria Spies – University of Iowa 3:10 p.m. - 3:30 p.m. “Single-molecule Studies of FeS-containing DNA Helicases: Kinetics, Conformational Dynamics and Molecular Mechanisms”

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PROGRAM - continued

MONDAY, AUGUST 4, 2014 ALICE CAMPBELL ALUMNI CENTER

3:30 p.m. - 3:50 p.m. Coffee break

3:50 p.m. - 4:10 p.m. Talk 9: Eric Tomko – Washington University in St. Louis “ATP- and NTP-dependent Promoter Opening by the Yeast RNAP II Pre-initiation Complex” 4:10 p.m. - 4:30 p.m. Talk 10: Alfonso Brenlla – Wayne State University “Mechanism of Aromatic Carcinogen Bypass by the Y-family Polymerase Dpo4”

4:30 p.m.- 6:30 p.m. POSTER SESSION II (Posters II-1 to II-25)

7:00 p.m.- 9:00 p.m. RECEPTION – BREAD COMPANY, 706 S. GOODWIN AVENUE, URBANA

TUESDAY, AUGUST 5, 2014 ALICE CAMPBELL ALUMNI CENTER

8:50 a.m. - 9:00 a.m. Introduction & announcements SESSION III: “Single molecules in live cells” Chair: Maria Spies – University of Iowa

9:00 a.m. - 9:20 a.m. Talk 11: Kenneth Ritchie – Purdue University “Mobility of TonB and FepA in the Membranes of E. coli”

9:20 a.m. - 9:40 a.m. Talk 12: Wenting Li, University of Wisconsin-Madison “Single Molecule Study of RelA During the Stringent Response in Live E. coli Cells”

Talk 13: Rudra Kafle, University of Michigan 9:40 a.m. - 10:00 a.m. “Dynamics of Chromosomal DNA in Escherichia coli”

10:00 a.m. - 10:20 a.m. Coffee break

10:20 a.m. - 10:40 a.m. Talk 14: Taekjip Ha – University of Illinois at Urbana-Champaign “Single Molecules and Cellular Mechanics”

10:40 a.m. - 11:00 a.m. Talk 15: Yan Mei Wang – Washington University in St. Louis “Single-molecule Investigation of Intraflagellar Transport Mechanisms”

Talk 16: Qiong Yang – University of Michigan 11:00 a.m. - 11:20 a.m. “From Molecules to Development: Revealing Simple Rules of Biological Clocks”

11:20 a.m. - 11:50 a.m. Business meeting 11:50 a.m. - 12:00 p.m. Poster awards 12:00 p.m. - 1:00 p.m. Lunch

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PROGRAM - continued

TUESDAY, AUGUST 5, 2014 ALICE CAMPBELL ALUMNI CENTER

SESSION IV: “New methods” Chair: Taekjip Ha – University of Illinois at Urbana-Champaign

Talk 17: Wei Cheng – University of Michigan 1:00 p.m. - 1:20 p.m. “Optical Trapping and Multi-parameter Analysis of Single HIV-1 in Culture Media Reveal the Positive Cooperativity of Envelope Spikes in Mediating Viral Infection”

1:20 p.m. - 1:40 p.m. Talk 18: Richelle Teeling-Smith – The Ohio State University “Exploring Dynamics with Single-Molecule Electron Paramagnetic Resonance”

Talk 19: Randall Goldsmith – University of Wisconsin-Madison 1:40 p.m. - 2:00 p.m. “What to do when the (fluorescent) lights go out: toward single molecule spectroscopy with optical microresonators”

Talk 20: Margaret Rodgers – University of Wisconsin-Madison 2:00 p.m. - 2:20 p.m. “A dual-functioning genetic tag for simultaneous isolation and observation of single fluorescent complexes from whole cell extract”

2:20 p.m. - 2:40 p.m. Talk 21: Aleksei Aksimentiev – University of Illinois at Urbana-Champaign “Probing DNA-protein association through atomistic and coarse-grained simulations”

Talk 22: Michael Hudoba – The Ohio State University 2:40 p.m. - 3:00 p.m. “Design of Force-Sensitive DNA Origami Components”

3:00 p.m. Closing remarks & departure

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ORAL PRESENTATION ABSTRACTS

KEYNOTE LECTURE

Single-Molecule Visualization of Protein-DNA Complexes: Understanding the Physics and Chemistry of Biology, One Molecule at a Time

Stephen Kowalczykowski University of California, Davis

It is now possible to image individual proteins acting on single molecules of DNA. Such imaging affords unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, a single DNA molecule is attached to a polystyrene bead, which is captured in an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow in a micro-flow cell, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Proteins and DNA are visualized via fluorescent reporters. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Molecules are introduced and supramolecular complexes are built, one component at a time, using microfluidic flowcells. Using these approaches, we have watched proteins functioning in the repair, replication, and manipulation of DNA. We have imaged unwinding of DNA by helicases, translocation along DNA by motor proteins, self-assembly of protein filaments on DNA as well as regulation of nucleation and growth, the search for DNA sequence homology protein-DNA filaments, replication of DNA, and nucleosome structure and it’s remodeling. I will summarize how these experiments were done, what we’ve learned, and prospects for the future.

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MONDAY AUGUST 4, 2014

SESSION I: SINGLE-MOLECULE INTERACTIONS

Talk 1: Conformational Switching in Single Prion Proteins Promotes Oligomerization

Sanjeevi Sivasankar Iowa State University

Transmissible spongiform encephalopathies which include neurodegenerative diseases like Creutzfeldt–Jakob disease, bovine spongiform encephalopathy, chronic wasting disease and scrapie, are characterized by the misfolding and oligomerization of prion proteins into protease resistant neurotoxic aggregates. However, the molecular mechanisms of prion misfolding and aggregation are unclear. Here we report, at the single molecule level, the role of the structured and disordered regions of prion proteins and different divalent ion co-factors in their protease resistance and oligomerization. Using a single molecule fluorescence based protease resistance assay, we demonstrate that prion monomers misfold to a protease resistant conformation before oligomer assembly; the intrinsically disordered N-terminal region and Cu2+ ions are obligatory for this conformational switching. Using single molecule force measurements with an Atomic Force Microscope (AFM) we show that the protease resistant conformation has a 900-fold higher association constant compared to the native conformation. The high binding affinity of protease resistant prions indicates that they serve as monomeric seeds for the subsequent formation of neurotoxic aggregates.

Talk 2: Direct Observation of TALE Protein Search Dynamics Along DNA

Charles M. Schroeder University of Illinois at Urbana-Champaign

In this talk, we discuss the direct observation of transcription activator-like effector (TALE) protein dynamics along DNA templates using single molecule techniques. TALE proteins are robust, programmable DNA-binding proteins that can be fused to a nuclease domain to generate the TALEN system, a leading technology in the field of genetic engineering. In recent years, powerful methods for gene editing have been developed, including zinc finger nucleases, the CRISPR/Cas9 system, and TALENs. Despite great promise for treating human disease, however, we still lack a complete understanding of the mechanisms governing TALE search dynamics and the role of off-target binding events that threaten to inhibit clinical implementation. From this view, our work aims to develop a molecular-level understanding of TALE binding and target sequence search on DNA, which will facilitate the design of new and efficient TALEN systems. In this work, we developed a single molecule assay to directly visualize the binding and 1-D search dynamics of TALE proteins along stretched, dual-tethered DNA templates. We implemented an efficient method for specific labeling of TALE proteins using an aldehyde-based bioorthogonal labeling scheme relying on formylglycine generating enzyme. Single molecule data on TALE search dynamics reveal a previously unknown two-state “search and bind” model, wherein rapid periods of 1-D diffusion along DNA are interspersed with stagnant periods of protein binding. The two-state kinetic model reconciles the ability for TALE proteins to quickly locate their target sequence amongst thousands of potential binding sites. We further generated a series of truncated TALE variants and observed the dynamics of these proteins at the single molecule level. In this way, we are able to identify the role of TALE subdomains on protein search, thereby further advancing the understanding of TALE dynamics. Overall, our work provides a “first-of- its-kind” view of the 1-D diffusion of TALE proteins on DNA, which will be critically important for the engineering of improved TALE proteins for precise genomic editing.

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Talk 3: Nucleosomes Accelerate Transcription Factor Dissociation

Yi Luo, Justin A. North, Cai Chen, Gary Kash, Sean Rose, Ralf Bundschuh, Michael G. Poirier The Ohio State University

Transcription factors (TF) bind DNA target sites within promoters to activate gene expression. TFs achieve high binding specificity on their recognition DNA sequence by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single-molecule Total Internal Reflection Fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA, which can be explained by a competitive partial binding model. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA-protein interactions not only by blocking DNA-protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA target sites.

Talk 4: G-quadruplex Formation in Telomeres Enhances POT1/TPPq Protection Against RPA Binding

Sujay Ray, Ray, Jigar N. Bandaria, Mohammad H. Qureshi, Ahmet Yildiz and Hamza Balci Kent State University

Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). POT1/ TPP1 heterodimer, a part of the shelterin complex, binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K+, POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA’s access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na+, in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

Talk 5: Mechanical Insight into Lipid-protein Interactions Using Bee Venom

Krishna P Sigdel, Nagaraju Chada, Tina R. Matin, Stephen White, Martin Ulmschneider and Gavin King University of Missouri

Lipid-protein interactions play vital roles in the stability and activity of membrane proteins. Melittin, a small linear peptide consisting of 26 amino acid residues, is a major toxic component in the venom of the European bee Apis mellifera. This cationic peptide is soluble but also has amphiphilic properties which make it suitable for monitoring lipid-protein interactions in membranes. In this work melittin was functionally and covalently tethered to an AFM tip and advanced towards a supported lipid bilayer and then retracted from that lipid surface. The goal of these force spectroscopy experiments was to directly measure the mechanical interaction between melittin and a POPC lipid bilayer. These measurements shed light upon the structure/function relationship of melittin in a near-native lipid environment.

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SESSION II: MOLECULAR MACHINES

Talk 6: Structured mRNA Induces the Ribosome into a Hyper-rotated State

Peter Cornish University of Missouri

During protein synthesis, mRNA and tRNA are moved through the ribosome by the process of translocation. The small diameter of the mRNA entrance tunnel only permits unstructured mRNA to pass through. However, there are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here, we show using single molecule Förster resonance energy transfer and small angle X-ray scattering experiments a new global conformational state of the ribosome. In the presence of the frameshift inducing dnaX hairpin, the ribosomal subunits are driven into a hyper-rotated state and the L1 stalk is predominantly in an open conformation. This previously unobserved conformational state provides structural insight into the helicase activity of the ribosome and may have important implications for understanding the mechanism of reading frame maintenance.

Talk 7: Dissecting the Functions of RNA Helicases in Splicing by Single-Molecule FRET

Julia R. Widom, Matthew L. Kahlscheuer and Nils G. Walter University of Michigan

The process of splicing is a critical step in gene expression, and defects in the splicing pathway are associated with many human genetic diseases. The spliceosome is unlike many macromolecular machines in that it lacks a pre-formed catalytic core and is built through sequential steps of assembly and rearrangement on the template of the pre-mRNA. These rearrangement steps require the activity of numerous RNA helicases and ATPases. Prp22 is an RNA helicase found in yeast (with a human homologue, HRH1) that is required for the release of the mRNA product after the two chemical reactions of splicing are complete. I will present experiments in which single- molecule FRET is used to investigate the unwinding activity of Prp22 on model substrates, with the goal of understanding how this unwinding activity is functionally utilized in the spliceosome. I will also describe earlier experiments that applied smFRET to the study of the entire spliceosome, using the technique of single-molecule pulldown FRET (SiMPull-FRET) to isolate complexes stalled before the first reaction of splicing. The study of Prp22 will be extended to the spliceosome using these techniques, which will allow us to isolate from yeast extract spliceosomal complexes that have been stalled after the second step of splicing by a dominant negative mutation in Prp22. This will permit investigation of the effects of Prp22 on mRNA dynamics after both steps of splicing and during mRNA release.

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Talk 8: Single-molecule Studies of FeS-containing DNA Helicases: Kinetics, Conformational Dynamics and Molecular Mechanisms

Maria Spies University of Iowa

DNA helicases are integral components of molecular machines that orchestrate and regulate a broad range of vital cellular processes including DNA replication, repair, and recombination. XPD is a DNA helicase with a critical role in nucleotide excision repair (NER). Two biochemical activities of XPD are critical for NER: it separates duplex DNA at the site of DNA damage and verifies that the damage is indeed NER-reparable. XPD-like helicases are composed of a Superfamily II motor core and two family-specific auxiliary domains: FeS cluster domain and ARCH domain. Practical utility of FeS clusters lays in their ability serve as endogenous quenchers of a wide spectral range of fluorophores. We exploited this phenomenon to characterize helicase-substrate and helicase-partner interactions as well as protein domain dynamics. I will discuss our findings regarding the mechanism by which XPD auxiliary domains define translocation polarity, ability to translocate on the protein- coated DNA and to signal the presence of DNA damage. To probe the motions of the XPD auxiliary domains, we developed a dual illumination single-molecule assay and a data analysis routine which enabled direct visualization of the binding and dissociation of single, fluorescently labeled DNA molecules while simultaneously observing changes in the distance between the ARCH and FeS domains. We show that ARCH domain undergoes thermally driven open-close transitions. The presence of CPD, a prototypical UV lesion, stabilizes a closed state of the ARCH. Direct access to the microscopic dynamics of XPD revealed how DNA binding and ARCH domain conformational transitions are connected to kinetically enhance the damage detection process and to recruit downstream factors in the NER pathway.

Talk 9: ATP- and NTP-dependent Promoter Opening by the Yeast RNAP II Pre-initation Complex

Eric J. Tomko, James Fishburn, Steven Hahn, and Eric A. Galburt Washington University in St. Louis

Transcription initiation in Eukaryotes depends on the formation of the multi-protein complex known as the Pre- Initiation Complex (PIC). In S. cerevisiae, transcription initiation on TATA-dependent promoters has at least four phases. First, PIC-formation is nucleated by the binding of TATA-box binding protein (TBP) to the TATA-box element on the promoter. Second, the Ssl2/XPB subunit of TFIIH catalyzes DNA unwinding and establishes a DNA bubble. Third, the PIC performs transcription-start-site scanning (TSS scanning) where DNA sequences downstream of the initial site of DNA opening are surveyed for potential initiation sites. Lastly, RNAP II escapes the PIC and enters into processive transcription elongation. The mechanism of DNA opening and subsequent transcription-start-site scanning is unknown. Here, using single-molecule magnetic tweezers approaches, we measure the distributions of DNA bubble size and lifetime formed in the presence of reconstituted PICs with varying ATP and NTP concentrations on both negatively and positively supercoiled DNA. Our data allow us to place constraints on the mechanism of transcription initiation in Eukaryotes and serve as a foundation for future single-molecule studies of this complex and critical step in eukaryotic transcription regulation active site to the 3’-5’ exonuclease site.

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Talk 10: Mechanism of Aromatic Carcinogen Bypass by the Y-family Polymerase Dpo4

Alfonso Brenlla, David Rueda and Louis J. Romano Wayne State University

Y-family polymerases such as Dpo4 perform the majority of trans-lesion DNA synthesis in vivo. In this work, we characterize Dpo4 binding dynamics, conformational rearrangements and catalytic activity in the presence and absence of carcinogenic DNA adducts. Our single-molecule fluorescence setup enables monitoring polymerase movement on a DNA substrate with single-nucleotide resolution. We observe that in the absence of nucleotides the binary complex shuttles between two different conformations within ~1 s. We term these two conformers as the preinsertion complex, in which the nucleotide-binding site is occupied by the terminal base pair and the insertion complex, where Dpo4 has translocated one base pair, thus making the dNTP binding site available. Interestingly, only the preinsertion complex was captured in a crystal structure. Binding of either correct or incorrect dNTPs result in the formation of an insertion ternary complex. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a dNTP-stabilized misalignment is observed, in which the template base at the n position is no longer base paired. In the second part of this work, we used a DNA template with a single adduct modification corresponding to either 2-aminofluorene (AF) or N-acetyl-2- aminofluorene (AAF), two well characterized carcinogenic arylamines. We find that in the absence of nucleotides both adducts distort polymerase binding, but addition of dNTPs induces the formation of a ternary complex with a conformation similar to the one observed with a natural DNA substrate. Finally, we observed that misincorporation pathways for both adducts present significant differences. While AF induces primer-template slippage, its acetylated counterpart AAF presents a dNTP-stabilized misalignment mechanism.

TUESDAY, AUGUST 5, 2014

SESSION III: SINGLE MOLECULES IN LIVE CELLS

Talk 11: Mobility of TonB and FepA in the Membranes of E. coli

Kenneth Ritchie Purdue University

Iron uptake is essential for most pro- and eukaryotic cells. Cells attempt to sequester environmental iron, both for their own use and as a defense against pathogens, by producing iron sequestering proteins such as transferrin, lactoferrin and ferritin. In response, Gram-negative bacteria chelate iron in the high-affinity siderophore ferric enterobactin (FeEnt). Transport of FeEnt across the Escherichia coli outer membrane occurs through a TonB- dependent process, where it is hypothesized that the inner membrane proteins TonB-ExeBD transfer energy across the periplasm to the outer membrane iron transporter FepA. Using a green fluorescent protein-TonB conjugate (TonB-GFP) and labeling FepA by Alexa-555 (A555-FepA), we have monitored the mobility of these molecules at the single molecule level. Both proteins display strongly confined motion in their respective membranes. Depolarization of the inner membrane, deletion of ExeBD or depolymerization of cytoskeletal MreB had minimal effect on the mobility of either protein. Addition of FeEnt modifies the confinement of a fraction of the TonB to mimic that of FepA, implying interaction in the presence of ligand.

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Talk 12: Single Molecule Study of RelA during the Stringent Response in Live E. coli Cells

Wentling Li, Heejun Choi, Yan Zhang, Emmanuelle Bouveret, James C. Weisshaar University of Wisconsin-Madison

The stringent response is a physiological response that occurs when bacterial cells encounter nutritional stresses such as amino acid starvation or fatty acid starvation. The most marked outcome of this response is an immediate accumulation of the effector nucleotides, guanosine tetra- and pentaphosphate (ppGpp and pppGpp).The RelA protein of Escherichia coli is a (p)ppGpp synthetase that is activated by amino acid starvation. Here, we use single molecule tracking method (sptPALM) to investigate the RelA protein association and dissociation behavior before and after the stringent response. In contrast to an earlier work in which RelA was found to diffuse like ribosomes in normal growth conditions and to diffuse freely following the stringent response, we find RelA diffusion under both conditions to be heterogeneous. And during the stringent response, RelA diffuses more slowly than in the normal growth condition. Analysis of all single molecule tracking trajectories of RelA by a hidden Markov model was consistent with two diffusion states where amino acid starvation increases the dwell time of RelA on the ribosome and promotes the accumulation of RelA in the state with slow mobility. These observations show that during the stringent response, RelA tends to bind to ribosomes more often compared to the normal growth condition, suggesting that RelA needs to be “on” ribosomes to synthesize (p)ppGpp.

Talk 13: Dynamics of Chromosomal DNA in Escherichia coli

Rudra P. Kafle, Molly Liebeskind, Thaige Gompa, and Jens-Christian Meiners University of Michigan

We investigate intracellular dynamics associated with the conformational fluctuations of the chromosomal DNA in live Escherichia coli cells by Fluorescence Correlation Spectroscopy (FCS). These fluctuations move the bound fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam in a confocal microscope. From the time correlation functions of the measured fluorescence intensity, we quantify the fluctuations of the DNA as measured by its time-dependent mean square displacement and the viscoelastic moduli of the nucleoid. These quantities in live cells significantly differ from the ATP-depleted dead cells on longer time scales, indicating that the fluctuations on longer time scale may be driven by active processes involving molecular motors that generate forces by ATP hydrolysis. On shorter time scales, we see little difference between live and dead cells, suggesting that the processes on corresponding short length scales rely primarily on thermally-driven diffusive mechanisms. We note that the rheological properties of E. coli nucleoid significantly change when the ATP hydrolysis in cells is inhibited. We compare our results to that of the in vitro experiments with the concentrated solution of lambda-DNA.

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Talk 14: Single Molecules and Cellular Mechanics

Taekjip Ha University of Illinois at Urbana-Champaign

The living cell is an ensemble of molecules that are not individually “alive”, but through their hierarchical association are capable of sustaining, repairing, and duplicating the cell, rendering it alive. As we aspire to build a quantitative narrative of the living cell, the next frontier in single-molecule techniques will lie at the interface between the molecular and cellular. I will describes an approach we are developing in order to bring the clarity and precision of modern single molecule biophysical approaches to cell biology. Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions. Applications of TGT to cell-cell adhesion and tumor repopulating cells will also be discussed.

Talk 15: Single-molecule Investigation of Intraflagellar Transport Mechanisms

Yan Mei Wang Wayne State University

In the past decade, flagella/cilia have come to be known as essential sensory organelles for cells. Flagella/cilia are hair-like surface projections of many eukaryotic cells. They provide motility and sensory functions for the cells, and defects in cilia cause a growing number of human disorders ranging from polycystic kidney disease to Bardet-Biedl Syndrome. The growth and signaling functions of flagella/cilia are maintained by intraflagellar transport (IFT), where kinesin motors move cargo from the cell body into the flagella and dynein motors remove material from the flagella to the cell body. The proper functioning of this process requires that the IFT machinery, which in Chlamydomonas is composed of the kinesin-2 and cytoplasmic dynein 1b motors, IFT trains, and BBSomes, to exchange motors and reorganize these proteins at the flagellar base and tip. How and where these reorganizations occur remains elusive. We use single-molecule fluorescence imaging methods to study three IFT reorganization mechanisms in Chlamydomonas: (i) Upon arrival at the flagellar tip, BBSomes, IFT trains, and dynein motors together dissociate from the microtubules and diffuse along the flagellar membrane for an average of 2.3 sec before initiating retrograde IFT. This result identifies the flagellar membrane as the site of IFT machinery reorganization at the flagellar tip, rather than the lumen as suggested previously. (ii) Kinesin motors, however, remain on the microtubule and dissociate into the flagellar lumen after an average of 1.6 sec. This result contradicts the long-held model that kinesin exits flagella by IFT, and proposes a new motor switching mechanism for eukaryotic cells. (iii) All IFT machinery components are organized in the cytoplasm before entering flagella, rather than recycling within flagella at the flagellar base.

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Talk 16: From Molecules to Development: Revealing Simple Rules of Biological Clocks

Qiong Yang University of Michigan

Organisms from cyanobacteria through vertebrates make use of biochemical and genetic oscillators to drive repetitive processes like cell cycle progression and vertebrate somitogenesis. Despite the complexity and diversity of these oscillators, their core design is thought to be shared. Notably, most of them contain a core positive-plus- negative feedback architecture. Here we use the early embryonic mitotic cycles in Xenopus as a motivating example and discuss how the positive feedback functions as a bistable switch and the negative feedback as a time- delayed, digital switch (Yang and Ferrell, Nat Cell Biol, 2013; Ferrell, Tsai, and Yang, Cell, 2011). I will next discuss our ongoing and future research projects on essential biological clocks in early embryos. We employ mathematical modeling, microfluidic techniques, and optical imaging for a quantitative understanding of self- organizing behaviors of single cells and single molecules during early embryo development. Interested students are encouraged to contact me ([email protected]) and to visit our webpage (www.umich.edu/~qiongy) for more details.

SESSION IV: NEW METHODS

Talk 17: Optical Trapping and Multiparameter Analysis of Single HIV-1 in Culture Media Reveal the Positive Cooperativity of Envelope Spikes in Mediating Viral Infection

Wei Cheng University of Michigan

Optical tweezers use the momentum of photons to trap and manipulate microscopic objects contact-free in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, direct optical trapping of viruses has been very limited largely due to the small size of these nanoparticles. Using optical tweezers that can simultaneously resolve two-photon fluorescence at single-molecule level, here we show that individual HIV-1 can be optically trapped and manipulated, which allows multi-parameter analysis of single virions in culture fluid under native conditions. We show that the number of envelope spikes that are essential for viral infection varies widely on the surface of individual virions. The efficiency of virus infection varied with the envelope content in a distinct sigmoidal dependence, which revealed a Hill coefficient of 2.9±0.8 (95% confidence). These results suggest that multiple envelope spikes cooperate on virion surface to mediate HIV-1 infection. Individual virions differ in their ability to infect host cells as a result of the molecular heterogeneity of spike content. We hypothesize that HIV-1 virions with high envelope spike content are preferentially transmitted in human populations, consistent with recent finding (Parrish et al., PNAS 2013) that transmitted founder viruses contained 1.9-fold more envelope per particle than chronic control viruses (Supported by NIH Director’s New Innovator Award 1DP2OD008693).

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Talk 18: Exploring Dynamics with Single Molecule Electron Paramagnetic Resonance

Richelle M Teeling-Smith, Ezekiel Johnston-Halperin, Michael G. Poirier, P. Chris Hammel The Ohio State University

Electron paramagnetic resonance (EPR) is an established and powerful tool for studying the dynamics of biomolecular systems. EPR measurements on bulk biomolecular samples using a commercial X-band spectrometer provide insight into atomic-scale structure and dynamics of ensembles of biomolecules. Separately, single molecule measurements of biomolecular systems allow researchers to capture heterogeneous behaviors that have revealed the molecular mechanisms behind many biological processes. We have merged these two powerful techniques to perform single molecule EPR. In this experiment, we selectively label single double-stranded DNA molecules with nitrogen-vacancy (NV) center nanodiamonds and experimentally demonstrate optical detection of the magnetic resonance of the single NV nanodiamond probe. Changes in the EPR spectrum reveal the dynamics and the orientation of the attached DNA molecule relative to the applied magnetic field. Using this new technique, we have successfully measured the first EPR spectrum of a single biomolecule. This research provides the foundation for an advanced single molecule magnetic resonance approach to studies of complex biomolecular systems.

Talk 19: What To Do When the (Fluorescent) Lights Go Out: Toward Single Molecule Spectroscopy with Optical Microresonators

Randall Goldsmith University of Wisconsin-Madison

Single-particle spectroscopy is a powerful tool for the mechanistic investigations of chemical and biological dynamics because unsynchronized processes can be directly observed. However, the traditional reliance upon fluorescence for single-particle measurements limits such investigations to systems where the target is fluorescent. Ultrahigh-Q optical microresonators offer a way of eliminating the need for fluorescence by enabling additional sensitive means of interaction with individual particles. Frequently, the interaction, either resonant or non-resonant, between an adsorbed analyte species and the propagating mode of the resonator can allow sensitive single-particle detection. We present a two-beam experimental geometry, where the goal is observation of spectral dynamics of a known target molecule. Our experiment relies on ultrahigh-Q toroidal optical microresonators as platforms for photothermal spectroscopy. Transitions are optically driven in the particle of interest, while the thermalization of the excitation energy is detected by the resonator. The relevant heat flows are explored with numerical simulations. We demonstrate implementation of the concept by making measurements on individual carbon nanotubes. Future augmentation to enable spectroscopy on individual non-fluorescent molecules will be discussed.

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Talk 20: A Dual-functioning Genetic Tag for Simultaneous Isolation and Observation of Single Fluorescent Complexes from Whole Cell Extract

Margaret L. Rodgers, Joshua Paulson and Aaron A. Hoskins University of Wisconsin-Madison

Single-molecule colocalization methods excel in detecting and analyzing biomolecular complex formation. Since large complexes often cannot be reconstituted from purified systems, these studies are often done in cell extracts. We have developed a new variation of the SiMPull approach1 to isolate and visualize native complexes using a single genetically incorporated tag. Our SNAP-SiMPull tag utilizes a SNAP tag for fluorophore conjugation fused to an E. coli biotin acceptor peptide, which can be biotinylated in vivo. Tagged proteins can be pulled down from cell extract directly onto a streptavidin-coated single-molecule slide. Using this method, we have isolated a number of complexes involved in yeast pre-mRNA splicing including both U1 and U6 snRNPs comprising 17 and 8 proteins respectively. By introducing multiple fluorescent labels, we have analyzed protein stoichiometry in these isolated complexes. Finally, we have performed functional assays on the immobilized particles to study their interactions with other components of the RNA splicing machinery. We predict that this approach will be broadly applicable for studies of other macromolecular machines. 1. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

Talk 21: Probing DNA-protein Association Through Atomistic and Coarse-grained Simulations Aleksei Aksimentiev University of Illinois at Urbana-Champaign

The fundamental role played by DNA in biology mandates that its sequence be preserved by an organism throughout its life cycle and be reproduced exactly in its progeny. Experimental studies have already identified all components of the DNA replication and repair machinery for several model organisms. Furthermore, the structure and function of the individual components have been characterized in extensive detail. However, just as knowing the specifications of all players in a team-sport is not sufficient to predict the outcome of the game, knowing the structure and function of all components of the DNA replication and repair machinery is not sufficient to describe how the actual process of DNA replication and repair proceeds. In collaboration with single molecule experimentalists at UIUC, we have been developing a physics-based model to quantitatively describe the behavior of protein-DNA systems as it emerges from the intricate interactions of the system’s components. We have designed our model to satisfy the conflicting requirements of being able to handle very large systems at physiological times scale while retaining the atomic level of details in description of interactions between DNA and proteins. In this talk, I will describe the applications of our model to studies of DNA systems under mechanical stress and interactions of single stranded DNA with single-stranded DNA binding proteins of the DNA replication machinery.

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Talk 22: Design of Force-Sensitive DNA Origami Components

Michael W. Hudoba, Michael Poirier and Carlos E. Castro The Ohio State University

Scaffolded DNA origami is powerful design and fabrication tool for the creation of nanoscale objects via bottom up self-assembly. These objects have ~nm level geometric complexity and spatial accuracy, which is comparable to biological machinery. DNA origami has been used to create different a wide range of objects such as drug delivery containers or platforms to guide molecular robots. Current applications of DNA origami exploit the large stiffness of bundles of dsDNA to create structures that maintain a well-defined and static geometry. However, DNA origami nanostructures with mechanically functional components, such as springs or actuators have remained largely unexplored. We aim to make DNA origami devices that are responsive to force magnitudes typically seen in biomolecular system (~picoNewtons). We have currently developed two approaches to make force-sensitive DNA origami components. The first (analog approach) uses curved bundles of DNA ‘beams’ that act as springs to continually measure forces. Conceptually, these springs function similar to a macroscopic leaf spring, where the equilibrium configuration is curved, and changing the curvature results in a restoring force. This approach can be used to design springs that deform continuously under an applied force. The second (binary approach) incorporates structures similar to DNA hairpins into DNA origami designs. The hairpin-like structures undergo a conformational change at a specific force threshold that can be tuned according to the design. These two approaches to create force sensitive components are demonstrated through the design of two sensors: an analog force sensor, and a binary force sensor. These devices are currently being calibrated with the use of magnetic tweezers, single molecule fluorescence, and flow-based techniques. Ultimately we aim to use these devices to measure forces of molecular interactions in cellular systems, for example cellular traction forces applied during cell migration.

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POSTER LIST

POSTER SESSION I

I-1 Polyamine Mediates Sequence and Methylation-dependent Chromatin Compaction Jejoong Yoo, Hajin Kim, Taekjip Ha and Aleksei Aksimentiev I-2 Constructing an Energy Landscape for the Hybridization of Short Oligonucleotides Kevin Whitley, Matthew J. Comstock and Yann R. Chemla I-3 Force-dependent Melting of Supercoiled DNA at Thermophilic Temperatures. Eric Tomko, James Fishburn, Steven Hahn, Eric Galburt I-4 A Single Molecule Perspective of Sequence Dependence Elasticity in DNA Julia T. Bourg, Krishnan Raghunathan, Alan Kandinov, Joshua Milstein and Jens-Christian Meiners I-5 G-quadruplex Conformation and Dynamics Are Determined by Loop Length and Sequence Ramreddy Tippana, Weikun Xiao, Sua Myong I-6 Probing the Effect of Different Ligands on the Conformational Dynamics of a Transcriptionally Acting preQ1 Riboswitch using Single Molecule FRET Microscopy Jiarui Wang, Krishna Chaitanya Suddala, and Nils Walter I-7 Single Molecule Study of Repair-specific Functions of Replication Protein A Ran Chen, David Beyer, Maria Spies, and Marc S. Wold I-8 Diffusion of Human Replication Protein A along Single Stranded DNA Binh Nguyen, Joshua Sokoloski, Roberto Galletto, Elliot L. Elson, Marc S. Wold, and Timothy M. Lohman I-9 Mechanical Activation of the Hha/H-NS Protein Complex to Condense DNA Haowei Wang, Samuel Yoshua, Sabrina S. Ali, William W. Navarre and Joshua N. Milstein I-10 Transcription Factor Binding inside Nucleosomes is Controlled by Core Histone PTMs Matthew Brehove, Yi Luo and Michael Poirier I-11 DNA Sequence and Modifications Control Nucleosome Mechanical Stability Thuy Ngo, Qiucen Zhang and Taekjip Ha I-12 The Influence of linker histones on transcription factor binding within nucleosomes. Morgan Bernier, Kingsley Nwokelo, Pei Zhang, Mark Parthun and Michael Poirier I-13 Quantifying the role of steric constraints in nucleosome positioning Tomas Rube and Jun Song I-14 The Influence of Histone H3 with Trimethylated Lysine 36 on the Stability of the Nucleosome Matthew D. Gibson, Jovylyn Gatchalian, Catherin A. Musselman, Justin A. North, Tatiana G. Kutateladze and Michael G. Poirier I-15 Gas-Liquid Phase Transitions in Sub Cellular Level Molecular Mechanisms by Single Molecule FRET Younghoon Kim, Christian Eckmann, Clifford P. Brangwynne and Sua Myong I-16 Bloom Helicase Unfolds G-quadruplex in the Absence of ATP Jagat B. Budhathoki, Sujay Ray, Vaclav Urban, Pavel Janscak, Jaya G. Yodh and Hamza Balci I-17 RTEL1 Binds and Remodels G-quadruplex DNA David Beyer and Maria Spies I-18 The Effect of Single-stranded DNA Binding Protein RPA2 on XPD Helicase Processivity Barbara Stekas, Zhi Qi, Masayoshi Honda, Maria Spies and Yann R. Chemla I-19 Single-Molecule Measurement of Mtr4 RNA Helicase Activity Using High-Resolution Optical Trapping Eric M. Patrick, Sukanya Srinivasan, Eckhard Jankowsky, and Matthew J. Comstock

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I-20 Single-molecule Imaging Reveals the Translocation Dynamics of Hepatitis C Virus NS3 Helicase Chang-Ting Lin, Felix Tritschler, Kyung Suk Lee, Meigang Gu, Charles M. Rice and Taekjip Ha I-21 Differential mechanism of unwinding trinucleotide repeat by yeast Srs2 and Sgs1 Yupeng Qiu, Hengyao Niu, Patrick Sung and Sua Myong I-22 Investigating the hyper-rotated state of the ribosome, and its possible involvement in frameshifting Bassem Shebl and Peter Cornish I-23 Correlation of the L1 Stalk Motion and Subunit Rotation Drew E. Menke and Peter V. Cornish I-24 Single molecule FRET study of the Dpo4 behavior at DNA containing Benzo[a]pyrene adduct Pramodha Liyanage, David Rueda and Louis Romano I-25 The spliceosome transiently undocks the splicing substrate to promote catalysis, proofreading, and alternative splicing Daniel Semlow, Mario Blanco, Nils Walter and Jonathan Staley I-26 Intramolecular head to tail distance in 2D reveals that kinesin walks with its cargo upright Kai Wen Teng, Marco Tjioe, Carol S. Bookwalter, Kathleen M. Trybus and Paul R. Selvin I-27 Characterization of Kinesin-1 Motor Domain Residues Important for Localization within Neurons Michael Kelliher, Aaron Hoskins and Jill Wildonger

POSTER SESSION II

II-1 Folding Dynamics of a WW Domain Protein Using Single Molecule Force Spectroscopy Miles L. Whitmore, Dena Izadi, Lisa J. Lapidus and Matthew J. Comstock II-2 Resolving Prion Protein Aggregation at the Single Molecule Level Chi-Fu Yen, Anumantha Kanthasamy and Sanjeevi Sivasankar II-3 Resolving the Molecular Mechanism of Cadherin Catch Bond Formation Kristine Manibog, Hui Li, Sabyasachi Rakshith and Sanjeevi Sivasankar II-4 Resolving the Molecular Mechanism of Cadherin Ideal Bond Formation Sunae Kim, Kristine Manibog, Sabyasachi Rakshith and Sanjeevi Sivasankar II-5 Characterizing the Interaction of Desmosomal Cadherins at Single Molecule Level Omer M. Shafraz, Sabyasachi Rakshith, Molly Lowndes, W. James Nelson and Sanjeevi Sivasankar II-6 Multiple Conformations of a Single SNAREpin between Two Nanodisc Membranes Reveal Diverse Pre-fusion States Jaeil Shin, Xiaochu Lou, Dae-Hyuk Kweon and Yeon-Kyun Shin II-7 Dynamics of the General Secretory System Viewed in Near-native Conditions Via Atomic Force Microscopy Raghavendar Reddy Sanaganna Gari, N.C. Frey, L.L. Randall and Gavin M. King II-8 The Synaptotagmin 1 Linker May Function as an Electrostatic Zipper That Opens for Docking but Closes for Fusion Pore Opening Xiaochu Lou, Ying Lai, Yongseok Jho, Tae-Young Yoon and Yeon-Kyun Shin II-9 Real Time Observation of Lipid-Protein Interactions in Crude Cell Lysates and with Single-Molecule Resolution Edwin Arauz, Vasudha Aggarwal, Ankur Jain, Taekjip Ha, and Jie Chen

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II-10 Single-molecule Studies of Membrane Proteins on Glass Substrates Using Atomic Force Microscopy Nagaraju Chada, Krishna P. Sigdel, Tina R. Matin, Raghavendar Reddy Sanganna Gari, Chunfeng Mao, Linda L. Randall, and Gavin M. King II-11 Cellular Pathways for Productive HIV-1 Entry and Molecular Mechanisms of its Inhibition Hanna Song and Wei Cheng II-12 Cell Geometry Affects the Distribution of Measured Diffusion Coefficients in Bacteria David J. Rowland and Julie S. Biteen II-13 Spatiotemporal Dissection of Cytoplasmic and Nuclear miRNA Function Laurie Heinicke, Sethuramasundaram Pichiaya, Elizabeth Cameron and Nils Walter II-14 Single-molecule Fluorescence Imaging of RecO Localization and Dynamics in Bacillus subtilis Hannah H. Tuson, Yi Liao, Lyle A. Simmons and Julie S. Biteen II-15 Single-molecule Mechano-memory Isaac T.S. Li, Taekjip Ha, and Yann R. Chemla II-16 Single-molecule Tracking of Elongation Factor P in Live E. coli Cells Heejun Choi, Sonisilpa Mahapatra, Suparna Sanyal and James C. Weisshaar II-17 The Princess and the Pea: A Story of Cell Mechanics Mehdi Roeimpeikar, Qian Xu and Taekjip Ha II-18 Somitogenesis in Zebrafish Zhengda Li, Ye Guan and Qiong Yang II-19 Virion Immobilization Post Diffusion Limits HIV-1 Infectivity Revealed by Real-time Single Particle Tracking Michael C. DeSantis, Jin H. Kim, Jamie L. Austin, and Wei Cheng II-20 Electrical Current Measurement and Manipulation of Single Geobacter Cells Via Optical Trapping Jess L. West, Adam J Gros, Rebecca J Steidl, Gemma Reguera and Matthew J Comstock II-21 Optical Trapping and Characterization of Single HIV-1 in Culture Media Yuanjie Pang and Wei Cheng II-22 Combined Multi-Color Fluorescence and Ultra-High Resolution Optical Tweezers Cho-Ying Chuang, Miles L Whitmore, Jess L West, and Matthew J Comstock II-23 Three Dimensional Localization of Single Biomolecules with Nanometer Resolution Patrick D. Schmidt, Chi Fu Yen, John Lajoie and Sanjeevi Sivasankar II-24 Site-specific Labeled HIV-1 Neutralization Antibodies for Single-molecule Fluorescence Measurement Jin H. Kim and Wei Cheng II-25 Mechanical Modulation of Enzyme Activity by Dynamic DNA Tweezers Soma Dhakal, Minghui Liu, Matthew R. Adendorff, Mark Bathe, Hao Yan and Nils G. Walter

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POSTER PRESENTATION ABSTRACTS

POSTER SESSION I

Poster I-1: Polyamine Mediates Sequence and Methylation-dependent Chromatin Compaction

Jejoong Yoo, Hajin Kim, Taekjip Ha and Aleksei Aksimentiev University of Illinois at Urbana-Champaign

All cellular activities, including cell development and differentiation, are consequences of gene regulations. In the conventional view, transcription factors directly regulate gene expressions by activating or repressing the binding of RNA polymerase to specific target genes. However, a more general and global mechanism of gene regulations based on compaction level of chromatin is becoming accepted thanks to high-throughput and high-resolution experimental techniques revealing the chromatin conformations in molecular details. For example, AT-rich heterochromatin segments of human genome are known to cluster one another forming compact spatial topologically associated domains (TADs) whereas GC-rich euchromatin segments form separate relatively less compact TADs. More surprisingly, high level of DNA methylation results in compaction of a specific locus or an entire chromatin, enabling reversible regulation of gene expressions. Although the correlation between the chromatin compaction and genomic sequence (GC and methylation contents) is well established, the underlying principle is poorly understood. Here, we demonstrate that the differential compaction of DNA by sequence can occur only in the presence of sub-mM polyamine using highly optimized computer simulations and novel single- molecule techniques, suggesting that a rather simple physical principle of polyamine-mediated DNA-DNA attractions might govern the global chromatin compaction in the cells. Consistent to the chromatin compaction in vivo, we find that polyamine-mediated DNA-DNA attraction is significantly stronger for AT-rich and methylation-rich DNA segments than GC-rich segments. Further, we show that this sequence-dependent DNA attraction originates from the DNA structure encoded by sequence in atomistic details.

Poster I-2: Constructing an Energy Landscape for the Hybridization of Short Oligonucleotides

Kevin Whitley, Matthew J. Comstock and Yann R. Chemla University of Illinois at Urbana-Champaign

The hybridization of short oligonucleotides plays a critical role in many biological systems, from DNA replication to gene silencing. Despite extensive studies, details on the mechanism of this process on the shortest length scale (~10 bp) remain poorly understood. We use high-resolution optical tweezers with simultaneous fluorescence microscopy to investigate the hybridization of single oligonucleotides under tension. We measure the change in end-to-end extension upon annealing and melting as well as the unbinding kinetics of short (7-12 bp), fluorescently labeled oligonucleotides of DNA and RNA hybridizing to a complementary DNA sequence tethered between trapped beads. Our results allow us to construct an energy landscape of oligonucleotide hybridization along a well-defined reaction coordinate. Interestingly, our measurements of DNA duplex and RNA-DNA melting as a function of oligonucleotide length indicate that the transition state for both has the same end-to-end extension as a ~6 bp duplex. Based on these results and prior studies, we propose that melting occurs through a common transition state in which the two complementary strands are partially aligned to each other with a minimal base-paired “core.” Additionally, we find that the change in extension upon hybridization deviates from extensible wormlike-chain model at forces >10 pN. We propose a model combining shear deformation and fraying of the terminal base pairs of the oligonucleotides to explain these results.

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Poster I-3: Force-dependent Melting of Supercoiled DNA at Thermophilic Temperatures

Eric Tomko, James Fishburn, Steven Hahn and Eric Galburt Washington University in St. Louis

Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a balance between stability and accessibility. For example, while mesophilic life forms maintain negatively superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the force- dependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles. We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to different general mechanisms of DNA opening in different environments.

Poster I-4: A Single Molecule Perspective of Sequence Dependence Elasticity in DNA

Julia T. Bourg, Krishnan Raghunathan, Alan Kandinov, Joshua N. Milstein and Jens-Christian Meiners University of Michigan

DNA looping is a ubiquitous and vital regulatory process found in all organisms to maintain proper function and viability. In protein-mediated looping, the DNA’s sequence dictates both protein binding sites and the local biomechanical properties. These mechanical properties, notably its elasticity and intrinsic curvature, govern the ease at which loops can form or breakdown. To probe the effects of sequence dependence on elasticity and the loop formation process, single molecule experiments were performed on short segments (~150bp) of DNA with varying amounts of AT and GC content between the protein-binding operators. Tethered particle motion (TPM) microscopy was employed to observe protein-mediated DNA loop formation in this system, and an analytical model of the loop formation process was used to calculate the elasticity of the DNA from the observed loop formation rates. Axial constant-force optical tweezers were used to directly stretch the same DNA molecules mechanically to determine their persistence length as a measure for their elasticity. Our results indicate that the intraoperator sequence has a larger effect on elasticity in the loop formation experiments than in the stretching experiments, which we attribute to different elasticity regimes when the DNA is strongly bent as in a DNA loop, compared to the thermally induced small curvature fluctuations in stretched DNA.

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Poster I-5: G-quadruplex Conformation and Dynamics Are Determined by Loop length and Sequence

Ramreddy Tippana, Weikun Xiao and Sua Myong University of Illinois at Urbana-Champaign

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N- methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.

Poster I-6: Probing the Effect of Different Ligands on the Conformational Dynamics of a Transcriptionally Acting preQ1 Riboswitch using Single-Molecule FRET Microscopy

Jiarui Wang, Krishna Chaitanya Suddala and Nils Walter University of Michigan

Non-coding RNAs play crucial roles in a multitude of biological processes such as translation, messenger RNA (mRNA) splicing and regulation of gene expression. Riboswitches are regulatory elements found mainly in the 5’ untranslated regions of numerous bacterial mRNAs capable of modulating gene expression in response to the binding of cellular metabolites. Riboswitches comprise two functional components: a ligand binding aptamer domain and an expression platform whose conformation decides the fate of gene expression. Despite its smallest size among all reported aptamers, the Bacillus subtilis (Bsu) preQ1 (pre-queuosine) riboswitch boasts precise control of the expression of genes involved in the biosynthesis of queuosine. The co-crystal structure of the Bsu aptamer domain bound to its ligand preQ1 showed a pseudoknot structure. However, the nature of folding pathway remained elusive, until recently. We have recently used single molecule fluorescence resonance energy transfer (smFRET) and computational simulations to show that the ligand-free Bsu aptamer adopts a pre-folded conformation in which the single stranded A-rich tail interacts with stem-loop P1-L1. Previous studies indicated that the Bsu riboswitch binds closely related ligands preQ0 and guanine with similarly favorable affinities. The effects of these ligands on the conformational dynamics, however, were not previously probed and can reveal to what extent near-cognate ligands stabilize the folded state to affect gene regulation. Here, we use smFRET to study the effects of preQ0 and guanine in comparison to preQ1 on the kinetics of Bsu aptamer folding. Our preliminary data show that at saturating concentration and in the presence of Mg2+, guanine stabilizes the folded state as potently as preQ0. However, surprisingly, in the absence of Mg2+, guanine tends to stabilize the folded state more efficiently than preQ0. We anticipate this comparative study to elucidate the ligand-mediated folding pathway of the Bsu aptamer.

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Poster I-7: Single Molecule Study of Repair-specific Functions of Replication Protein A

Ran Chen, David Beyer, Maria Spies and Marc S. Wold University of Iowa

Replication Protein A (RPA), the major eukaryotic single-strand DNA (ssDNA) binding protein, is essential for replication, repair, recombination, and cell cycle progression. Defects in RPA activities lead to genomic instability, a major contributor to the development of cancer and other diseases. The large subunit of RPA (RPA1) mediates binding to ssDNA. The RPA-DNA interface contains a series of polar residues and four conserved aromatic residues. We have used a combination of biochemical analysis in vitro and knockdown- replacement studies in vivo to characterize the contribution of these aromatic residues to RPA function. Mutation of these aromatic residues results in a separation-of-function phenotype. Cells expressing the aromatic mutants supported DNA replication, had normal checkpoint activation after DNA damage but were defective in DNA repair and accumulated double-strand breaks. Also, the aromatic residue mutants were unable to support nucleotide excision or double strand DNA break repair. We are using single molecule total internal fluorescence microscopy (smTIRF) and ensemble assays to determine the affinity and kinetics of binding to different DNA structures including single strand intermediates found at sites of damage and replication. Mutation of the aromatic residues altered the stability of the RPA-DNA complex and decreased the affinity for short ssDNA regions. Our results show that DNA replication and DNA repair require different RPA-DNA interactions and that functions in repair depend on the high affinity DNA-binding domains of RPA1. These studies are contributing to understanding how human cells maintain genome integrity.

Poster I-8: Diffusion of Human Replication Protein A along Single Stranded DNA

Binh Nguyen, Joshua Sokoloski, Roberto Galletto, Elliot L. Elson, Marc S. Wold and Timothy Lohman Washington University in St. Louis

Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single molecule fluorescence approaches to examine human RPA diffusion along ssDNA and find that an hRPA hetero-trimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one- dimensional diffusion coefficient of hRPA on ssDNA of D1 ~5000 nucleotide2s-1 at 37˚C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA. This work was supported in part by NIH grants GM030498 (T.M.L.), GM044721 (M.S.W.), GM098509 (R.G.) and HL109505 (E.L.L.).

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Poster I-9: Mechanical Activation of the Hha/H-NS Protein Complex to Condense DNA

Haowei Wang, Samuel Yoshua, Sabrina S. Ali, William W. Navarre and Joshua N. Milstein University of Toronto

The bacterial chromosome must be under varying levels of mechanical stress due to a high degree of crowding and repeated protein-DNA interactions experienced within the nucleoid. DNA tension is difficult to measure in cells and it is not known if its effects have any functional significance. However, in vitro experiments have implicated a range of biomechanical phenomena for DNA. The histone-like nucleoid structuring protein, H-NS, is a key regulator of DNA condensation and gene expression in enterobacteria and is affected by a variety of cofactors with which it may form a complex, such as the protein Hha. By combining tethered particle motion (TPM) and optical tweezers experiments we probed the effects of tension on DNA in the presence of the Hha/H- NS complex. We find that a brief fluctuation in DNA tension, induced by optical tweezers, causes the rapid and irreversible compaction of DNA when in the presence of H-NS and Hha. Our results imply that the Hha/H-NS complex may selectively condense bacterial DNA based upon the level of mechanical tension that is experienced along different regions of the chromosome.

Poster I-10: Transcription Factor Binding inside Nucleosomes Is Controlled by Histone PTMs

Matthew S. Brehove, Yi Luo and Michael Poirier The Ohio State University

Genomic DNA in a cell is stored wrapped around a histone octamer core 147 base pairs at a time into repeating units called nucleosomes. DNA wrapped in nucleosomes is generally inaccessible to DNA binding proteins and thus nucleosomes control access to the genome. Post translational modifications (PTM’s) to these histones serve as epigenetic marks that can signal for transcription, repression, and repair. Some of these PTM’s are on the histone-DNA interface and can alter the accessibility of transcription factors to the nucleosomal DNA. Here our smFRET data shows the effect of these PTMs on transcription factor binding inside of a nucleosome using mutants that mimic common modifications.

Poster I-11: DNA Sequence and Modifications Control Nucleosome Mechanical Stability

Thuy T.M. Ngo, Qiucen Zhang and Taekjip Ha University of Illinois at Urbana-Champaign

Understanding the physical basis of how DNA sequence and modifications affect nucleosome dynamics and nucleosomal DNA exposure will help elucidate how genomic and epigenetic modifications regulate cellular functions, cell differentiation and cancer development. Here, we used single-molecule force fluorescence spectroscopy and single-molecule DNA cyclization measurement to investigate local conformational dynamics of the nucleosome under tension and its modulation by DNA sequence and modifications. First, we observed that the nucleosome can unwrap asymmetrically and directionally under force. Second, we showed that nucleosome mechanical stability is controlled by local DNA flexibility. We demonstrated the correlation between DNA flexibility and unwrapping force by varying DNA sequence, DNA methylation and DNA mismatches. DNA methylation decreases DNA flexibility and reduces nucleosome mechanical stability while DNA mismatches have opposite effects. Our work elucidates the fundamental physical principles for regulation role of DNA sequence and modifications on nucleosome mechanical and directional stability which controls nucleosome accessibility for replication, transcription, repair and remodeling.

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Poster I-12: The Influence of Linker Histones on Transcription Factor Binding Within Nucleosomes Morgan W. Bernier, Kingsley Nwokelo, Pei Zhang, Mark Parthun and Michael Poirier The Ohio State University

The Linker Histone, H1, is involved in the compaction of chromatin into a higher order structure. This compacted structure inhibits DNA accessibility and thus regulates gene expression. While core histones have been shown to reduce transcription factor binding, certain post translational modifications of core histones in the entry-exit region of nucleosomes tend to increase nucleosome unwrapping thus allowing transcription factors to bind more easily. The effect of H1 on this binding is not well known particularly in the presence of core histone modifications. Additionally, H1 has several isoforms whose various functions are also not understood. Using a combination of ensemble and smFRET assays, we explore the binding of H1 and its isoforms and how they inhibit the binding of the transcription factor, Gal4, within nucleosomes. We also present data on how post translational modifications contribute to H1’s effect on Gal4 binding. Because of the extremely low Kd of Gal4, ensemble assays are not enough to observe how H1 affects binding rates. The smFRET studies allow us to observe individual binding events of both H1 and Gal4 and provide us with rate constants of the interaction between the proteins and nucleosomes.

Poster I-13: Quantifying the Role of Steric Constraints in Nucleosome Positioning

Tomas Rube and Jun Song University of Illinois at Urbana-Champaign

Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5' end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in 50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.

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Poster I-14: The Influence of Histone H3 with Trimethylated Lysine 36 on the Stability of the Nucleosome Matthew D. Gibson, Jovylyn Gatchalian, Catherin A. Musselman, Justin A. North, Tatiana G. Kutateladze and Michael Poirier The Ohio State University

The fundamental unit of chromatin, the nucleosome, consists of DNA wrapped around a histone protein octamer core. Histones contain a large number of post translational modifications (PTMs), which often function as protein binding sites. The idea of a histone code has emerged which attempts to link modifications to biological functions. These PTMs are typically thought to work by directly or indirectly recruiting transcription factors (TFs). However, proteins which bind these PTMs can also mechanically alter nucleosome dynamics to increase DNA accessibility. Using Fluorescence Resonance Energy Transfer we investigated the influence of PHF1, which specifically binds trimethylated Lysine 36 on histone H3 (H3K36me3), on TF affinity to its target sequence buried within nucleosomes. We find that PHF1 serves to increase TF access to nucleosomal DNA. We measure the affinity increase due to PHF1s binding domain as about a factor of 3, and find that the domains of PHF1 contribute multiplicatively. These results suggest that PTM H3K36me3 may serve to facilitate transcription by increasing DNA accessibility. We plan to expand these studies to single molecule measurements which will elucidate the effect of binding on TF rates and compaction of nucleosome arrays.

Poster I-15: Gas-Liquid Phase Transitions in Sub Cellular Level Molecular Mechanisms by Single Molecule FRET

Younghoon Kim, Christian Eckman, Clifford P. Brangwynne and Sua Myong University of Illinois at Urbana-Champaign

In germ cell development, ribonucleoprotein (RNP) complex termed p-body plays a critical role in mRNA storage, splicing, degradation and translation repression. Many proteins within p-body contain RNA binding domains and low complexity (LC) sequences of unknown function. We employed single molecule fluorescence to characterize LAF1 helicase of C. elegans as a model system to investigate p-body assembly process. LAF-1 is a DEAD box helicase which possess N-terminal RGG box and C-terminal poly-glutamine tract and a helicase core. Our results reveal that LAF-1 specifically binds single strand (ss) RNA and induces unexpected compaction and dynamics of the RNA strand. LAF-1 displays no unwinding of double stranded RNA, yet it promotes annealing of complementary ssRNA. Series of truncation mutants reveal that the N-terminal RGG box is responsible for providing the dynamic interaction and RNA annealing whereas other domains contribute to the compaction of RNA and oligmerization. Native gel analysis indicates that LAF-1 and the mutants form higher order structure, reflecting their inherent propensity to oligomerize. Together, we unravel the molecular mechanism how LAF-1 may contribute to gas-liquid transition-like behavior of p-granule.

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Poster I-16: Bloom Helicase Unfolds G-quadruplex in the Absence of ATP

Jagat B. Budhathoki , Sujay Ray, Vaclav Urban, Pavel janscak, Jaya G. Yodh and Hamza Balci Kent State University

RecQ family helicases, such as BLM and WRN, have been shown to unfold G-quadruplex (GQ)structures in the presence of ATP. Eliminating BLM and WRN have been shown to cause genomic instability, retardation of replication machinery, and DNA breaks in potent1ially GQ forming sites of the genome. In addition to helicases, single strand DNA (ssDNA) binding proteins have also been shown to possess GQ unfolding activity, which indicates the significance of unfolding these structures for cellular proper functioning of metabolic activities such as replication, transcription and repair. Motivated by efficient unfolding of GQ by ssDNA binding proteins, we studied GQ-helicase interactions in the absence of ATP in order to probe possible GQ destabilization due to binding of a protein in the vicinity of GQ. Using single molecule Förster Resonance Energy Transfer (smFRET) and bulk biochemical assays, we found that binding of BLM, WRN, and RECQ5 to an ssDNA overhang in the vicinity of GQ leads to varying degrees of GQ destabilization, including unfolding, in the absence of ATP. We also observed that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to the ssDNA overhang, as modulated by the nucleotide state, ionic conditions and overhang length. Furthermore, increasing GQ stability, via shorter loops or higher ionic strength reduces BLM-mediated GQ unfolding.

Poster I-17: RTEL1 Binds and Remodels G-Quadruplex DNA

David Beyer and Maria Spies University of Iowa

The inactivation of tumor suppressor genes commonly occurs at chromosomal fragile sites, often-repetitive regions of the genome characterized primarily by a tendency to form secondary structures that impede essential interactions between the replisome and template DNA. Similarly, the repeated TTAGGG sequence arrays that compose the human telomere can readily form G-quadruplex (G4) secondary structures in the lagging strand that can stall replication and lead to drastic changes in telomere stability and length. Many fragile sites across the human genome have the capacity to form G4-like structures. Essential human DNA helicase RTEL1 co-localizes with the replisome during telomeric replication and prevents the global generation of fragile sites. As is the case with most human DNA helicases, RTEL1 likely has several cofactors that target and tune its general biochemical activities to confer a specific adaptive advantage. It is currently unclear whether RTEL1 prevents the accumulation of both genomic and telomeric fragile sites by assisting the replisome through difficult structures or via some other mechanism. We have developed a set of total internal reflection fluorescence (TIRF) assays to explore interactions between RTEL1 and G4 DNA. Our system employs a biotinylated full-length helicase construct purified directly from human cell culture. Fluorescence trajectories of RTEL1 incubated with G4 DNA constructs in the presence of ATP show binding and remodeling behavior. Studies performed in concert with likely cofactors may clarify the role these behaviors play in a cellular context. Mapping the interaction network between RTEL1 and both replisomal and telomeric cofactors will elucidate the mechanism of RTEL1’s demonstrated role in promoting gainful, robust cell cycle progression.

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Poster I-18: The Effect of Single-stranded DNA Binding Protein RPA2 on XPD Helicase Processivity

Barbara Stekas, Zhi Qi, Masayoshi Honda, Maria Spies and Yann R. Chemla University of Illinois at Urbana-Champaign

FacXPD helicase is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human superfamily 2B helicase XPD involved in transcription initiation and nucleotide excision repair. Previous work has shown that the unwinding of double-stranded DNA by FacXPD is regulated by the single-stranded DNA binding protein FacRPA2. The mechanism by which this occurs is unknown. Here, we present a single molecule study of this regulation using combined optical traps and fluorescence. We show that XPD is a weak helicase as a monomer, only able to unwind short distances (~12 bp) under tension applied by the optical traps, with a strong dependence on DNA sequence. In the presence of RPA2, however, XPD monomers are able to unwind more processively (>90 bp). We show that RPA2 on its own can melt ~6 bp of DNA under tension, suggesting that RPA2 may assist XPD unwinding by binding ahead of the helicase and disrupting the duplex ahead. Alternately, RPA2 may form a complex with XPD, activating it for processive unwinding. To distinguish these possible mechanisms, we use a combination optical trap and FRET assay to correlate the binding of RPA2 with processive unwinding events by XPD.

Poster I-19: Single-Molecule Measurement of Mtr4 RNA Helicase Activity Using High- Resolution Optical Trapping

Eric M. Patrick, Sukanya Srinivasan, Eckhard Jankowsky and Matthew J. Comstock Michigan State University

We present single-molecule high resolution optical trapping measurements of the Mtr4 helicase unwinding an RNA duplex. RNA helicases act as RNA structure remodelers and play key roles during RNA metabolism, from transcription, native structure adoption to degradation. During aberrant RNA degradation, the exosome works in conjunction with other protein factors to get rid of misfolded RNA molecules. These protein factors include helicases such as Mtr4 that ensure that the RNA to be degraded has the correct structure. To elucidate how Mtr4 unwinds such RNAs, we have performed high resolution optical trapping experiments utilizing a 16-base pair duplex RNA with an adenosine-rich 3´ end (which has sequence homology to its natural substrate) between two 1.5 kb dsDNA handles tethering a pair of trapped beads. We find that Mtr4 unwinds these RNA duplexes in one or two step bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4 remains stably bound after unwinding the duplex, preventing re-annealing. Closely linked to Mtr4 are two additional proteins Trf4, which introduces adenosine tags to the target RNA, and Air 1/2, which together make the TRAMP complex. Future experiments utilizing combined fluorescence and optical trapping measurements may reveal how Mtr4 activity is coordinated within the TRAMP complex.

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Poster I-20: Single-molecule Imaging Reveals the Translocation Dynamics of Hepatitis C Virus NS3 Helicase

Chang-Ting Lin, Felix Tritschler, Kyung Suk Lee, Meigang Gu, Charles M. Rice and Taekjip Ha University of Illinois at Urbana-Champaign

Worldwide, over 185 million people are chronically infected with hepatitis C virus (HCV), facing risks of developing liver diseases, such as hepatocarcinoma. There is no vaccine available. New therapies without side effects are highly needed. HCV encodes a superfamily 2 helicase (NS3h) in the C-terminal portion of nonstructural protein 3. This enzyme is essential for virus replication. Previous studies have well characterized the unwinding properties of the helicase. The ensemble approaches, however, have largely limited the understanding of the translocation dynamics. Here, we used optical traps to stretch kilobase-size single-stranded DNA (ssDNA), the single-molecule tracking of fluorescence-labeled NS3 enables us to directly determine the translocation speed, processivity, binding duration and the stoichiometry of translocating complex. Interestingly, we observed NS3h-mediated repetitive looping of ssDNA in the range of hundreds nucleotides. We further applied single-molecule fluorescence resonance energy transfer (smFRET) to the analysis of repetitive looping behavior. By tuning the fluorophore pair position between protein and nucleic acids, more structural information has been revealed. The dual-ways of movements observed by single molecule analysis may play roles in HCV life cycle.

Poster I-21: Differential Mechanism of Unwinding Trinucleotide Repeat by Yeast Srs2 and Sgs1 Yupeng Qiu, Hengyao Niu, Patrick Sung and Sua Myong University of Illinois at Urbana-Champaign

Trinucleotide repeat (TNR) expansion causes many known inherited neurological and muscular disorders in human including Huntington’s disease and Friedreich’s ataxia. One source of expansion is the replication defect at TNR sequence that leads to the hairpin formation and subsequent lengthening of the TNR segment. The Srs2 and Sgs1 are two helicases in yeast that display varying degree of resolving TNR hairpin during replication to prevent expansion. Using single molecule fluorescence, we investigated the unwinding mechanism by which Srs2 and Sgs1 resolve TNR hairpin and compared it to unwinding of duplex DNA. While Sgs1 unwinds both structures indiscriminately, Srs2 displays a repetitive unfolding of TNR hairpin without fully unwinding it. Further, the repetitive unfolding of Srs2 shows dependence on the hairpin folding strength and the total length of TNR. Our results reveal an exquisite mechanism of Srs2 that may contribute to efficient resolving of the TNR hairpin.

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Poster I-22: Investigating the Hyper-rotated State of the Ribosome, and Its Possible Involvement in Frameshifting Bassem Shebl and Peter Cornish University of Missouri

Ribosomes play a pivotal role in the central dogma, translating encoded mRNA to functional proteins. Yet, the underlying dynamics of translation is not fully understood. We observed a novel translocation intermediate; the hyper-rotated state (HRS), of the ribosome.1 Along with other local and global conformational changes of the ribosome, HRS could provide further insight into the intricacies of regular translation and recoding deviations such as Programmed Ribosomal Frame shifting (PRF). Further investigations would aid in the conquest to find new drug targets, and thus more effective antibiotics and antivirals. Emerging resistant bacteria as Methicillin- resistant Staphylococcus aureus (MRSA) has proven to be a serious hazard with a wide pathological and economical impact on the health-sector and general population.2, 3. We aim to investigate this novel state and its possible involvement in the intrinsic helicase activity of the ribosome, and frameshifting using single molecule Forster Resonance Energy Transfer (smFRET). In addition, we are developing a bicistronic fluorescence construct to quantify PRF with future applications in drug screening and evolutionary studies.

Poster I-23: Correlation of the L1 Stalk Motion and Subunit Rotation

Drew E. Menke and Peter Cornish University of Missouri

Examination of the dynamic motions of the ribosome during translation elongation has provided critical insight into the mechanism of protein synthesis. Previously, a number of studies have examined these key dynamic motions and have provided a detailed analysis of both the motions of the L1 stalk and inter-subunit rotation. The L1 stalk an rRNA/protein protrusion positioned by the Exit site on the large subunit populates three states: open, half close and closed. Subunit rotation the counter clockwise rotation of the large and small subunits opposite one another populated the classical (nonrotated) and hybrid (rotated) states. Current results cannot directly correlate the contribution of these two dynamic motions together. Using single molecule Fluorescence Resonance Energy Transfer (smFRET) and a hybrid constructs labeled with three Fluorescent dyes we will examine both subunit rotation and the L1 stalk simultaneously.

Poster I-24: Single-molecule FRET Study of the Dpo4 Behavior at DNA Containing Benzo[a]pyrene Adduct Pramodha S. Liyanage, David Rueda and Louis Romano Wayne State University

Benzo[a]pyrene (BP) is a polyaromatic hydrocarbon (PAH) which can be metabolically activated to highly reactive benzo[a]pyrene diol epoxides (BPDE) in mammals. BPDE readily reacts with DNA generating N2-dG covalent adducts. These BP adducts are known to be extremely carcinogenic and tumorigenic in animal models. Normally, high fidelity polymerases are unable to synthesize DNA across the BP lesions, and get stalled at the adduct position. Translesion synthesis (TLS) is the process by which bulky adducts and other DNA damage are bypassed, rescuing the stalled replisome. Y-family DNA polymerases play an important role in TLS. In this study we use the Y-family DNA polymerase Dpo4 as a model system to investigate the bypass mechanism of a BP adduct. We characterized Dpo4 binding to damaged DNA and subsequent conformational rearrangements by single molecule fluorescence resonance energy transfer (smFRET).

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Poster I-25: The Spliceosome Transiently Undocks the Splicing Substrate to Promote Catalysis, Proofreading, and Alternative Splicing

Mario Blanco, Daniel Semlow, Jonathan Staley, Nils Walter and Yi Zeng University of Chicago

Gene expression requires high fidelity at all stages. In contrast to fidelity mechanisms in transcription and translation, fidelity mechanisms in splicing remain poorly understood. To ensure fidelity, the spliceosome employs DExD/H-box ATPases to discriminate against suboptimal splice sites, but it has remained unclear how these factors promote intron excision with high specificity. By assaying substrate conformation by single molecule fluorescence energy resonance transfer, we revealed that the DExD/H-box ATPases Prp16 and Prp22 proofread and promote splice site selection at the catalytic stage through a common mechanism of substrate undocking. Although Prp16 is canonically required only for exon ligation, we unexpectedly discovered that in the Prp16-dependent rejection of suboptimal branch sites, undocking allowed re-docking and selection of alternative branch sites, establishing that spliceosomal DEAH-box ATPases can function as RNA chaperones to facilitate alternative splicing. Our observation that Prp16 promotes alternative branch site selection also permitted us to investigate the mechanism of Prp16 function in a manner that is uncoupled from 3’ splice site recognition and exon ligation and readily dissect the substrate requirements for Prp16-dependent branch site undocking. Our preliminary results indicate that Prp16, like the spliceosomal DExD/H-box ATPases Prp2 and Prp22, acts on single stranded substrate RNA downstream of the catalytic core. These data strongly suggest a common mechanism for DExD/H-box ATPases at the catalytic stage involving 3’ to 5’ translocation along the splicing substrate to transiently disrupt the catalytic core and facilitate splice site selection. The function of spliceosomal DEAH-box ATPases at the catalytic stage can therefore be likened to the resolution of non-native folding intermediates that impede activation of a catalytic ribozyme.

Poster I-26: Intramolecular Head-to-tail Distance in 2D Reveals That Kinesin Walks With Its Cargo Upright

Kai Wen Teng, Marco Tjioe, Carol S. Bookwalter, Kathleen M. Trybus and Paul R. Selvin University of Illinois at Urbana-Champaign

In the cell, kinesin works with different motors in order to carry various cargoes ranging from proteins to cellular organelles towards the plus end of the microtubule. Cargo shared by kinesin, other motors, and accessory proteins has to be carefully positioned due to steric factor. The question we wish to answer is: On a single molecule level, how does kinesin carry its cargo across the microtubule? A glance at the structure of kinesin shows that immediately upsteam from the motor domain is the neck coiled-coil region, which was found to orient tangential to the microtubule in previous studies. Do the stalk and the cargo binding region of kinesin continuously extend from the neck coiled-coil region? Or do flexible elements (hinges) play a role in positioning the cargo? Through the use of single molecule high resolution colocalization (SHREC) we track the position of the cargo and motor domain as kinesin is actively walking under low ATP concentration. What we found was that the cargo is not oriented tangential to the microtubule, but it sits mostly above the motor domain. Distance between the cargo and motor domain inferred that majority of the stalk sits upright in the z-axis. We also found that kinesin has the freedom to position its cargo either in front or in the back of its motor domain, resulting in a wider distribution of cargo position in the front and back, versus sideways.

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Poster I-27: Characterization of Kinesin-1 Motor Domain Residues Important for Localization within Neurons

Michael Kelliher, Aaron Hoskins and Jill Wildonger University of Wisconsin-Madison

Work with kinesin in cultured neurons showed that a constitutively active kinesin-1 construct (K560) is capable of localizing to the distal tips of developing neurites that ultimately become axons. Other work has implicated residues within the β5-L8 and α4-L12-α5 regions of the kinesin-1 motor as being important for proper localization. In this work we introduce the mutations made to the K560 constructs into the Drosophila melanogaster kinesin-1 and characterize the effects on neuronal morphology and polarized transport within neurons in live fruit flies. We are also carrying out companion single-molecule motility assays on the K560 mutants to gain insight into how these mutations affect the behavior of individual motors.

POSTER SESSION II

Poster II-1: Folding Dynamics of a WW Domain Protein Using Single Molecule Force Spectroscopy

Miles L. Whitmore, Dena Izadi, Lisa J. Lapidus and Matthew J. Comstock Michigan State University

We present high-resolution force spectroscopy measurements of the protein folding dynamics of a single WW domain. Protein folding, the process by which the polypeptide chains acquire the correct three-dimensional structure is still a poorly understood process. Force is a natural technique for denaturing proteins in order to observe the process of refolding in detail, but the time resolution of standard instruments typically preclude investigation of fast-folding model proteins. In this study we added cysteine residues to both N and C termini of the human Yes-associated protein (hYAP), which typically folds in less than 1 ms. The peptide is then connected to two 1.5 kb thiol-modified double stranded DNA handles. The protein-DNA chimeras were suspended between a pair of polystyrene beads held in high-resolution dual optical traps. Reversible protein folding and unfolding was observed both during force-extension pulling/relaxation experiments as well as under constant force feedback conditions. We also labeled the N and C termini of the WW domain protein with donor and acceptor fluorophores and performed simultaneous and independent FRET measurements of folding and unfolding using combined high-resolution trapping and single molecule fluorescence spectroscopy.

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Poster II-2: Resolving Prion Protein Aggregation at the Single Molecule Level

Chi-Fu Yen, Anumantha Kanthasamy and Sanjeevi Sivasankar Iowa State University

Transmissible Spongiform Encephalopathies (TSEs) are a class of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (PrP) aggregates in the brain. A key step in this aggregation process is the conversion of proteinase-K (PK) sensitive PrP (PrPsen) into a pathological isoform that resists PK digestion (PrPres). Metal ions are known to play an important role in promoting PrP misfolding and oligomerization; however, the underlying mechanisms are poorly understood at molecular level. To address these questions, we developed single molecule assays to monitor the conversion of PrPsen into PrPres and to compare their interaction kinetics. Using single molecule fluorescence based PK-resistance assay, we demonstrate that PrPsen monomers convert to a PrPres conformation before oligomer assembly. The unstructured N-terminal region of PrPsen and Cu2+ ions are essential cofactors for structural transition. Using single molecule force measurements with an Atomic Force Microscope (AFM), we show that the association rate between monomeric PrP is reduced upon removing the N-terminal region. Compared to the PrPsen isoform, PrPres monomers show a 900 fold higher affinity (KA), indicating their potential as seeds for subsequent formation of prion protein aggregates.

Poster II-3: Resolving the Molecular Mechanism of Cadherin Catch Bond Formation

Kristine Manibog, Hui Li, Sabyasachi Rakshith and Sanjeevi Sivasankar Iowa State University

Classical cadherin Ca2+-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here, we use single molecule force clamp spectroscopy with an Atomic Force Microscope along with Molecular Dynamics and Steered Molecular Dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca2+ ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that they form long-lived, force induced hydrogen bonds that lock X-dimers into tighter contact. When Ca2+ concentration is decreased, fewer of these hydrogen bonds are formed and catch bond formation is eliminated.

Poster II-4: Resolving the Molecular Mechanism of Cadherin Ideal Bond Formation

Sunae Kim, Kristine Manibog, Sabyasachi Rakshith and Sanjeevi Sivasankar Iowa State University

Classical cadherin cell-cell adhesion proteins play an essential role in maintaining tissue integrity in the presence of mechanical stress. Cadherins bind in multiple adhesive conformations; by switching between these conformations, cadherins control the mechanical properties of their interactions. We had previously shown that while one cadherin conformation forms ‘catch’ bonds which strengthen with force, the second conformation forms ‘slip’ bonds which weaken when pulled. We had also determined that cadherins form a third, previously unknown, type of interaction called an ‘ideal’ bond that is insensitive to force. While we have recently resolved the molecular mechanisms of cadherin catch and slip bond formation, the molecular determinants by which ideal bonds form are unknown. To resolve this, we use single molecule force clamp measurements with an Atomic Force Microscope to characterize the molecular basis of cadherin ideal bond formation.

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Poster II-5: Characterizing the Interaction of Desmosomal Cadherins at Single Molecule Level

Omer M. Shafraz, Sabyasachi Rakshith, Molly Lowndes, W. James Nelson and Sanjeevi Sivasankar University of Illinois at Urbana-Champaign

Desmosomes are cell-cell adhesion complexes that are present in tissues that resist mechanical stress. They are mainly composed of two adhesive proteins which are members of the cadherin superfamily of cell adhesion proteins, desmocollin (Dsc) and desmoglein (Dsg). However, the role of these proteins in desmosomal adhesion is unclear. Here, we use the single molecule force spectroscopy with an Atomic Force Microscope (AFM-FS) to characterize the interactions of type-2 isoforms of desmocollin (Dsc2) and desmoglein (Dsg2). We show that Dsc2 forms Ca2+ dependent homophilic bonds by swapping a conserved Tryptophan residue between opposing binding partners; mutating this Trp inhibits Ca2+ dependent homophilic binding. In contrast, Dsg2 forms Ca2+ independent heterophilic bonds with Dsc2 via a mechanism that does not involve Trp strand-swapping. Previous studies suggest that desmosome formation requires the presence of classical cadherins at the site of desmosome assembly. This suggests a cross-talk between desmosomal and classical cadherins at cell-adhesion contacts. We therefore used AFM-FS to test if Dsc2 and Dsg2 interact with E-cadherin, a classical cadherin present in the epithelium. Our data shows that while Dsc2 does not bind to E-cadherin in the presence of Ca2+, Dsg2 forms Ca2+ independent complexes with E-cadherin. Using cadherin mutants we show that the interactions between Dsg2 and E-cadherin occur via a previously uncharacterized binding interface that does not involve either Trp strand-swapping or X-dimer formation (two well established classical cadherin binding mechanisms).

Poster II-6: Multiple Conformations of a Single SNAREpin between Two Nanodisc Membranes Reveal Diverse Pre-fusion States

Jaeil Shin, Xiaochu Lou, Dae-Hyuk Kweon and Yeon-Kyun Shin Iowa State University

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized pre-fusion conformations have been elusive. We used single molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. While the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure the membrane-proximal C-terminal half shows either high FRET representing a helical bundle (45%), low FRET reflecting a frayed conformation (39%), or mid FRET revealing an yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1.

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Poster II-7: Dynamics of the General Secretory System Viewed in Near-native Conditions via Atomic Force Microscopy

Raghavendar Reddy Sanaganna Gari, N.C. Frey, L.L. Randall and Gavin M. King University of Missouri

In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for protein transfer across and into membranes. Further, it is known that SecA is the ATPase of the general secretory system and it binds SecYEG to perform translocation. In so doing, SecA makes large surface area contact with the unstructured cytoplasmic loops spanning transmembrane helices 6-7 and 8-9 of SecY. Despite their functional significance, measurements of flexible and disordered protein domains remain a significant experimental challenge. Recently, atomic force microscopy (AFM) has emerged as an important complementary tool in biophysics and is well suited for studying membrane protein dynamics in near-native conditions (i.e., in a native lipid environment, at physiologically relevant temperature and ionic strength). We have studied purified SecYEG that was reconstituted into liposomes via AFM. After confirming activity, changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude and were attributed to the aforementioned loops of SecY. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. Finally, we have imaged SecA engaged on SecYEG and related the structural states observed to the activity of the translocase. This work provides a novel and near-native vista of central components of the general secretory system.

Poster II-8: The Synaptotagmin 1 Linker May Function as an Electrostatic Zipper That Opens for Docking but Closes for Fusion Pore Opening

Xiaochu Lou, Ying Lai, Yongseok Jho, Tae-Young Yoon and Yeon-Kyun Shin Iowa State University

Synaptotagmin 1 (Syt1), a major Ca2+ sensor for fast neurotransmitter release, contains tandem Ca2+-binding C2 domains (C2AB), a single transmembrane a-helix, and a highly charged 60-residue-long linker in between. Using the single vesicle docking and content mixing assay we found that the linker region of Syt1 is essential for its two signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. The linker contains the basic amino acid-rich N-terminal region and the acidic amino acid-rich C-terminal region. When the charge segregation was disrupted, fusion pore opening was slowed while docking was unchanged. Intramolecular disulfide cross-linking between N- and C-terminal regions of the linker or deletion of 40 residues from the linker reduced docking while enhancing pore opening. The EPR analysis showed Ca2+-induced line broadening reflecting a conformational change in the linker region. Thus, the results suggest that the electrostatically bipolar linker region might have the capacity to extend for docking and fold to facilitate pore opening.

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Poster II-9: Real Time Observation of Lipid-Protein Interactions in Crude Cell Lysates and with Single-Molecule Resolution

Edwin Arauz, Vasudha Aggarwal, Ankur Jain, Taekjip Ha, and Jie Chen University of Illinois at Urbana-Champaign

Lipid-protein interactions play key roles in signal transduction. Obtaining new mechanistic insights of these interactions is obligatory for a better understanding of biological processes. Here we use a single-molecule pull- down assay (SiMPull) to probe lipid-protein interactions in crude cell lysates. We demonstrate the applicability of this assay by showing specific interaction between several signaling lipids and their lipid-binding partners. We perform intensive single-molecule data analysis to quantitatively describe the assembly lipid-binding proteins on their target lipids. Importantly, this assay is applicable to full-length proteins expressed in crude cell lysates, as show for the protein kinase AKT which binds to PI(3,4,5)3 lipid specifically. This new assay lays the foundation to study the interaction of large macromolecular complexes with lipids second messengers in cell lysates, avoiding the need of harsh and lengthy procedures used during protein purification.

Poster II-10: Single-molecule Studies of Membrane Proteins on Glass Substrates Using Atomic Force Microscopy Nagaraju Chada, Krishna P. Sigdel, Tina R. Matin, Raghavendar Reddy Sanganna Gari, Chunfeng Mao, Linda L. Randall, and Gavin M. King University of Missouri

Since its invention in the mid-1980s, the atomic force microscope (AFM) has become a valuable complementary tool for studying membrane proteins in near-native environments. Historically, mica is the most common substrate utilized for biological AFM. Glass being amorphous, transparent, and optically homogeneous has its own set of advantages over mica and has the potential to broaden the overlap of AFM with techniques that require high quality non-birefringent optical access. The use of silanized glass as an AFM substrates has been reported as a means to fine tune surface chemistry. However, such coatings usually require hours of additional preparation time and can lead to increased surface roughness. In this work, we present a simple technique for preparing borosilicate glass as a substrate for two membrane protein systems: non-crystalline translocons (SecYEG) of the general secretary system from E. coli, and bacteriorhodopsin (BR) from H. salinarum. For both these membrane proteins, quantitative comparisons of the measured protein structures on glass versus mica substrates show agreement. An additional advantage of glass is that lipid coverage is rapid (< 1 minute) and complete (occupying the entire surface). A goal is to study the bacterial export system using recently developed precision measurement techniques such as ultra-stable AFM.

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Poster II-11: Cellular Pathways for Productive HIV-1 Entry and Molecular Mechanisms of its Inhibition

Hanna Song and Wei Cheng University of Michigan

Productive entry of the human immunodeficiency virus type I (HIV-1) into CD4+ T cells is initiated by binding of the viral envelope gp120 to CD4 receptor. This binding causes a cascade of conformational changes in both the gp120 and gp41 that eventually lead to viral-cell membrane fusion and HIV-1 entry. Early studies have suggested that HIV-1 can enter target cells via direct fusion at the plasma membrane. In contrast, recent studies have suggested that the direct fusion at the plasma membrane is not productive. Instead, HIV-1 may enter cells via dynamin-dependent endocytosis. To examine the extent to which endocytosis leads to productive infection of HIV-1, we have used several inhibitors of dynamin to investigate whether there is a correlation between inhibition of HIV-1 infection and the inhibition of cell endocytosis. Transfection of TZM-bl indicator cells by dyn1(K44A), the dominant-negative mutant of dynamin I, decreased HIV-1 infection by ~30%, which correlated with the decrease of endocytosis as monitored via transferrin uptake, suggesting that dynamin-dependent endocytosis contributes to the productive infection of HIV-1. Dynasore, a noncompetitive inhibitor of dynamin, inhibited HIV-1 infection in various cell lines, but this inhibition is not correlated with the reduction in transferrin uptake, suggesting that dynasore inhibits HIV-1 infection through an off-target effect. T20, the membrane- impermeable inhibitor of HIV-1 entry, potently inhibited HIV infection in all cell lines investigated. Interestingly, endocytosed HIV-1 can be clearly observed in host cells even in the presence of saturating T20, suggesting that T20 may be endocytosed together with HIV-1 and exert its effect inside an endosome. This suggestion is further supported by the fact that inhibition of viral endocytosis doesn’t change the efficacy of T20. Ongoing colocalization studies of HIV-1, endosomal marker, and fluorescent-labeled T20 at single-molecule resolution will definitely test this hypothesis (Supported by March of Dimes Foundation, 5-FY10-490; WC).

Poster II-12: Cell Geometry Affects the Distribution of Measured Diffusion Coefficients in Bacteria David J. Rowland and Julie S. Biteen University of Michigan

Fast diffusion in small volumes is a phenomena important to many biophysical studies dealing with single molecules confined to micron-sized volumes such as membrane domains or bacteria. Presented here is the simulated transition from free to confined diffusion in a half-micron diameter bacterial cell as measured by mean squared displacement (MSD) analysis. Partially confined motion in an asymmetric volume can separate a single diffusing population into two, prompting the use of a two-population diffusion model. The use of double- exponential fits to cumulative step size probability curves underestimates the diffusion coefficient, however, and we show that geometrically separating the diffusion into axial and transverse directions more precisely predicts the real diffusion coefficient. Two orthogonal methods of calculating the MSD curve are used. We compare the traditional single-molecule super-resolution technique of fitting particle point spread functions with spatiotemporal image cross correlation spectroscopy (STICS) which does not suffer from particle blurring as a result of long integration times or fast diffusion. Both show that geometric separation of step sizes more precisely predicts the true diffusion coefficient than does a two-term fit to the whole data set and STICS more accurately measures fast diffusion.

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Poster II-13: Spatiotemporal Dissection of Cytoplasmic and Nuclear miRNA Function

Laurie Heinicke, Sethuramasundaram Pichiaya, Elizabeth Cameron and Nils Walter University of Michigan

Endogenous microRNA (miRNA) genes are transcribed in the nucleus as primary miRNA transcripts (pri- miRNA) and processed via multiple steps to generate mature miRNAs in the cytoplasm. These small non-coding RNA (ncRNAs) associate with components of the RNA-induced silencing complex (RISC) and engage mRNA targets and regulate gene repression via translational inhibition and/or mRNA degradation. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, the intracellular dynamics and assembly of miRNA-associated complexes and the spatiotemporal modulation of miRNA-regulated gene expression are still unclear. To uniquely probe intracellular RNA silencing pathways, our lab has developed a method termed intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) to determine the localization, diffusion constant and assembly state of single miRNA complexes inside living human cells at 30 nm spatial and 100 ms temporal resolution. We have used iSHiRLoC to probe nuclear functions of miRNAs. We found that a significant fraction of microinjected mature let-7-a1 (~20-30%) localizes to the nucleus, in contrast to a control cxcr4 miRNA whose whereas nuclear localization was minimal (~5-10%). Inhibition of transcription reduced nuclear localization of let-7-a1 to ~5-10%, the level of cxcr4 in the absence or presence of transcription inhibitor, strongly suggesting that only let-7-a1 binds RNA targets in the nucleus. These observations are consistent with a recent report that describes autoregulation of let-7-a1 biogenesis in the nucleus by mature let-7- a1. We are currently pursuing time course experiments, subcellular fractionation followed by Northern blotting and knock down of miRNA-associated proteins to better understand the import mechanism and target engagement of nuclear miRNAs. Together, these studies highlight the versatility of iSHiRLoC by providing biological insight regarding assembly and localization of intracellular miRNAs.

Poster II-14: Single-molecule Fluorescence Imaging of RecO Localization and Dynamics in Bacillus subtilis

Hannah H. Tuson, Yi Liao, Lyle A. Simmons and Julie S. Biteen University of Michigan

In all organisms, the high fidelity of DNA replication is essential for maintenance of chromosome integrity. DNA damage can be caused by polymerase errors or by external factors (e.g., X-rays or mutagenic chemicals). Thus, the cell has evolved a number of repair mechanisms to respond to different types of damage. In B. subtilis, repair of double-strand breaks (DSBs) in the DNA occurs through RecA-mediated homologous recombination. This role for RecA in DSB repair in B. subtilis is analogous to that of Rad51 in eukaryotes, making B. subtilis an excellent model system for studying cellular response to DNA damage. The mechanism by which RecA finds DSBs in vivo is not well described, but is believed to involve the proteins RecF, RecO, and RecR. Previously, bulk fluorescence studies have shown that RecO forms foci after the induction of double-strand breaks. However, RecO in undamaged cells can only be visualized when over-expressed, leaving questions about its true localization at wild type expression levels. Here, we have created cells in which PAmCherry-RecO is natively expressed from the RecO promoter as the only RecO source. We use single-molecule fluorescence microscopy in live B. subtilis to show that RecO is generally diffuse throughout the cell. This result suggests that, unlike several other proteins involved in DNA repair, RecO is not associated with the replisome prior to DSB recognition. Furthermore, RecO dynamics change when cells are treated with the DNA-damaging agent phleomycin. Additionally, we are examining the co-localization of RecO with SSB (which recruits RecO) and RecA (which is recruited by RecO). Understanding the real-time dynamics, positioning, and interactions of RecO will yield a model for the early stages of DSB recognition and repair and further inform our general understanding of DNA damage repair.

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Poster II-15: Single-molecule Mechano-memory

Isaac T.S. Li, Taekjip Ha, and Yann R. Chemla University of Illinois at Urbana-Champaign

Understanding the spatial distribution of individual adhesion bonds and the tension exerted on them is crucial for understanding whole cell adhesion behavior. Here, we introduce a new class of molecular force sensors to record cellular adhesion events at the single-molecule level. A DNA structure was designed that responds to mechanical perturbation above certain threshold tension and maintains a memory of that perturbation. We name this feature “single-molecule mechano-memory” (smMM). The smMM sensor undergoes conformational changes under tension and is kinetically trapped under a new conformation. Single-molecule force spectroscopy and fluorescence spectroscopy were performed to characterize the activation force as well as memory life time. We show that in the absence of mechanical perturbation the smMM sensor is well folded and stable. In the presence of tension above ~35 pN, the sensor is converted to the unfolded “memory” state in which it remains kinetically trapped for an average of 25 seconds. Both activation force and life time of the sensor can be tuned by its DNA sequence. As a proof of concept for this class of sensors, smMM sensors were coated on a surface where cell adhesion takes place. Individual adhesion events are detected using fluorescently-labeled oligonucleotide probes to mark unfolded sensors.

Poster II-16: Single-molecule Tracking of Elongation Factor P in Live E. coli Cells

Heejun Choi, Sonisilpa Mahapatra, Suparna Sanyal and James C. Weisshaar University of Wisconsin-Madison

Elongation Factor P (EF-P) is essential for efficient peptidyl transfer for cellular growth by enhancing the rate of polyproline addition in peptide elongation. In vitro studies showed that EF-P is associated with ribosome at its exit site. We are interersted in understanding the ribosome-EF-P interaction in live cells. Here, we constructed a translational fusion of mEos2 appended to C-terminus of efp in chromosome. Using single molecule tracking PALM and superresolution imaging, we are able to study the subcellular diffusion and distribution of EF-P in live cells. Tracking of single molecules of EF-P-mEos2 showed that EF-P diffuses at 1.4 µm2/s and showed signs of ribosome-association. Implication of translation and transcription halting drugs on association of EF-P to ribosome will be discussed.

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Poster II-17: The Princess and the Pea: A Story of Cell Mechanics

Mehdi Roeimpeikar, Qian Xu and Taekjip Ha University of Illinois at Urbana-Champaign

Single molecules of integrins, a class of trans-membrane proteins involved in cell adhesion, pull on their ligands with a force of approximately 40 pN. Measurements of these forces were previously performed using a series of double stranded (ds) DNA, each with a different rupture force, conjugated to the ligand. DNA types with rupture forces below 40pN are easily pulled apart by cells (through engagement with integrins). Above 40 pN, cells remain attached. In this research, using this series of different types of DNA tethers, two-dimensional force spectroscopy can be performed through multiplexing two different types of DNA tethers.

Poster II-18: Somitogenesis in Zebrafish

Zhengda Li, Ye Guan and Qiong Yang University of Michigan

Biological clock is a vital component of organisms, which determines the specific timing of biological event sequences. During embryogenesis, robust time tuning not only helps coordinated differentiation, but also leads to accurate morphogenesis. Understanding the interplay of clocks in developments will not only help investigation of fundamental questions like organism size control, pattern formation or cell fate determination, but also cast light on smarter medicine usage schemes or better drugs dealing with abnormally differentiated cells. However, at present, even though under intense investigation, the specific role of biological clocks in development is still far from clear. Our research is to investigate the segmentation process of zebrafish. Somitogenesis is a common phenomenon for nearly all vertebrates. Segments, also called somites, form early and develop to skeleton muscle, epithelial cells and many other structures. The study of somite formation may trace back to 1970s when many possible mechanisms are proposed. Further research unveiled a conserved molecular mechanism among vertebrates, and presented a model: clock and wavefront model, which prevailed for decades. However, with the development of microscopy and molecular biology, more evidence are emerging which proposed new functional components or even challenged the classical model. We build preliminary model based on current information, and find that to explore the interplay of cell cycles and segmentation clocks would be of great interests. Using in toto imaging, we can track the phase of different oscillators as well as the motion and proliferation of each cell, and our final goal is to understand the how global regulation and local interaction works together to produce stable patterns in organisms, which would rely on the further development of the single molecular imaging. Our research will help to understand the mechanism of somitogenesis and to expand our knowledge on coupled oscillators in biological systems.

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Poster II-19: Virion Immobilization Post Diffusion Limits HIV-1 Infectivity Revealed by Real-time Single Particle Tracking

Michael C. DeSantis, Jin H. Kim, Jamie L. Austin, and Wei Cheng University of Michigan

HIV-1 is known to have low infectivity. Although virion-cell interactions that take place prior to viral entry have been suggested to limit the infectivity of the virus, the underlying mechanisms are not completely understood. Furthermore, receptor binding, in addition to nonspecific interactions at the cell surface, might influence the subsequent steps, i.e., entry pathways, of infection. Using single-particle tracking methods with spatial and temporal resolutions of ~20 nm and 40 ms, respectively, we have quantified the dynamics of mCherry labeled HIV-1 virions with varied envelope (Env) glycoprotein incorporation interacting with TZM-bl cells, a HeLa- derived cell line that is commonly used for quantitation of HIV-1 infection. Our analysis revealed that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, most virions only have transient interactions with the cell surface before permanent dissociation. As a result, the probability for a single virion to become immobilized upon encounter with a cell is at least an order of magnitude lower than the frequency of initial collision. The majority of these immobilized virions enter cells via endocytosis with efficiencies as high as 75%. However, highly efficient endocytic uptake is independent of either viral Env or coreceptors on target cells such that it can effectively reduce the pool of infectious virions. DEAE-dextran, a reagent known to enhance viral titers, dramatically increased the rate of immobilization in a manner independent of viral Env content. Moreover, the presence of DEAE-dextran decreased endocytic uptake by twofold which may afford virions greater opportunity to bind specifically with receptors thereby increasing HIV-1 infectivity. In summary, these studies suggest that the low probability for immobilization upon target cell encounter as one potential bottleneck to HIV-1 infectivity. Reagents that can increase immobilization or decrease coreceptor- independent endocytosis can dramatically enhance HIV-1 infectivity. (Supported by NIH DP2-OD008693 and F32-GM109771)

Poster II-20: Electrical Current Measurement and Manipulation of Single Geobacter Cells Via Optical Trapping Jess L. West, Adam J Gros, Rebecca J Steidl, Gemma Reguera and Matthew J Comstock Michigan State University

The ability of Geobacter bacteria to respire to extracellular electron acceptors such as uranium and electrodes shows promise for applications in bioremediation and bioenergy. Key to these technologies is a better understanding of how individual cells transfer electrons outside the cell to extracellular electron acceptors. Bulk measurements of cells attached to microfabricated electrodes indicate that a single cell may be able to generate current ranging from 100 to 1,000 fA. We have constructed an experimental setup integrating optical tweezers and sample chambers equipped with a micron-scale gold electrode that allow in situ current measurements down to 50 fA. We have trapped individual Geobacter sulfurreducens cells and we can precisely manipulate them onto a gold electrode while simultaneously measuring current. With this technique, we are interrogating the cellular components responsible for current production in Geobacter.

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Poster II-21: Optical Trapping and Characterization of Single HIV-1 in Culture Media

Yuanjie Pang and Wei Cheng University of Michigan

Optical trapping uses the momentum change of photons to impart forces on microscopic objects, thereby immobilizing and manipulating these objects in three dimensions. Optical trapping may have great potential in microbiology applications because of its ability to trap tiny biological particles in solution. Here we demonstrate trapping of single HIV-1 virions in culture media using the single-beam gradient optical tweezers, and simultaneous multi-parameter characterizations of the trapped virion based on its thermal motion recorded by an ultra-sensitive position sensing diode (PSD) at the back-focal-plane (BFP) of the optical tweezers. By fitting the virion’s thermal motion power spectrum to a Lorentzian function; we obtained two important parameters, Dvolt, a proportionality factor, and fc, the corner frequency, of the Lorentzian. Both parameters conform to different statistical distributions for single virions as opposed to apparent aggregates. We then calibrated the electronic signal from the PSD using spatial distance values by sinusoidally oscillating the sample chamber at a known amplitude and frequency. This calibration allowed us to convert Dvolt into the hydrodynamic diffusion coefficient, and to subsequently calculate the size and the trap stiffness of the virion. The size information further revealed the aggregation status of the virions in the culture media that was hidden behind Dvolt and fc distributions, and virion aggregation was found to be concentration-dependent. Furthermore, by fitting the size- stiffness relation to a theoretical model, we were able to deduce the refractive index of the virion, which is an important parameter for modelling optical biosensors. We believe the single virion manipulation technique will open a new path in virology studies. For example, a single virion to a single cell infection assay can be set up, which can potentially reveal the heterogeneity of infection among individual virions from a viral population (Supported by NIH Director’s New Innovator Award 1DP2OD008693).

Poster II-22: Combined Multi-color Fluorescence and Ultra-High Resolution Optical Tweezers

Cho-Ying Chuang, Miles L Whitmore, Jess L West, and Matthew J Comstock Michigan State University

We present a single-molecule instrument that combines ultra-high resolution optical tweezers with multicolor confocal fluorescence microscopy. Timeshared dual optical traps were interlaced and synchronized with three fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting detectors (one for each excitation laser). Our new instrument enables the simultaneous measurement of DNA tether extension changes (e.g., from helicase or polymerase motion) and multiple fluorescently labeled observables (e.g., internal protein conformation dynamics via FRET or precise stoichiometry of complexes via multi-colored fluorophore counting). We demonstrated our instrument by measuring the binding and unbinding of fluorophore-labeled single stranded DNA oligonucleotides to a complementary tethered strand of DNA. Further, we combined multi- channel sample chambers with precise computer control of fluorescence measurement and triggered chamber motion to implement an automated ‘molecular assembly line.’ This allowed us to precisely add individual molecules of different types to a single DNA tether while conserving fluorescence photons and reducing photobleaching. In the future, these instrumentation advancements should enable the precise single-molecule assembly and measurement of complex, multi-component molecular machine systems.

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Poster II-23: Three Dimensional Localization of Single Biomolecules with Nanometer Resolution Patrick D. Schmidt, Chi Fu Yen, John Lajoie and Sanjeevi Sivasankar Iowa State University

Super-resolution fluorescence methods are widely used to image single biomolecules with sub-diffraction resolution. While these techniques can localize fluorophores with nm resolution in the x- and y- directions, their resolution along the optical (z) axis is poor. To overcome this limitation, we recently developed a fluorescence technique called Standing Wave Axial Nanometry (SWAN) that can localize single molecules along the z-axis of a fluorescence microscope with nm accuracy and precision. Here, we present our progress in integrating SWAN with 2D fluorescence localization methods to achieve previously unprecedented, nm localization accuracy in all three spatial dimensions. We will focus on our recent progress in designing fast feedback loops that stabilize the microscope over long periods of time and eliminate nm scale instrumental drift that occurs on the relevant timescale for data acquisition. Compensating for thermal and mechanical drifts should enable us to image single molecules in three dimensions with nm localization accuracy in each dimension.

Poster II-24: Site-specific Labeled HIV-1 Neutralization Antibodies for Single-molecule Fluorescence Measurement Jin H. Kim and Wei Cheng University of Michigan

To quantify the copy number of proteins using single-molecule fluorescence, specific labeling of the protein with a fluorophore in high efficiency is required. An antibody containing only a single antigen binding site conjugated with a single fluorophore provides the ideal condition for correlating the copy numbers between antigens of interest and the fluorophores on the antibody. In general schemes to achieve such a labeled antibody, sulfhydryl groups in mildly reduced half antibody molecules were used for labeling with fluorophores via maleimide chemistry. However, the uncertainty in preparation of such a half antibody and the nonspecific reduction of multiple sulfhydryl groups in an antibody molecule render this scheme highly unreliable. Here, we have developed a method to conjugate a single Alexa fluor 594 dye to an antibody containing only a single antigen binding site. To prepare an antibody with only a single antigen binding site and a single free sulfhydryl group available for maleimide conjugation, the heavy chain genes of two HIV-1 neutralizing antibodies, VRC01 and PGT145, were modified by a truncation of the Fc portions and an insertion of a hexahistidine tag after the first cysteine in the hinge region of the IgG. The modified antibody, named VRC01 Fab” and PGT145 Fab”, were successfully expressed in the suspension culture of 293-F cells, and purified using Ni-NTA column, followed by conjugation with Alexa fluor 594 and further purified using gel filtration column. The final labeled VRC01 Fab” and PGT145 Fab” exhibited higher than 95% of purity and 98% of labeling efficiency. In addition, these antibodies neutralized HIV-1 infection in cell culture assays at levels comparable to wild type IgG antibodies. Therefore, these site-specific labeled antibodies, VRC 01 Fab” and PGT145 Fab” will provide efficient assay tools for investigating the copy number of envelope glycoproteins on the surface of HIV-1 virions.

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Poster II-25: Mechanical Modulation of Enzyme Activity by Dynamic DNA Tweezers

Soma Dhakal, Minghui Liu, Matthew R. Adendorff, Mark Bathe, Hao Yan and Nils G. Walter University of Michigan

Switchable nanomachines provide a universal platform to control dynamic systems by altering distances at the nanoscale on-demand. Recently, a tweezers-like DNA device has been used to control the activity of an enzyme/cofactor pair juxtaposed on the two arms of the tweezers. Initial studies focused on bulk properties of the tweezers-mediated reactions and hence lacked insight into the mechanism of enzymatic activation. Here, we used site-specifically fluorophore-labeled DNA tweezers and monitored the arm-to-arm distance through single- molecule fluorescence resonance energy transfer (smFRET). The tweezers are composed of DNA double-helix arms joined by a Holliday junction ‘hinge’. A DNA strand that can cycle between hairpin and double helical structures through a strand-displacement reaction is incorporated between the arms to control the arm-to-arm distance. Both smFRET and AFM measurements consistently showed that these previously designed tweezers only partial close in the “closed” state (to an arm-to-arm distance of ~6.5 nm). We improved the design by varying the hairpin stem-length from 3 to 5 base pairs (bp). Consistent with our smFRET results, molecular dynamics simulations showed bending and twisting motion of the arms. Further, smFRET experiments on the isolated Holliday junction hinge suggested that the “correct” isomer II needed to close these tweezers is relatively disfavored, rationalizing the only partial closing of the tweezers. The performance of each tweezers design was quantitatively assessed by juxtaposing the enzyme glucose-6-phosphate dehydrogenase (G6pDH) with its cofactor NAD+ on the tweezers arms and measuring the G6pDH activity through a coupled enzyme cascade. Using our optimized tweezers, we were able to enhance the activity of G6pDH by up to ~9-fold. Our strategy for improving a DNA device using feedback from single-molecule experiments may represent a general approach to designing refined nanodevices for future applications.

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GRADUATE & POSTDOCTORAL PARTICIPANTS

Name Institution Email address

Aggarwal, Vasudha University of Illinois at Urbana-Champaign [email protected] Bernier, Morgan The Ohio State University [email protected] Beyer, David University of Iowa [email protected] Boehm, Elizabeth University of Michigan [email protected] Bourg, Julia University of Michigan [email protected] Brehove, Matthew The Ohio State University [email protected] Brenlla, Alfonso Wayne State University [email protected] Budhathoki, Jagat Kent State University [email protected] Cameron, Elizabeth University of Michigan [email protected] Casy, Wilder University of Missouri [email protected] Chada, Nagaraju University of Missouri [email protected] Chen, Ran University of Iowa [email protected] Choi, Heejun University of Wisconsin- Madison [email protected] Chowdhury, Farhan University of Illinois at Urbana-Champaign [email protected] Chuang, Cho-Ying Michigan State University [email protected] Cuculis, Luke University of Illinois [email protected] DeSantis, Michael University of Michigan [email protected] Dhakal, Soma University of Michigan [email protected] Gibson, Matthew The Ohio State University [email protected] Gros, Adam Michigan State University [email protected] Hao, Linxuan Washington University in St. Louis [email protected] Hejna, Miroslav University of Illinois at Urbana-Champaign [email protected] Hudoba, Michael The Ohio State University [email protected] Izadi, Dena Michigan State University [email protected] Kafle, Rudra University of Michigan [email protected] Kapil, Dave University of Illinois at Urbana-Champaign [email protected] Ke, Haixin Washington University in St. Louis [email protected] Kelliher, Michael University of Wisconsin-Madison [email protected] Kim, Jin H. University of Michigan [email protected] Kim, Sunae Iowa State University [email protected]

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Name Institution Email address

Kim, Younghoon University of Illinois at Urbana-Champaign [email protected] Kruk, Katie University of Michigan [email protected] Lee, Stephen University of Michigan [email protected] Lenhart, Justin University of Michigan [email protected] Li, Isaac University of Illinois [email protected] Li, Wenting University of Wisconsin-Madison [email protected] Li, Zhengda University of Michigan [email protected] Lin, Chang-Ting University of Illinois at Urbana-Champaign [email protected] Liyanage, Pramodha Wayne State University [email protected] Lou, Xiaochu Iowa State University [email protected] Luo, Yi The Ohio State University [email protected] Manibog, Kristine Iowa State University [email protected] Marsh, Brenden University of Missouri [email protected] Menke, Drew University of Missouri [email protected] Mohapatra, Sonisilpa University of Wisconsin-Madison [email protected] Ngy, Thuy University of Illinois at Urbana-Illinois [email protected] Nguyen, Benh Washington University in St. Louis [email protected] Norman, Zenia University of Missouri [email protected] Numez, Marcos University of Michigan [email protected] Ordabayev, Yerdos Washington University in St. Louis [email protected] Pang, Yuanjie University of Michigan [email protected] Park, Seongin University of Illinois [email protected] Patrick, Eric Michigan State University [email protected] Pennella, Min University of Missouri [email protected]

Qiu, Yupeng University of Illinois at Urbana- Champaign [email protected] Radzinski, Nikolai University of Wisconsin-Madison [email protected] Ramreddy, Tippana University of Illinois at Urbana-Champaign [email protected] Regan, Emma University of Michigan [email protected] Rodgers, Margaret University of Wisconsin-Madison [email protected] Roeinpeikar, Mehdi University of Illinois at Urbana-Champaign [email protected] Rowland, David University of Michigan [email protected] Rube, Tomas University of Illinois at Urbana-Champaign [email protected]

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Name Institution Email address Sanaganna Gari, R.R. University of Missouri [email protected] Savage, Alan Ohio State University [email protected] Schimert, Kristin University of Michigan [email protected] Schmidt, Patrick Iowa State University [email protected] Shafraz, Omer Iowa State University [email protected] Shebl, Bassem University of Missouri [email protected] Sherani, Aiman University of Michigan [email protected] Shin, Jaeil Iowa State University [email protected] Sigdel, Krishna University of Missouri [email protected] Simenson, Angelynn University of Missouri [email protected] Skootsky, Joshua University of Michigan [email protected] Sokoloski. Joshua Washington University in St. Louis [email protected] Stekas, Barbara University of Illinois at Urbana-Champaign [email protected] Su, Xin University of Michigan [email protected] Teeling-Smith, Richelle The Ohio State University [email protected] Teng, Kai wen University of Illinois at Urbana-Champaign [email protected] Tomko, Eric Washington University in St. Louis [email protected] Tuscon, Hannah University of Michigan [email protected] Wang, Jiarui University of Michigan [email protected] West, Jess Michigan State University [email protected] Whitley, Kevin University of Illinois in Urbana-Champaign [email protected] Whitmore, Miles Michigan State University [email protected] Widom, Julia University of Michigan [email protected] Wu, Colin University of Iowa [email protected] Wurm, Sarah The Ohio State University [email protected] Yang, Zhilin University of Wisconsin-Madison [email protected] Yen, Chi-Fu Iowa State University [email protected] Yoo, Jejoong University of Illinois in Urbana-Champaign [email protected] Yoshua, Samuel University of Toronto [email protected] Young, Isaac Iowa State University [email protected] Zeng, Yi University of Chicago [email protected]

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FACULTY PARTICIPANTS

Name Institution Email address

Aksimentiev, Aleksei University of Illinois in Urbana-Champaign [email protected] Chemla, Yann University of Illinois in Urbana-Champaign [email protected] Cheng, Wei University of Michigan [email protected] Comstock, Matthew Michigan State University [email protected] Cornish, Peter University of Missouri [email protected] Galburt, Eric Washington University in St. Louis [email protected] Goldsmith, Randall University of Wisconsin-Madison [email protected] Ha, Taekjip University of Illinois in Urbana-Champaign [email protected] Hoskins, Aaron University of Wisconsin-Madison [email protected] King, Gavin University of Missouri [email protected] Kowalczykowski, Stephen University of California, Davis [email protected] Lohman, Timothy Washington University in St. Louis [email protected] Meiners, Jens-Christian University of Michigan [email protected] Ritchie, Kenneth Purdue University [email protected] Schroeder, Charles University of Illinois in Urbana-Champaign [email protected] Sivasankar, Sanjeevi Iowa State University [email protected] Spies, Maria University of Iowa [email protected] Wang, Yan Mei Washington University in St. Louis [email protected] Washington, Todd University of Iowa [email protected] Yang, Qiong University of Michigan [email protected] Yodh, Jaya University of Illinois in Urbana-Champaign [email protected]

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